Arthritis & Rheumatism, 2001 Annual Scientific

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THE DISEASE COURSE OF PATIENTS WITH RHEUMATOID ARTHRITIS IN REMISSION; A PROSPECTIVE 2 YEAR FOLLOW-UP STUDY. Esmeralda T Molenaar, Alexandre E Voskuyl, Huib J Dinant, Dick P Bezemer, Ben A Dijkmans Amsterdam, The Netherlands

LOSS OF CORTICAL BONE AND ASSOCIATED FACTORS IN A RHEUMATOID ARTHRITIS COHORT. Jose F Roldan, Inmaculada del Rincon, Agustin Escalante San Antonio, Texas

Objectives: To assess the prevalence of persistent remission and factors associated with persistent remission in a large cohort of RA patients in remission. Methods: RA patients in remission according to the ACR/Pinals criteria were evaluated every 3 months for disease activity during 2 years. Factors measured at baseline and possibly associated with persistent remission were evaluated. Results: Of the 180 RA patients included 93 (52%) experienced persistent remission. Those patients had significantly (p⬍0.05) less tender joints, lower Ritchie score, lower VAS score, lower serum IgM-RF and IgA-RF levels, lower HAQ score at baseline when compared to patients suffering an exacerbation. DMARD use was not associated with persistent remission. In a multivariate analysis the following variables were independently associated with persistent remission: DAS⬍1.6 (odds ratio with 95% Cl: 3.7, 1.5-9.5) and VAS⬍12 mm (1.1, 1.0 –1.2). The area under the curve of the DAS⬍ 1.6 in the first year was associated with remission in the second year (9.9, 4.1–24). Conclusion: Persistent remission occurs frequently and is associated with a low disease activity score (DAS), in particular with a low cumulative DAS. The use of a cumulative DAS may be a tool in taking therapeutic decisions. Disclosure:

BACKGROUND: The combined cortical thickness of the second metacarpal bone (CCT-MC) can be used to measure diaphyseal cortical bone from a plain X-ray of the hands. Prior cross-sectional studies have shown that the CCT-MC is associated with the severity of rheumatoid arthritis (RA). OBJETIVE: To identify factors associated with CCT-MC and its loss over time in a cohort of patients with RA. METHODS: We studied 507 RA patients. Their mean age was 54.4, 71% were women, 35.6% had rheumatoid nodules, 82 % had a positive rheumatoid factor (RF) and 72.87% had shared epitope (SE) positive HLA-DRB1 alleles. 47% were taking prednisone. We measured CCT-MC on digitized X-rays as the distance between the endosteal and periosteal diameters at the midpoint of the second metacarpal bone, expressed in millimeters. An average of both sides was taken. We used generalized estimating equations (GEE) to assess factors associated cross-sectionally with the CCT-MC. To measure the rate of loss of CCT-MC over time, we included the time between X-rays as a covariate in GEE. To evaluate the effect of demographic, clinical and genetic predictors on the rate of loss of CCT-MC, we tested an interaction term between each variable of interest, and the time between X-rays. RESULTS: 811 hand radiographs were available for study. The mean interval between the first and followup X-ray was 1.5 years (range 0 to 23 years). The mean CCT-MC on the baseline X-ray was 4.09 mm (95% C.I. 3.99, 4.19). Factors associated cross-sectionally with the CCT-MC included age (P ⱕ 0.001), sex (P ⱕ 0.001), race ( P ⱕ 0.001), body mass index (P ⱕ 0.001), ESR (P ⫽ 0.001), rheumatoid nodules, (P ⱕ 0.001), the number of joint deformities(P ⱕ 0.001), and the duration of prednisone use (P ⱕ 0.001). In longitudinal analyses, the mean CCT-MC rate of loss was 0.09 mm/year (95% C.I. 0.08, 0.10). Factors associated with accelerated loss of cortical bone included Hispanic ethnicity (rate of loss ⫽ 0.11 mm/yr vs. 0.07 mm/yr, P ⫽ 0.04), SE-positive HLA-DRB1 allele (rate of loss 0.11 vs. 0.08, P ⫽ 0.01), and use of prednisone (0.11 vs. 0.07, P ⫽ 0.04). CONCLUSIONS: The CCT-MC in RA is associated with demographic and anthropometric characteristics and disease severity. Cortical bone is lost at an accelerated rate among Hispanic RA patients, those with HLA-DRB1 allleles containing the SE, and patients taking prednisone. Future studies should evaluate the role of the CCT-MC as a predictor of outcome in RA, and the mechanisms for cortical bone loss in RA. Disclosure: This research was supported in part by NIH grants HD37151, HL04481 and AR47530, a Clinical Science Grant from the Arthritis Foundation, and a Beginning Grant-in-Aid from the Texas Affiliate of the American Heart Association.

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DISCONTINUATION OF DMARD AFTER REMISSION IN PATIENTS WITH RHEUMATOID ARTHRITIS. Yong-Beom Park, Jung-Min Kim, Soo-Kon Lee Seoul, Republic of Korea

NO REDUCED BONE MINERAL DENSITY IN PATIENTS WITH EARLY RHEUMATOID ARTHRITIS. Christina Book, Kristina Akesson, Inga Redlund-Johnell, Lennart TH Jacobsson Malmo, Sweden

Background: The goal of rheumatoid arthritis (RA) treatment is remission. Many RA patients have experienced remission after disease-modifying anti-rheumatic drugs (DMARD) therapy. However, drug therapy after the remission has not been established yet. Although successful low-dose methotrexate maintenance therapy was introduced recently, the discontinuation of DMARD is always a desire for patients and physician. In this study, we attempted to determine how percentage of patients was maintained without relapse after the discontinuation of DMARD and to evaluate the factors related to the relapse of RA among patients who experienced relapse. Methods: Eighteen patients with RA who were volunteers for discontinuation of DMARD were enrolled in this study. All patients were diagnosed by ACR criteria (1987) and they all discontinued DMARDs after the stable period of RA remission. The RA remission was defined by ACR preliminary remission criteria (1981). RA-related clinical and laboratory disease activity variables at diagnosis and clinical course of patients were examined and HLA typing for the susceptible alleles of RA were performed. Results: Four male and fourteen female patients with RA were studied. Their mean age was 41.5 ⫾ 9.8 years old and mean follow-up duration after the discontinuation was 8.6 ⫾ 3.3 months.Twelve patients (66.7%) experienced RA relapse after the mean duration of 3.6 ⫾ 1.8 months. However, six patients did not experience the relapse. The patients with relapse group had higher tender joint counts at diagnosis and longer duration from the diagnosis to the remission than non-relapse group. HLA DR␤1 genotyping showed no difference between the two groups. Conclusion: The remission of RA is sustained in only one-third patients after the discontinuation of DMARD. More severe disease at diagnosis and longer period to achieve the remission may be related to the relapse of RA after the discontinuation of DMARD.

Objects. Several reports have demonstrated a reduced bone mineral density (BMD) in patients with prevalent rheumatoid arthritis (RA). To examine whether RA patients have low BMD from onset of disease we examined a cohort of patients with early RA. Methods. One hundred and sixty-eight consecutive patients [118 women (70%) and 50 men (30%)] with RA and disease duration ⱕ12 months within a defined catchment area were recruited from 1995 through 2000. The mean age at onset of disease was 61 years (range 21-84). Ninety-nine patients (59%) had positive rheumatoid factor (RF). The examination included radiographs of hands and feet and a structured clinical evaluation. Dual energy X-ray absorptiometry (DXA) was performed on 156 patients. Results. There was no statistically significant BMD reduction in this group of RA patients compared to age matched controls. The mean Z score for the femoral neck was for men -0.45, for women -0.12 and for all patients -0.21. The mean Z score for L2-L4 was for men 0.21, for women -0.25 and for all patients -0.12. BMD was not associated with measures of disease activity, apart from a weak association between RF seropositivity and BMD in L2-L4 (P ⬍0.04). There was no correlation between BMD and radiological erosions or periarticular bone loss of the hands or feet. Conclusions. At onset of disease we could not detect any statistically significant bone loss within this group of patients with early RA compared with age matched controls. This suggests that prevention for osteoporosis is feasible in early RA. Disclosure:

Disclosure:

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APPENDICULAR AND GENERALIZED BONE LOSS IN RA OF RECENT ONSET - A ONE YEAR PROSPECTIVE STUDY. Hanne Merete Lindegaard, Kim Horslev-Petersen, Bo Abrahamsen, Peter Junker Odense and Graasten, Denmark

THE RELATION BETWEEN DISEASE ACTIVITY AND RADIOLOGIC PROGRESSION OF JOINT DAMAGE IN RHEUMATOID ARTHRITIS. Paco MJ Welsing, Hilde L Swinkels, Anke M van Gestel, Piet LCM van Riel Nijmegen, Netherlands Objective:To investigate the relation between disease activity and radiologic progression of joint damage in RA in an open study and factors that might confound or modify this relation. Methods: The follow-up data of 190 patients included in an open prospective study of early RA with a follow-up time from 3 to 9 years were used for this study. Disease activity was measured by the Disease Activity Score (DAS), and radiologic progression was measured using the modified Sharp score. Patients were classified as having continuously high or low disease activity over 6 years, and radiologic progression scores were compared. To further analyse this relation, a Generalised Estimated Equations (GEE) model was used. As outcome variable 3-yearly progression scores and as independent variables the mean DAS, and it’s standard deviation (sd) over 3-year periods, age, gender, and rheumatoid factor (RF) were used. The influence of a time lag between disease activity and radiologic progression and of disease duration was studied using an interperiod correlation matrix. To investigate if the relation between disease activity and radiologic progression was more pronounced in joint groups than in aggregated measures, correlations were compared. Correlations between disease activity and progression of cartilage destruction and bone erosions were compared. Effect modification by DMARD treatment and the effect of measurement frequency were investigated in a treatment trial comparing Sulfazalazine Methotrexate and the combination. Results: 21 and 11 patients had continuous low (DAS 1.9 sd 0.35) and high disease activity (DAS 4.2 sd 0.37) respectively, their median progression scores were 39 and 82 (p⫽0.13). In the GEE model it was shown that the 3-yearly progression scores decreased with time, the mean DAS, and especially the sd of the DAS, a positive RF and higher age increased the progression score. No time lag between disease activity and radiologic progression or an influence of disease duration was found. The correlation between swelling and pain and radiologic progression was higher in joint groups than in aggregated measures. Correlations between cartilage destruction and erosions and DAS were comparable. In the treatment trials the correlations between DAS and radiologic progression did not differ between treatment groups or with different measurement frequencies. Conclusion: There is a longitudinal relation between disease activity (the mean DAS and its fluctuation) and radiologic progression of joint damage. Disclosure:

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PLACE ART HERE 10.0p x 3.1p Wednesday, November 14

AIM: To study the correlation between markers of connective tissue metabolism and the rate of appendicular/generalized bone mineral loss within one year after a diagnosis of RA. METHODS: Forty-five patients fulfilling the 1987 ACR criteria for RA, with disease duration less than one year were included. All were DMARD and glucocorticosteroid naive. None received bisphosphonates. Patients were categorized into high-risk (HR) and low-risk (LR) subsets according to the number of severity predictors. DXA-scans (Hologic QDR-2000 or Norland XR-36) of the lumbar spine, left hip, non-dominate forearm and hand were performed at the time of diagnosis and after 6 and 12 months treatment with Methotrexate. 19/26 patients belonged to the HR/LR subsets. RESULTS: Serial BMD measurements showed that the rate of bone loss in the forearm and 3rd MCP joint was higher in HR as with compared with LR patients (p ⬍0.05). This finding contrasts with equal decreases of BMD in the lumbar spine, hip and ultradistal part of radius and ulna in the two risk categories. Serum CRP, Hyaluronan and the N-terminal collagen III propeptide (PIIINP) were positively correlated to the annual percent change in BMD of wrist and finger joints. Bone formation as judged by C- and N-terminal procollagen I peptides (PICP and PINP) remained stable within the normal range whereas bone degradation estimated by a cross-linked collagen I fragment (CTx) rose transiently within the first three months of follow up. CONCLUSION: Despite optimal disease control as estimated by clinical and serological indices, RA of recent onset is associated with progressive loss of trabecular and cortical bone, particularly at appendicular sites. Current antiinflammatory medication is unable to prevent appendicular osteopenia, which is primarily due to enhanced bone degradation. This raises the possibility that combination therapy with antiinflammatory and antiresorptive agents could be beneficial in early RA, particularly in high-risk patients.

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ENHANCED EXPRESSION OF OSTEOPONTIN (OPN) IN RHEUMATOID JOINTS -A POSSIBLE MARKER FOR DISEASE ACTIVITY IN RHEUMATOID ARTHRITIS. Shiro Ohshima, Jorg Schedel, Norihoko Yamaguchi, Katsuhiro Nishioka, Toru Mima, Masatoshi Shimizu, Yoshinori Katada, Shigeyuki Wakitani, Norikazu Murata, Hiroaki Matsuno, Rie Katayama, Shigeyuki Kon, Toshimitsu Uede, Peter Petrow, Beat A Michel, Renate E Gay, Steffen Gay, Yukihiko Saeki Osaka, Japan and Zurich, Switzerland

DEPRESSION MEDIATES THE RELATIONSHIP BETWEEN PAIN AND COGNITIVE FUNCTION IN RHEUMATOID ARTHRITIS PATIENTS. Scott C Brown, Jennifer M Glass, Denise C Park, Leslie J Crofford Ann Arbor, MI

Objective: To investigate the involvement of osteopontin (OPN) in the pathogenesis of rheumatoid arthritis (RA), the expression of OPN was examined in RA. Methods: Expression and localization of OPN in RA synovium were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). Moreover, both synovial fluid (SF) and plasma OPN levels were determined in patients with RA by a novel ELISA assay, which enables us to measure both thrombin-cleaved and non-cleaved OPN. The same assays were also done in patients with osteoarthritis (OA) and healthy controls (HC). Results: IHC showed strong expression of OPN predominantly in the synovial RA lining cells, while less and scattered OPN expression was observed in OA synovium. ISH showed a similar expression pattern of OPN in RA synovium. ELISA revealed that OPN levels in SF of both RA and OA patients were markedly increased compared with those in plasma (RA, p⬍0.05: OA, p⬍0.05), although there were no significant differences in plasma OPN levels among the samples of RA, OA, and HC. Most remarkably, levels of OPN in RA SF were significantly higher than those in OA SF (p⬍0.05) and correlated with serum CRP levels (r⫽0.798, p⬍0.0001). Interestingly, the ratio of thrombin-noncleaved versus cleaved OPN levels was significantly decreased in both SF and plasma of the RA patients compared with either those of the OA patients or HC (SF; RA vs OA; p⬍0.05, plasma; RA vs OA or HC, p⬍0.05). OPN (ng/ml)

RA (n⫽23) OA (n⫽17) Healthy controls (n⫽10)

SF 10825⫾2285 4405⫾815

Plasma 655⫾98 812⫾88 663⫾48

Thrombin-non-cleaved/cleaved ratio SF 0.969⫾0.244 1.975⫾0.435 -

Plasma 0.938⫾0.312 2.266⫾0.412 2.576⫾0.281

Conclusions: The present study revealed enhanced local expression of OPN in the rheumatoid joint. In addition, the ratio of thrombin-cleaved versus non-cleaved OPN might be a novel marker to assess the disease activity of RA.

BACKGROUND: Rheumatoid arthritis is a systemic disease whose cognitive effects are largely unknown. Although much work has examined the impact of cognitive activity on pain and depression, relatively few studies have considered the possible impacts of chronic physical and psychological distress on cognitive function in arthritis patients. OBJECTIVE: The purpose of this study was to assess the hypothesis that pain and depression negatively impact the cognitive functioning of rheumatoid arthritis patients. METHODS: One hundred twenty-one community-dwelling rheumatoid arthritis patients (ages 34 to 84) completed a battery of cognitive tasks and multiple measures of pain and depression. Structural equation modeling techniques were used to assess the relative contributions of age, pain, and depression to cognitive performance. RESULTS: Individuals who performed poorly on cognitive tasks reported more pain and depression and were older than those individuals who performed well on cognitive tasks. Moreover, high levels of pain were associated with depression. Further analyses revealed that depression mediated the relationship between pain and cognition. That is, when depression was entered into the analyses after pain, the previously significant effects of pain on cognition were no longer found. Interestingly, depression still mediated the pain-cognition relationship, even after controlling for age. The hypothesized model explained 55% of the variance in cognition. CONCLUSIONS: High levels of pain were associated with poor cognitive performance in rheumatoid arthritis patients. However, pain’s impact on cognition appeared to occur indirectly, through pain’s impact on depression, which in turn caused cognitive dysfunction. In other words, pain had no direct effect on cognition, after controlling for depression. These findings suggest the importance of both pain and depression in understanding cognitive function in rheumatoid arthritis and may have important implications for treatment of this disease.

Disclosure: S. Ohshima is supported by TOYOBO Biotechnology Foundation. All others by their respective institutions.

Disclosure:

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HYALURONIC ACID (HA) LEVELS IN RHEUMATOID ARTHRITIS: CORRELATION WITH VARIOUS MEASURES OF DISEASE ACTIVITY. Luis R Lopez, Rafael G Grau, Luis R Espinoza Westminster, CO; Indianapolis, IN and New Orleans, LA

PSYCHOLOGICAL STRESS INDUCED BY DEEP EMOTION WITH TEARS EFFECTS ON NEUROENDOCRINE AND IMMUNE RESPONSE IN PATIENT WITH RHEUMATOID ARTHRITIS. Hiroyasu Ishii, Gayo Iwakawa, Masakazu Nagashima, Shin-ichi Yoshino Tokyo, Japan

Objective: To study the correlation of serum Hyaluronic Acid (HA) levels with serologic and clinical measures of disease activity in patients with rheumatoid arthritis (RA). Methods: 189 RA patients (155 females, 34 males) and age/sex matched controls were enrolled. The mean age for RA patients was 54.9 years (range 26-88 years). The mean disease duration was 11.8 years. Patients with liver disease were excluded. Serum HA levels were measured by an enzyme-linked protein binding assay using hyaluronic acid binding protein as a capture reagent. HA, RF, CRP and ESR were measured and clinical measures of disease activity were recorded at enrollment. Results: Serum HA levels were positive (⬎100 ng/mL cut-off) in 23.8% of RA patients compared to 2.2% of the controls. The RA mean HA level (75.4 ng/mL) was significantly elevated (controls mean 20.1 ng/mL, p⬍0.001). In this group of RA patients, 90.3% had elevated RF titers, 55.6% increased CRP levels and 71.6% elevated ESR. Disease duration (years) did not correlate with HA levels (p⬎0.237). The mean AM stiffness was 90.1 minutes and no statistical correlation (p⬎0.207) with HA levels was found. ACR (I-IV) functional class showed a positive correlation with HA levels (class I ⫽ 50.2 ng/mL, II ⫽ 75.0 ng/mL and III ⫽ 77.2 ng/mL) but did not reach statistical significance (p⫽0.147). The physician’s global disease assessment (scale 0 to 10) showed a significant correlation (p⬍0.05) with HA levels (scores 0-3.3 ⫽ 62 ng/mL, 3.6-6.6 ⫽ 78 ng/ml and 6.7-10 ⫽ 112 ng.mL). The patient’s global disease assessment showed a significant correlation (p⬍0.001) with HA levels (scores 0 –3.3 ⫽ 71 ng/mL, 3.6-6.6 ⫽ 74 and 6.7-10 ⫽ 85 ng/ml). The swollen/painful joint count also showed a significant correlation (p⬍0.001) with HA levels. When RF, CRP and ESR levels were correlated with these disease activity measures, weak positive correlations that did not reach statistical significance were observed with the last 4 parameters. Conclusions: HA levels are elevated in RA and correlated with most measures of disease activity in this study. The correlation with HA was stronger than that for standard serologic measures of disease activity i.e. RF, CRP and ESR. HA is normally produced in the synovium and its synthesis increased during joint inflammation, making HA a useful serologic marker for disease activity/synovium involvement in RA.

OBJECTIVE It has been hypothesized that psychological stress affects the condition of patients with rheumatoid arthritis (RA). We have recently demonstrated that the effects of mirthful laughter or general anesthesia on neuroendocrine and immune system of patients with RA. In this study, we evaluated the relationship between neuroendocrine and immune responses in serum levels and psychological stress induced by deep emotion with tears in patients with RA. METHODS Ten RA patients with well-controlled, serum C-reactive protein level (CRP⬍1.0) “controlled RA patients” and 10 RA patients with uncontrolled, serum CRP level (CRP⬎1.0) “uncontrolled RA patients” were compared with 20 healthy subjects after listening to a sob story that moved them to tears. Neuroendocrine and immune responses of RA patients and normal subjects were evaluated by measuring levels of serum cortisol, catecholamines, cortisol releasing hormone (CRF) and interleukine-6 (IL-6), as well as CD4/CD8 ratio and NK cell activity. RESULTS Two-way analysis of variance (group/time) revealed a significant difference in serum cortisol level, CD4/CD8 ratio, NK cell activity and serum IL-6 level between controlled and uncontrolled RA patients during the stress session. The uncontrolled RA patients were much more sensitive than the controlled RA patients. CONCLUSIONS Our study reveals that neuroendocrine responses to psychological stress were significant in the two groups of RA patients. In conclusion, psychological stress induced by deep emotion with tears has a more influence on neuroendocrine and immune responses in uncontrolled RA patients. Disclosure:


Disclosure: Supported by Corgenix Inc. Dr. Lopez is employee of Corgenix and stock owner.

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PROGNOSTIC VALUE OF ANTIPERINUCLEAR AND ANTIKERATIN ANTIBODIES IN EARLY RA. AN 8 YEAR PROSPECTIVE STUDY. Stephane Genevay, Gilles Hayem, Patrice Verpillat, Olivier Cyrille Meyer

SERUM YKL-40 LEVELS IN PATIENTS WITH RHEUMATOID ARTHRITIS. Julia S Johansen, Michael Hansen, Mette Klarlund, Paul A Price, Birgitte Volck, Ib Lorenzen, DRD1 Group, TIRA Group Hvidovre, Denmark and La Jolla, CA

Objective : Antikeratin (AKA) and antiperinuclear (APF) antibodies (antifilaggrin antibodies) are specific antibodies found very early in rheumatoid arthritis (RA). We designed an 8 year prospective study on early RA patients in order to assess the prognostic significance of AKA and APF on functional disability and cartilage damage. Method : A cohort of 64 patients tested for AKA and APF in 1992 early in the course of a symmetrical oligo or polyarthritis were contacted in 2000. Twenty-nine patients were able to be re-evaluated. The diagnosis of RA was confirmed in 25 out of 29. Clinical, biological (including HLA-DR genotyping) data along with hands and feet X-ray were obtained. Health Assessment Questionnaire (HAQ), Disease Activity Score (DAS) and radiological damage (Larsen score) were primary endpoints. Non parametric tests were used for statistical comparisons. Results : Among the 25 patients with RA, 9 were positive for rheumatoid factor (RF), 9 for AKA and 6 for APF during the first year of their disease. APF were associated with a higher Larsen score (22.5 vs 6.7 ; p⬍0.02) and with a higher DAS score (3.98 vs 2.26; p⬍0.04) evaluated at a mean of 8.55 years after disease onset. AKA were associated with a higher DAS score only (3.75 vs 2.16; p⬍0.04). All APF patients had significant erosions (defined as a Larsen score greater than 4). Neither RF nor the presence of the shared epitope did influenced the disease outcome. Conclusion: RA patients initially tested positive for APF had a higher score of disease activity and a higher score of radiological destruction, all of them having significant erosions at the time of evaluation. AKA positive patients were found to have a more active disease. These data suggest that early determination of antifilaggrin antibodies may help to identify patients with RA with a more active and a more destructive disease after a long term (⬍8 years) duration.

Objectives: YKL-40 (human cartilage glycoprotein-39) is secreted by chondrocytes, synovial cells, macrophages and neutrophils. YKL-40 is an autoantigen in rheumatoid arthritis (RA), and high serum levels of YKL-40 are found in diseases characterized by inflammation or fibrosis. The aim was to evaluate changes in serum YKL-40 in patients with RA during different treatments and seek associations with changes in swollen joints, the acute phase reactants and joint destruction. Methods and Patients: Serum YKL-40 was determined by RIA in 395 patients with RA (female/male: 305/90; median age 60 years (range 20-85); disease duration 3.5 years (0.1-44)). The patients were followed for one year during treatment with DMARD’s ⫹/- glucocorticoids. Larsen score of the hands were determined initially and after 1 year. Results: Patients with active RA (n⫽310) at inclusion had significantly (p⬍0.001) higher serum YKL-40 (median 197 ng/ml, range 34 –1280) than patients with inactive RA (150 ng/ml, range 40 –736) and age-matched controls (103 ng/ml). Serum YKL-40 was elevated compared to the upper normal level in 45% of the patients with active RA and in 16% of the patients with inactive RA. There was no relation between serum YKL-40 and disease duration. The percentage changes in serum YKL-40 during the study were significantly (p⬍0.0001) correlated with the percentage changes in the number of swollen joints (rho⫽0.49), serum CRP (0.52) and ESR (0.50). 49% of the MTX treated patients had a significant decrease (i.e. ⬎20% decrease in serum YKL-40 compared to the initial value) after 3 months of treatment and 46% had a decrease after 12 months. The corresponding values were for patients treated with sulfasalazine (50% had a decrease at 3 months and 79% at 12 months), leflunomide (35% at 3 months and 32% at 12 months), penicillamine (35% at 3 months and 43% at 12 months). High doses of prednisolone (15-30 mg p.o./day) resulted in a significant decrease in 53% of the patients after 1 day and in 67% after 4 weeks. The initial value of serum YKL-40 was not related to the number of bone erosions at inclusion or the progression in Larsen score. However, a persistently elevated serum YKL-40 during the 1 year study was related to a progression in Larsen score (Chi-square: p⫽0.029). Conclusion: Serum YKL-40 levels may be useful for follow-up of the treatment of RA patients. Disclosure:

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STUDY INCLUSION CRITERIA IN RA: PATIENT SELECTION SIGNIFICANTLY EFFECTS OUTCOME. Mark A Quinn, Philip G Conaghan, Paul Astin, Michael J Green, Paul Emery

PREVALENCE OF HEPATITIS C VIRUS INFECTION IN RHEUMATOID ARTHRITIS PATIENTS. Jean-Francis Maillefert, G Muller, JB Bour, Maxime Dougados, Christian Tavernier, Maxime Breban Dijon and Paris, France

Background: Clinical trials in RA have variable inclusion criteria. Limiting patients allowed into studies may have a marked influence on outcome, particularly when using modern composite outcome measures. Such inclusion criteria may limit the application of study findings to the general RA population. Although widely debated the ACR 20 response criteria has been advocated as the primary measure of efficacy in RA trials. In a large secondary care cohort of early RA patients treated with standard therapy, the affect of commonly applied inclusion criteria such as joint counts, elevated CRP and specified disease activity scores on ACR 20 response was studied. Methods: The Yorkshire Early Arthritis Register database was used. To date 230 consecutive patients with RA, symptom duration ⬍12 mo, DMARD naive, have been treated with sulphasalazine mono-therapy for 6 months. Outcome was measured using ACR 20 after application of set inclusion criteria. Results:

Inclusion criteria

n⫽

% RF⫹

Mean age (years)

Median symptom duration (mths)

ACR 20 response

All patients SJC⬎8/TJC⬎10 CRP⬎10mg/l SJC⬎8/TJC⬎10 ⫹ CRP⬎10mg/l DAS28 ⬎5.1 (severe) DAS28 ⬎3.2 ⬍5.1 (moderate) DAS28 ⬍3.2 (mild)

230 107 145 74 167 52 4

69 65 70 70 66 76 75

57 59 59 60 58 54 57

5 4 5 4 5 5 5

50% 60% 56% 65% (p⬍0.05) 60% 35% (p⬍0.05) 0% (p⬍0.05)

Conclusion: In patients with early RA, stipulating inclusion criteria for studies may significantly influence ACR 20 improvement at 6 months. Study inclusion criteria may be as important as outcome measures when interpreting results of clinical trials. If study results are to be compared, not only must conventional demographic data be consistent, but also measures of disease activity.

Background. A number of virus have been implicated in the etiology and pathogenesis of RA. Hepatitis C infection, which has been recognized has a cause of some autoimmune diseases, such as sicca syndrome, and which has been described as sometimes presenting with rheumatic manifestations indistinguishable from RA, might be a candidate Objective. To evaluate the prevalence of hepatitis C virus infection in patients with rheumatoid arthritis (RA). Methods. Consecutive RA patients hospitalized in 2 departments of rheumatology were prospectively included. Patients’ sera were screened for the presence of antibodies to hepatitis C (enzyme-linked immuno-absorbent assay). Cases with positive serology were further evaluated by hepatitis C RNA PCR. Results. Two hundred and twenty five patients (167 women, 58 men, mean age ⫽ 53.3 ⫾ 14.7 years) were included. The mean disease duration was 65.6 ⫾ 85 months. Rheumatoid factors and antinuclear antibodies were found positive in 64 and 36% of patients, respectively. Systemic vasculitis was observed in 9 patients. Mean ESR and CRP were 37.6 ⫾ 31 mm at the first hour (normal ⬍10 mm), and 38.4 ⫾ 44 mg/l (normal range ⬍5 mg/l), respectively, with 48% patients considered as having active disease. Aspartate transaminase were increased in 12 patients, and alkaline phosphatase in 9. A positive hepatitis C serology was observed in 2 patients, including 1 with a previously-diagnosed hepatitis C infection. Hepatitis C RNA PCR was positive in both patients. Conclusion. We found a 0.89% prevalence of hepatitis C infection in RA patients, which did not differ from that one observed in the general French population (1%). These results do not support the participation of hepatitis C infection in the pathogenesis of RA. Disclosure:

Disclosure: We acknowledge the Arthritis Research Campaign for funding this project

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BODY COMPOSITION AND INSULIN-LIKE GROWTH FACTOR-1 LEVELS IN RHEUMATOID ARTHRITIS. Servet Akar, Abdurrahman Comlekci, Merih Birlik, Fatos Onen, Yigit Goktay, Dinc Ozaksoy, Nurullah Akkoc Izmir, Turkey

TEN YEARS FOLLOWUP OF RENAL FUNCTION OF PATIENTS WITH RHEUMATOID ARTHRITIS. Hiroshi Kajiyama, Chihiro Terai, Yumi Koseki, Hirotaka Kaneko, Atsushi Maeda, Katsuko Yamashita, Naoyuki Kamatani Tokyo, Japan

Purpose: Change in body composition (reduced lean body mass, bone mineral content [BMC] and increased fat mass) in rheumatoid arthritis (RA) patients has been described previously. This has been explained with changes in cytokine and hormone production and also with physical activity. However in these studies age and sex but not body mass index (BMI) matched controls were used. Primary objective of this study was to evaluate body composition in RA patients and to compare it with age, sex and BMI matched controls. Secondary objective was to investigate the serum insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) levels and their correlations with body composition alterations, if any existed. Methods: Sixty five RA patients (ACR 1987) (55 female and 10 male; mean age 54.9⫾10,8) and 31 healthy controls (25 female, 6 male; 53.8⫾ 8.6) were included in this study. Mean disease duration for RA patient was 9.2⫾9.6 years and mean ACR functional class was 2.3⫾1.0. Body composition was measured by bioimpedance analysis and dual-energy X-ray absorbsiometry (DXA). Visceral adipose tissue (VAT) was assessed with computed tomography. Results: All body composition parameters except from BMC assessed by DXA were not significantly different from control group (results are shown in the table below). In contrary to previous reports, we found significantly high serum IGF-1 levels in RA patients (mean serum IGF-1 levels in patients were 237.4⫾58.0 ng/ml vs in controls 184.0⫾56.9 ng/ml; p⫽0.001). IGFBP-3 levels were also higher in patients compared with the controls; but this was not statistically significant (mean serum IGFBP-3 levels in patients were 3223.3⫾727.1 ng/dl vs in controls 3179.8⫾988.9 ng/dl; p⫽0.84). Conclusion: Body composition except from BMC is not different from age, sex and BMI matched controls.

RA Control

BMI (kg/m2)

% Body fat

%Lean

BMC (g)

VAT (cm2)

27.5⫾4,8 28.1⫾4.8

36.3⫾8.0 35.4⫾7.8

60.9⫾8.5 61.9⫾7.8

1805.2⫾358.8 2074.0⫾295.8

144.2⫾41.4 145.9⫾49.4

Abnormal urinary findings are often encountered in patients with RA. Serious renal dysfuction in patients with RA has been reported infrequently, while renal failure is the second or third major cause of death in patients with RA. This discrepancy may be partly derived from the confusion of evaluating renal involvements in patients with RA. Renal damage without abnormal urinalysis can be often overlooked and renal dysfunction in RA can be underestimated because of low daily creatinine production due to muscle atrophy of the patients. To overcome this problem, we followed the change of renal function during 10 years in 100 RA patients. Objective: To assess changes in the renal function of patients with RA during ten years. Methods: We reviewed clinical records of 100 patients with RA who have been attending to our outpatient clinic more than 10 years. Patients complicated with diabetes mellitus and nephritis were excluded. Serum levels of creatinine (Cr), blood urea nitrogen (BUN), hematuria, proteinuria, and blood pressure, as well as regular blood test and complete blood count of patients in 1991 and in 2001 were evaluated and compared with those of sex- and age-mathched healthy people who have been taking annual health checks for more than 10 years. Urinalysis was done using Ames dipsitcks test, and proteinuria and hematuria were defined positve when the dipstick showed ⫹ to 3⫹. Results: Average age of 91 female RA patients was 57.1 years and that of 9 male patients was 68.3 years. The average age and sex in controls were matched with the RA patients. The parameters of inflammation such as ESR and CRP as well as RAPA, Hb, and WBC in 1991 were not different from those in 2001. Proteinuria was positve in 4 RA patients in 1991 and another 4 patients developed drug-induced proteinuria during 10 years, however, no one showed proteinuria in 2001. Serum Cr and BUN of RA patients in 1991 (0.70⫾0.11 mg/ml and 16.3⫾4.57 mg/dl, respectively) were not different from those in 2001 (Cr 0.73⫾0.15 mg/ml and BUN 16.1⫾4.73 mg/dl). Cr and BUN of controls were 0.74⫾0.12 mg/ml and 14.2⫾2.97 mg/dl, respectively in 1991, and 0.76⫾0.12 mg/ml and 15.3⫾2.93 mg/dl, in 2001. No other variables of RA patients and controls were significantly changed during 10 years. Conclusion: No significant deterioration of renal function was demonstrated in RA patients, although several patients had experienced drug-induced renal damage. Disclosure:

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CONNECTIVE TISSUE METABOLISM IN PATIENTS WITH EARLY RHEUMATOID ARTHRITIS AND UNCLASSIFIED POLYARTHRITIS. Trine Jensen, Mette Klarlund, Michael Hansen, Stephan Christgau, Karl Erik Jensen, Henrik Skjodt, the TIRA Group Copenhagen, Denmark

INCIDENCE AND CHARACTERISTICS OF TUBERCULOUS INFECTION IN PATIENTS WITH AUTOIMMUNE DISORDERS. Cristina Vadillo, Cesar Hernandez-Garcia, Lydia Abasolo, Marina Salido, Cristina Lajas, Inmaculada C Morado, Esperanza Pato, Pilar Macarron, Benjamin Fernandez-Gutierrez, Antonio A Banarez, Juan A Jover Madrid, Spain

Objectives: Synovial inflammation and destruction of articular cartilage and subchondral bone are major manifestations of RA. Conventional markers of disease activity, e.g. swollen joints, ESR and CRP, assess the disease process. Biochemical markers, which more accurately reflect the pathological process in the joints are required. The aim was to determine and correlate the serum levels of biochemical markers of cartilage- and bone metabolism to early joints erosions in patients with early RA and unclassified polyarthritis. Methods: 51 patients with early RA and 21 patients with unclassified polyarthritis (UP) (median disease duration 3/3 months, range 1-24/1-24, respectively) with active joint disease were followed with monthly intervals up to 2 years. The synovial inflammation and cartilage degradation was assessed by: serum metalloproteinase 1 (MMP1) and metalloproteinase 3 (MMP3); cartilage and bone metabolism by serum pyridinoline (Pyd); bone formation by: serum N-terminal propeptide of type I collagen (PINP; and bone resorption by: serum C-telopeptide of type I collagen (CrossLaps™). Bone erosions in hands were determined by x-ray and MRI at entry and after 2 years. Results: At inclusion and the mean level during the first 6 months (based on 5 measurements) of serum MMP3 was significantly elevated in patients with RA compared to patients with UP. No differences in MMP1, Pyd, PINP and CrossLaps™ were found between the groups. The biochemical markers did not change significantly in patients, who became inactive at 24 months (n⫽18). No correlations were found between the initial values of MMP1, MMP3, Pyd, PINP and CrossLaps™ and the progressions in erosions, but the mean level of MMP3 correlated with radiological progression (rho⫽0.33, p⫽0.02). Patients with erosive disease (n⫽22) had elevated serum levels of MMP3 compared to patients with non-erosive disease (n⫽20) both at entry and during the study period (mean level: 49 vs. 16 ␮g/L, p⫽0.02). At inclusion serum MMP3 and Pyd correlated significantly with the number of swollen joints, ESR, CRP and to each other, while MMP1 and CrossLaps™ correlated significantly to swollen joints and ESR, respectively. Mean levels of MMP3 (based on 10 measurements) correlated significantly with the mean levels of swollen joints, ESR, CRP, MMP1, Pyd and CrossLaps™. Conclusion: Serum levels of MMP 3 seems to be correlated to disease activity and progression in radiological scores.

Objective. To describe the incidence and characteristics of tuberculous infection in patients with autoimmune disorders. Patients and Methods. We retrospectively identified and analyzed all new cases of tuberculosis diagnosed from Jan-91 to Dec-00 in patients with autoimmune disorders attending a rheumatology clinic in a tertiary hospital setting. The population at risk was obtained from a clinical database which included every patient attending the clinic. We calculated the total time of follow-up as the difference between first and last clinical appointment. In patients with rheumatoid arthritis (RA), total duration of treatment with a given drug was obtained from a database oriented at the longitudinal follow-up of patients with RA. We calculated the incidence rate as the fraction between the absolute frequency of tuberculous cases and the sum of individual at risk periods in each subgroup. Results. We identified 15 new cases of tuberculosis (13 females, mean age 61.1 years) in 1487 patients at risk followed for an accumulate period of 4501 years (global incidence rate 3.3/1000 patients-years). All these patients were therapies that included monotherapy or combined therapy with methotrexate (n⫽4), gold salts (n⫽1), azathioprine (n⫽8), cyclophosphamide (n⫽1), anti-TNF (n⫽2), and sulphasalazine (n⫽1). Eleven out of 15 patients were on corticosteroids. Tuberculosis location included lungs (n⫽5), genitourinary system (n⫽3), lymph nodes (n⫽3), splenic (n⫽1), central nervous system (n⫽1), and milliary tuberculosis (n⫽1). The incidence rates in patients with RA taken specific drugs was 2.03/1000 persons-year in patients treated with methotrexate, 43.5/1000 persons-year in patients treated with azathioprine, 156/1000 persons-year in patients treated with cyclophosphamide, and 49.2/1000 persons-year in patients treated with anti-TNF. Conclusions. The clinical characteristics of tuberculosis in patients with autoimmune disorders are atypical, with a higher frequency of extrapulmonar involvement and a variable rate as a function of treatments employed. Disclosure:

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THALIDOMIDE: EFFICACY AND SAFETY IN 30 PATIENTS WITH LUPUS AND SKIN INVOLVEMENT. M J Cuadrado, E Smith, P Gordon, Y Karim, M A Khamashta, G RV Hughes London, U.K.

PULSE METHYLPREDNISOLONE FOR SLE FLARES: A LOWER DOSE DECREASES THE RISK OF SERIOUS INFECTION WITHOUT REDUCING EFFICACY. Humeira Badsha, Kok Ooi Kong, Tsui Yee Lian, Christopher J Edwards, Hiok Hee Chng Singapore, Singapore

Background: Thalidomide can be very effective treatment for a variety of skin disease, including the cutaneous involvement in lupus, and often in cases that have been resistant to more conventional drugs. Objective: To study the efficacy and safety of thalidomide in systemic lupus erythematosus (SLE) patients with severe skin involvement refractory to conventional treatment. Methods: Patients with SLE and severe skin involvement, unresponsive to hydroxychloroquine, mepacrine, prednisolone or azathioprine betwen 1994 and 1999 were included. A starting dose of 50 or 100 mg daily for at least 4 weeks was used and reduced to 50mg on alternate days. The response to thalidomide was categorised as complete or partial remission or no visible improvement. All patients received a baseline electromyogram (EMG) followed by repeat EMG’s every 6 months or sooner if patients developed neuropathic symptoms. Results: Thirty patients (28 female, mean age: 38.3⫾7.4,range 36-56years) with either discoid lupus (n⫽7), subcutaneous lupus (n⫽5) or SLE with skin involvement (n⫽18) were included. The response rate was 93% with 18 (60%) patients in complete remission and 10 (33.3%) showing improvement of the skin lesions. Only 1 patient failed to respond. Improvement was apparent at 4 weeks of treatment with maximum benefit at 16 weeks. Eight patients developed peripheral neuropathy (6 patients were symptomatic with abnormal EMG, 2 asymptomatic with abnormal EMG), and 2 other patients complained of symptoms but EMG was normal. Four patients totally recovered after cessation of the drug but in the other 4, there was persistent neuropathy at 2 years. Other side effects included drowsiness (36.6%), bloating and abdominal pain (16.6%) and dermatitis (3.3%). The relapse rate after stopping the drug was 75%. Conclusions: Our results suggest that thalidomide is effective in the treatment of severe lupus with skin involvement. As it is teratogenic advice about contraception, where appropriate, is mandatory. Neurotoxicity is the main side effect. It is possibly dose related and a study to examine the efficacy and safety of lower dose thalidomide are currently underway in our unit. Disclosure:

Background:IV methylprednisolone (MEP) (1gm/day for 3 days) has been used to treat nephritis as well as other serious manifestations of systemic lupus erythematosus (SLE). However, its use has been complicated by an increased incidence of severe infections. In this study we sought to test our clinical impression that using lower doses of MEP (500mg/day for 3 days) was equally effective and associated with fewer serious infections. Methods: We conducted a retrospective study of SLE patients who had received either a low dose of pulse MEP (1-1.5 gms over 3 days) or a high dose of MEP (ⱖ3 gms over 3-5 days [1 patient received more than 3gms i.e. 5gms over 5 days]). 81 patients were identified as having received pulse MEP between 1989 and 2000. 26 patients were excluded because they were lost to follow-up, had insufficient data, or because their dose of MEP was neither the “high dose” nor “low dose”. 29 patients received the high dose while 26 patients had the low dose. SLEDAI scores and prednisolone doses were calculated at the time of MEP pulses and 6 months later. All serious infections (requiring admission and iv antibiotics) occuring during this 6 month period and their outcomes were recorded. In addition, demographic data, albumin, C3/C4, creatinine, glucose and immunosuppresive therapies were collected. Results: The two groups of patients were similar in demographic data, initial SLEDAI scores, use of IV cyclophosphamide, and organ involvement. Despite high- and low-dose MEP being equally efficacious (lowering of SLEDAI scores and prednisolone doses) there was a significantly lower incidence of serious infection in the low dose group (p⫽0.017) (table). There were 20 episodes of infection in 17 patients in the high-dose group compared to 9 episodes in 7 patients in the low-dose group. In both groups a majority of infections (75% and 77% in the high- and low-dose groups) occurred in the first month after IV MEP. Conclusion: “Low-dose” MEP (1-1.5 gms over 3 days) is effective in controlling SLE flares and is associated with fewer serious infectious complications than more traditional “high-dose” MEP (1gm/day for 3 days). Data

High-dose group (n⫽29)

Low dose group (n⫽26)

p-value

SLEDAI 0 months SLEDAI 6 months Serious infection : patients (episodes)

19.83 2.04 17 (20)

18.77 3.27 7 (9)

0.93 0.64 0.017

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HYDROXYCHLOROQUINE PREVENTS LATER DAMAGE IN SLE. M Petri Baltimore, MD

PROSPECTIVE STUDY OF REMISSION INDUCTION BY MYCOPHENOLATE MOFETIL IN LUPUS NEPHRITIS. Caroline Gordon, Jane Cross, Dwomoa Adu, David Jayne, for the BSR/RA UK Lupus Nephritis Study Group Birmingham, W.Midlands, United Kingdom

Hydroxychloroquine (Plaquenil) is known to reduce SLE flares and to improve lipid levels. We asked whether it had other long-term benefits in SLE. Methods: 933 SLE patients (pts.) enrolled in a longitudinal cohort were studied. They were 42% Af-Am (55% Cauc) and 92% female. Plaquenil was coded in 2 ways: present at first visit or taken at 50% or more of total visits. Clinical, laboratory, and damage variables were recorded at least yearly. Damage was measured using the SLICC/ACR Damage Index. Results: Damage Item

Plaquenil - 50% P-value

Plaquenil-Earliest Visit P-Value

Neuropsych Damage Cognitive Seizures CVA Renal Damage 2 GFR Proteinuria ESRD Gastrointestinal Premature Gonadal Failure Diabetes Total Damage Index

⬍0.0001 0.007 0.0009 0.02 ⬍0.0001 0.004 ⬍0.0001 0.0003 0.01 0.02 0.06 ⬍0.0001

0.02 0.02 NS NS 0.003 0.09 0.03 0.03 0.05 NS 0.03 0.0004


In addition, plaquenil was associated with fewer thrombotic events (p⫽0.04) and, as expected, lower cholesterol (p⫽0.001). Conclusion: The results suggest that early (first visit) or continued (50% of visits) use of Plaquenil might protect against later neuropsych or renal damage. However, only a clinical trial can determine if this is directly due to Plaquenil or due to a confounding variable, such as the subsets of SLE (skin, joints) for which Plaquenil is prescribed.

The optimal management of lupus nephritis remains uncertain. Intravenous (IV) pulse cyclophosphamide is widely used but there remains concern about toxicity and resistant cases. Mycophenolate mofetil (MMF) is a potential alternative to cyclophosphamide for the treatment of lupus nephritis. Case series of MMF in refractory lupus nephritis have shown benefit, and a small randomised trial found a similar therapeutic response to oral cyclophosphamide.This multi-centre, prospective, open pilot study was designed to assess the safety and tolerability of MMF with prednisolone for disease remission induction in lupus nephritis. Twenty four patients with WHO Class III, IV and V lupus nephritis, without previous exposure to cyclophosphamide or MMF, were enrolled in a 12 month study. MMF was administered at 2gm/day, with oral prednisolone (60mg/day reduced to 10mg/day by 3 months). For the purposes of the study, patients were assessed clinically and serologically every 3 months. Remission was defined as no evidence of disease activity (systems scored D or E) using the British Isles Lupus Assessment Group (BILAG) Index, a validated lupus disease activity index. Results for the 24 patients is documented below (mean ⫾ 1 standard deviation or % number of patients). Clinical features

Baseline

6 months

12 months

Proteinuria (g/day) Serum albumin (g/l)- normal ⬎36 Urinary (RBC/hpf) Creatinine(mmol/l)- normal ⬍120 C3 (g/l)- normal ⬍0.74 C4 (g/l)- normal ⬍0.14 Anti-dsDNA Abs IU (⬍74) All systems remission (BILAG) Renal remission (BILAG)

3.3 ⫾ 2.9 28.8 ⫾ 4.8 40 ⫾ 63 88.3 ⫾ 28.8 0.59 ⫾ 0.29 0.10 ⫾ 0.07 283 ⫾ 274 N/A N/A

1.3 ⫾ 1.8 36.5 ⫾ 6.9 18 ⫾ 26 85 ⫾ 20.4 0.74 ⫾ 0.34 0.13 ⫾ 0.07 57 ⫾ 50 36% 64%

0.7 ⫾ 1.1 40.3 ⫾ 5.5 4⫾5 87.5 ⫾ 25.2 0.96 ⫾ 0.27 0.18 ⫾ 0.08 43 ⫾ 23 57% 86%

There were 3 withdrawals due to refractory disease subsequently controlled by cyclophosphamide. There were 8 adverse effects: 3 respiratory tract infections, 2 mild gastro-intestinal side effects, 1 episode of thrombocytopaenia unrelated to MMF and 2 others (no amenorrhoea and no malignancy). No deaths have occurred and no patients have required MMF withdrawal. MMF and prednisolone therapy led to 86% renal remission and had few adverse effects. MMF is potentially superior to cyclophosphamide for the treatment of lupus nephritis and merits direct comparison in larger, randomised controlled trials with prolonged follow-up. Disclosure: Roche provided the mycophenolate mofetil for this study but this pharmaceutical company has not been involved in the design or the analysis of the results of this study.

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HIGH DOSE ARAVA IN LUPUS (HAIL). M Petri Baltimore, MD

IS THERE A ROLE FOR MYCOPHENOLATE MOFETIL IN THE TREATMENT OF REFRACTORY SLE? M Y Karim, P Alba, M J Cuadrado, I C Abbs, D D’Cruz, T Godfrey, M A Khamashta, G RV Hughes London, UK

Leflunamide (ARAVA) is a pyrimidine inhibitor with efficacy equal to that of methotrexate in rheumatoid arthritis. In a previous open-label trial in SLE (Smolen, A&R), efficacy was not proven. This study determined the response rate to a higher maintenance dose (40 mg instead of 20 mg). Patients: 20 SLE patients (95% female, 50% African-American) with active arthritis who had failed multiple therapies were enrolled. 75% were on prednisone, 35% on methotrexate, 95% on hydroxychloroquine and 95% NSAID. Methods: After screening and qualifying, 100 mg leflunamide was given for 3 days, followed by 40 mg daily. Patients were seen monthly for 3 months. At each visit, the Physician’s Global Assessment (0 to 3 VAS), SLEDAI, and tender/swollen joint count were assessed, in addition to monitoring for safety. Results: All 20 patients completed all visits. There were 4 dropouts for toxicity (1 rash, 2 diarrhea, 1 LFT), 1 dropout for CNS-SLE flare and 1 patient reduced from 40 to 20 mg because of diarrhea. 6 of the 20 (30%) had a complete response - usually by the 1 month visit. All of these patients maintained the complete response when, after 3 months, the leflunamide dose was reduced to 20 mg. Complement increased. Mean serum creatinine declined by 0.08 mg/dL (p⫽0.005). Mean weight declined by 3Ibs (p⫽0.001). Mean platelets declined by 23,000 (p⫽0.001). Conclusion: 30% of SLE patients with severe arthritis, failing multiple other therapies, achieve a complete, rapid (1 month) response to leflunamide 40 mg which can then be maintained on leflunamide 20 mg. The most striking finding is the dichotomy that 30% of SLE arthritis patients respond completely whereas the rest did not respond at all. This study demonstrates the benefit of leflunamide for 30% of resistant SLE arthritis patients. Randomized clinical trials of leflunamide in SLE, including organ involvement other than arthritis, are indicated. Disclosure:

Background: Mycophenolate mofetil (MMF) is an immunosuppressive drug widely used in solid organ transplantation, which may play an increasing role in autoimmune disease. Objectives: To evaluate the efficacy and safety of MMF in the treatment of active SLE refractory to other immunosuppressive agents. Patients and methods: We prospectively studied 21 patients treated with MMF who had previously failed on other immunosuppressive regimens. Twenty patients fulfilled at least 4 of the ACR classification criteria for SLE, and 1 fulfilled 3 criteria. There were 20 female, the median age was 33.6⫾7.2 years (range: 21-47), and median follow-up was 14⫾9.4 months (range: 2-33). Indications for treatment were uncontrolled disease activity in 8 patients and renal involvement in 13 patients. We measured disease activity (SLEDAI), parameters of renal function (serum creatinine, 24-hr urine protein), concomitant oral steroid dose, and immunological profile. Data were collected at baseline, and every 6 months. Results: Baseline SLEDAI was 12.6⫾3.6, and SLEDAI at the last clinic visit was significantly reduced to 3.5⫾1.5 (p⬍0.0001). In patients with renal disease, there was a reduction in 24-hour protein excretion from 3.7⫾2g to 1.14⫾1g (p⫽0.02). There was no significant change in serum creatinine, anti-dsDNA antibodies, or complement C3 and C4 levels. Prednisolone dose was significantly reduced from 19.5⫾7mg to 9.75⫾3.6 mg (p⬍0.0001). Three patients stopped MMF at 2 months, 2 patients due to gastrointestinal side effects and 1 patient due to severe skin lesions. Two patients in remission discontinued the treatment because they were planning to conceive. Two patients needed to increase MMF dose because of disease flares and 1 patient had a herpes zoster infection. Conclusion: MMF appears to be a safe and effective alternative immunosuppressant in SLE not responding to conventional immunosuppressive treatment. Disclosure:

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COMBINATION CYCLOPHOSPHAMIDE (CY) AND FLUDARABINE (FLU) IN PROLIFERATIVE LUPUS NEPHRITIS (LN): TOXICITY AND PRELIMINARY EFFICACY. Gabor G Illei, Cheryl H Yarboro, Ryan Schlimgen, Takashi Kuroiwa, Ellen M Vaughan, Thomas Fleisher, John H Klippel, Howard A Austin, James E Balow, Dimitrios T Boumpas Bethesda, MD

ANTI-dsDNA ANTI-IDIOTYPIC ACTIVITY AS THE MAIN MECHANISM OF IVIG EFFECT IN SLE. L Rauova, B Gilburd, M Blank, I Goldberg, J Kopolovic, M Ehrenfeld, J Rovensky, Y Shoenfeld Tel Hashomer, Israel and Piestany, Slovakia

Introduction: CY is among the most efficacious treatments for LN. However, late gonadal toxicity and risk of relapse of LN represent substantial shortcomings. We examined the prospect that combination low-dose pulse CY and FLU (which induces sustained lymphopenia) might overcome these problems of conventional CY treatment. Patients and study design: Phase I/II study to evaluate the safety of this combination and to collect preliminary data about its efficacy. Patients with active proliferative LN were treated with monthly oral boluses of low-dose CY (0.5gm/m2 on day 1) and subcutaneous fludarabine (30mg/m2 on days 1-3). Prednisone was aggressively tapered to a low-dose, alternate day schedule. Complete response (CR) was defined as stable creatinine, proteinuria ⬍1gm/day and inactive urine sediment (⬍10 RBC/hpf and no cellular casts). Results: Thirteen patients were enrolled in the study; 11 received three or more cycles. The study was terminated early because of the higher then expected rate of bone marrow suppression. One patient had severe aplastic reaction and died from transfusion associated graft versus host disease (TA-GVHD); two other patients had self-limiting grade 4 neutropenia not associated with any clinical sequalae. Three patients had mild leukopenia. Eight of the 10 patients with at least 18 months post-treatment follow-up had sustained significant response: 5 achieved and maintained CR, 3 had significant response but continued to have persistent stable mild hematuria (n⫽2) or low grade proteinuria (n⫽1) that did not require additional treatment. Additional treatment was started in 2 patients. Prolonged CD4 lymphocytopenia was noted in most patients (mean CD4: 98/uL at 7 months and 251/uL at 12 months); this was not associated with an increased rate of infections. Conclusion: Sustained clinical response was achieved with a short course of low dose pulse cyclophosphamide in combination with fludarabine. Further studies are needed to establish the optimal dosing regimen to reduce the potential for severe myelosuppression. All patients treated with FLU should receive irradiated blood products to prevent TA-GVHD.

Anti-idiotypic modulation by concentrated specific natural polyclonal anti-dsDNA anti-idiotypic antibodies affinity purified from IVIG was employed in the treatment of experimental SLE. Methods: Specific natural polyclonal anti-dsDNA anti-idiotypic antibodies (IVIG-ID) were affinity purified from IVIG on an anti-dsDNA-Sepharose column constructed with anti-dsDNA idiotypes affinity purified from 55 patients with SLE. The NZB/W F1 mice were treated with 3 weekly intravenous injections of IVIG-ID (2 mg/kg/inj) and regular IVIG (400 mg/kg/inj) at the age of 21 weeks (after developing of anti-dsDNA antibodies) and 8 weeks, respectively. Results: The IVIG-ID treated mice showed a decline in the titer of a-dsDNA antibodies during the treatment, reaching maximum effect one week after the last injection. This effect of treatment sustained as long as 2 month after completing the treatment. The incidence of proteinuria was significantly lower in IVIG-ID treated mice compared to non treated ones. The treatment with IVIG-ID at the age of 21 weeks capable to prevent the progression of IgG deposits in the glomerular basement membrane without affecting the mesangial deposits. An early treatment with IVIG-ID and with high dose IVIG at the age of 8 weeks resulted also in retardation of the kidney mesangial deposits. Conclusion: Treatment with concentrated specific anti-dsDNA-anti-ID prepared from commercial IVIG is more effective in suppression of humoral and clinical signs of SLE than regular IVIG. In agreement with the network theory, these results point to the considerable regulatory role of anti-idiotypes in the mechanism of action of IVIG in SLE. Disclosure:

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OUTCOME AND PROGNOSTIC INDICATORS OF DIFFUSE PROLIFERATIVE LUPUS GLOMERULONEPHRITIS (SLEDPGN) TREATED WITH SEQUENTIAL ORAL CYCLOPHOSPHAMIDE (CYC) AND AZATHIOPRINE (AZA). Chi Chiu Mok, Carmen TK Ho, Kwok Wah Chan, Chak Sing Lau, Raymond WS Wong Hong Kong

SUSTAINED FREEDOM FROM LUPUS ACTIVITY THREE YEARS POST STEM CELL TRANSPLANTATION; NORMALIZATION OF NEUROCOGNITIVE, CARDIAC & PULMONARY FUNCTION IN CRITICAL SLE: WHAT HAVE WE LEARNED? Ann E Traynor, Walter G Barr, Robert M Rosa, Steven K Baker, John A Kessler, Richard K Burt Chicago, IL

Objectives: To study the outcome and prognostic indicators of SLE-DPGN treated with sequential oral CYC and AZA. Patients and methods: From 1978 to 1998, patients with biopsy proven SLE-DPGN treated with prednisone (1mg/kg/day for 8 wks and then tapered) and sequential oral CYC (1-2mg/kg/day for 6 months) followed by AZA (1-2mg/kg/day) were studied. Remission was defined according to criteria suggested by Boumpas et al1. Those who remitted at 12 months were identified and predictors for remission were studied by regression. All patients were followed till they developed nephritis relapse or creatinine (Cr) doubling. Timing and risk factors for flares and Cr doubling were evaluated with Kaplan-Meier’s analysis and the Cox model. Results: 55 patients were studied (47 women, 8 men). The age at time of renal biopsy was 31.1⫾10.4 yrs. 25(46%) patients had a serum Cr level of ⱖ 106 umol/L and 29(53%) were nephrotic. At 12 months post-treatment, 37(67%) and 12(22%) patients had complete (CR) and partial remission (PR), respectively. 3 patients were treatment-resistant, 2 had early nephritis relapse and 1 died. The initial serum Cr level was an independent predictor for CR (RR 0.96 [0.93-0.99] per umol/L, p⫽0.004). Excluding those who were treatment-resistant or died (n⫽4), 21(41%) patients had renal flares (76% DPGN) during a median follow-up of 4 yrs. The cumulative risks of renal flare were 6% at 1 yr, 21% at 3 yrs, and 32% at 5 yrs. The median time to relapse was 43 months. The histological activity score (hazard ratio [HR] 1.13 [1.01-1.27] per point, p⫽0.04] and the mean daily CYC dose (HR 0.95 [0.92-0.98] per mg, p⫽0.001) were multivariate predictors for renal flares. During latest follow-up, 9/54(17%) patients had doubling of Cr and 6(11%) were dialyzed. The cumulative risks of Cr doubling were 6% at 3 yrs, 8.4% at 5 yrs, and 19.2% at 10 yrs. An increasing chronicity index at initial renal biopsy was an independent predictor for renal function deterioration (adjusted HR 2.1 [1.2-3.6] per point, p⫽0.009). Conclusions: Sequential oral CYC and AZA appears to be a reasonably effective treatment regimen for SLE-DPGN. Up to 2/3 of patients remain in remission after 5 yrs and 4/5 had stable renal function after 10 yrs. Predictors for treatment resistance and relapse include increasing Cr level, higher activity scores and a lower daily CYC dose being used. Increasing chronicity indices predicts for deterioration of renal function. 1Boumpas DT, Balow JE: Lupus 7:622-29, 1998

The original cohort of eight severely ill lupus patients has now been followed for a median of three years (12 months-60 months) post high dose cyclophosphamide, anti-thymocyte globulin and CD34 selected stem cell infusion. 75% of them remain with no evidence of lupus activity, and no use of immune suppressive medications including NSAID’s and prednisone. Moreover, each of these has shown a gradual and sustained resolution of neurocognitive dysfunction, sleep disturbance, pulmonary dysfunction, cardiac dysfunction and proteinuria. Two of the original eight patients have shown relapse of diseases activity, one at 14 months and one at 30 months following transplant. Both have been responsive to standard therapy, whereas prior to transplant, they were not. The most cogent observations emanating from this trial have been: the sustained ability of chronically diseased organs to normalize their function following immune remission, and the ability of the engrafted immune system to curtail expansion of detectable self reactive populations long term. These remarkable observations form the basis for the current Phase III study. They also are the basis for our evaluation of the role of hematopoietic stem cells in organ repair and the potential role for CAMPATH 1G and allografting to rescue those patients who develop active lupus following HSCT. In conclusion, patients with critical and refractory lupus can develop sustained self tolerance and normalize critically diseased organ systems. The current focus is to make this therapy accessable to a broader population, to enhance its efficacy for those who do relapse, and to understand better the contribution of hematopoietic stem cells to the normalization of involved organ function long term. Disclosure:

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A RANDOMISED CONTROLLED TRIAL OF AEROBIC EXERCISE ON FATIGUE IN SLE. Colin M Tench, Ian McCurdie, John McCarthy, Peter D White, David P D’Cruz London, United Kingdom

IMMUNOMODULATION OF LUPUS ASSOCIATED RESPONSES OF PERIPHERAL BLOOD LYMPHOCYTES OF PATIENTS BY PEPTIDES BASED ON HUMAN AND MURINE ANTI-DNA AUTOANTIBODIES. Zev Sthoeger, Molly Dayan, Alexandra Cherniak, Rephael Segal, Ori Elkayam, Edna Mozes Rehovot, Israel

Disclosure: This work was funded by the Arthritis Research Campaign.

Two peptides based on the sequence of the complementarity determining regions (CDR) 1 and 3 of a pathogenic murine monoclonal anti-DNA autoatibody that bears the 16/6 idiotype (Id) were shown to either prevent or treat induced or spontaneous animal models of systemic lupus erythematosus (SLE). To further investigate the role of CDR based peptides, two additional peptides based on the human monoclonal anti-DNA, 16/6Id were synthesized. The main objective of the present study has been to test the ability of the CDR based peptides to immunomodulate SLE associated responses of peripheral blood lymphocytes (PBL) of SLE patients. We have examined the ability of the peptides to inhibit the 16/6Id specific in vitro proliferative responses and IL-2 secretion by the PBL tested. The production of TGF␤ was assessed by ELISA. PBL of 24 out of the 62 SLE patients tested proliferated in vitro following stimulation with the human 16/6Id. Peptides based on the CDRs of both the human and murine anti-DNA autoantibodies inhibited efficiently the 16/6Id induced proliferarion and IL-2 production. The inhibition was specific since two control peptides (synthesized in the reverse sequence) had no inhibitory effect on the 16/6Id triggered responses. Further, the CDR based peptides did not affect the responses of the PBL tested to the mitogen, phytohemagglutinin. The effect of the various CDR based peptides on the PBL of healthy donors who responded to the 16/6Id was significantly less prominent than that on the patients’ PBL. The above inhibitions correlated with an upregulated production (by 2.5-3.5 fold) of the immunosuppressive cytokine, TGF␤. To our best knowledge this is the first demonstration of the potential of CDR based peptides to down regulate in vitro autoreactive T cell responses of SLE patients. The latter may be mediated by an up regulated secretion of TGF␤. Thus, these peptides are potential candidates for a novel specific treatment of SLE patients. Disclosure: This research was supported by Teva Pharmaceutical Industries Limited, Israel.

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Background. Fatigue is a common and debilitating symptom in SLE and its treatment is difficult. We studied the effects of aerobic exercise and relaxation on fatigue in SLE. Methods. 93 female outpatients with SLE were randomised into 3 groups: a 12 week exercise programme, a 12 week relaxation programme or to no intervention. The primary outcome measure was a self assessed clinical global change score at 12 weeks. Fatigue was measured at 0 and 12 weeks using the Fatigue Severity Score (FSS, score 1-7) with higher scores indicating increasing severity of fatigue. Aerobic fitness was measured at 0 and 12 weeks with a standardised treadmill exercise tolerance test. Disease activity and damage were assessed using the SLAM and SLICC. Results. The 93 patients had a median (interquartile range) age of 39 (33-45) years, a median disease duration of 30 (10-84) months, a median SLAM score of 5 (3-8), a median SLICC of 0 (0-0) and a median VO2peak of 22 (19-28) mls/kg/min. Four patients withdrew from the study (2 from the relaxation group, 1 each from the exercise and control groups). Analysis by intention to treat showed that 16/33 (49%) of the patients in the exercise programme were either very much better or much better after 12 weeks. This compared with 8/29 (28%) in the relaxation programme and 5/32 (16%) in the control group. Chi squared⫽8.3, df⫽2, p⫽0.02. The mean (SEM) FSS fell from 5.4 (0.2) to 4.8 (0.3) after exercise therapy (11% better), compared to a fall of 5.4 (0.2) to 5.3 (0,2) after relaxation therapy (2% better) and 5.5 (0.2) to 5.4 (0.2) in the control group (2% better). One-way ANOVA p⫽0.02 Bonferroni correction exercise v control p⫽0.02. Exercise duration increased by 1.7 (0.6) minutes (18%) after exercise therapy compared to an increase of 0.2 (0.4) minutes (2%) in the relaxation group and a reduction of 0.3 (0.4) minutes (3%) in the control group (p⫽0.01). There were no significant differences in disease activity, damage or aerobic fitness between the groups at 12 weeks. At 3 months follow up the mean FSS was 4.8 in the exercise group, 5.4 in the relaxation group and 5.1 in the control group. Comparing change in FSS from baseline One-way ANOVA p⫽0.02 Bonferroni correction exercise v relaxation p⫽0.03. Conclusions. Aerobic exercise therapy is a safe and effective treatment for fatigue in SLE and improves general well being. Exercise capacity increases with exercise therapy and the benefits of exercise persist at 3 months follow up.

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SYSTEMIC IMMUNE ACTIVATION AND GENERATION OF NEW AUTOANTIBODIES ARE FEATURES OF A SLE FLARE AFTER REMISSION INDUCED BY AUTOLOGOUS STEM CELL TRANSPLANTATION. Andreas Thiel, Oliver Rosen, Gero Massenkeil, Timo Alexander, Erika Gromnica-Ihle, Gerd-Ruediger Burmester, Renate Arnold, Andreas Radbruch, Falk Hiepe Berlin and Berlin-Buch, Germany

CHARACTERIZATION OF A HUMAN MONOCLONAL ANTIBODY THAT ANTAGONIZES B-LYMPHOCYTE STIMULATOR BIOACTIVITIES. Les Sekut, Bonnie Sturm, Carol Poortman, Ruth Wager, Chen Zhang, Donara Abramian, Todd Riccobene, Svetlana Sosnovtseva, Cynthia Sung, Viktor Roschke, Kevin P Baker, David M Hilbert Rockville, MD

Recently, autologous stem cell transplantation (ASCT) has been emerged as a new therapeutical approach in severe autoimmune diseases. Among them, patients with SLE who did not respond sufficiently to immunosuppressive regimens are considered to be candidates for ASCT. So far, tremendous efficacies of ASCT with achievement of long-term remissions were reported in SLE. Nevertheless the disease can reappear. To understand better the immunologic mechanisms leading to remission and relapse, respectively, after ASCT we have followed-up autoantibody patterns and titres as well as B- and T-cell subpopulations in five patients with SLE treated by ASCT including effective in vivo immunoablation and ex vivo depletion of lymphocytes. At first, ASCT induced response in all patients. While patients #1,#2, #4, and #5 have remained in clinical remission for now 37, 33, 5, and 2 months, respectively, reappearance of disease was diagnosed in #3 18 months post ASCT. The remissions were accompanied by disappearance or striking reduction of autoantibodies. Of note, at the presentation of relapse the autoantibody pattern was changed. Anti-Sm and anti-U1RNP antibodies arose in very high titres which had been never observed before. Interestingly, anti-dsDNA antibodies reappeared later 22 months post ASCT. Until 12 months post ASCT the B and T cell reconstitution revealed a similar behavior in all 3 patients exhibiting drastically increased numbers of naive B and Th cells. Afterwards, absolute counts of both peripheral naive B cells and Th cells decreased in patient #3. In parallel antigen-experienced B and Th cells started drastically to increase. Furthermore, the patient #3 evolved additional signs of disturbed B cell homeostasis. The increase of antigen-experienced B and Th cells and the opposite behaviour of naive cells may precede relapse of SLE after remission induced by ASCT. The change of the autoantibody pattern linked to the relapse leads to the speculation that the relapsing SLE is dependent on activation of new autoreactive B- and T-cells rather that old autoreactive plasma cells or autoreactive B-cells were not effectively eliminated by ASCT.

B-lymphocyte stimulator (BLyS; also known as BAFF, zTNF4, TALL1 and THANK), a member of the TNF ligand family, enhances B lymphocyte proliferation and Ig secretion. Studies of serum obtained from autoimmune patients have correlated elevated BLyS with the pathogenesis of SLE, RA and other autoimmune disorders suggesting that constitutive over-expression of BLyS may contribute to autoimmune pathologies. Furthermore, transgenic mice that overexpress BLyS develop a lupus/autoimmune-like phenotype. Thus, an antagonist of BLyS that blocks aberrant B cell activation associated with autoimmunity may be of therapeutic benefit. Accordingly, a fully human antibody with specificity for human BLyS (huBLyS) was isolated. In vitro, this antibody inhibited huBLyS-induced splenocyte proliferation. To study the in vivo pharmacology of this antibody, huBLyS and anti-huBLyS were administered to mice. Treatment with huBLyS alone resulted in increases in splenic weight, splenic B cell representation, and serum IgA concentration. Co-administration of the neutralizing anti-huBLyS inhibited the pharmacological effects of huBLyS administration in both a dose and schedule-dependent manner. The ability of the anti-huBLySspecific monoclonal antibody to effectively inhibit BLyS-driven B cell responses suggests that it may represent a novel therapy for patients with autoimmune disorders. Disclosure: All authors are employees of Human Genome Sciences Inc.

Disclosure: supported by grants from the Federal Ministry of Education and Research (Kompetenznetz Rheumatologie, C3.5) and the Deutsche Forschungsgemeinschaft (SFB 421,C4)

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NORMALIZATION OF PERIPHERAL B CELLS FOLLOWING TREATMENT OF ACTIVE SLE PATIENTS WITH HUMANIZED ANTI-CD154 MAB (5C8, BG9588). Amrie C Grammer, Satoshi Shinohara, Eduardo Vazquez, Hanan Gur, Gabor Illei, Peter E Lipsky Bethesda, MD and Tel Hashomer, Israel

EFFECT OF LJP 394 OR HIGH DOSE CORTICOSTEROIDS AND CYCLOPHOSPHAMIDE ON ANTI-dsDNA ANTIBODIES IN SLE PATIENTS. M D Linnik, R G Bagin, V Strand, LJP 394-90-05 Investigator Consortium San Diego and Palo Alto, CA

The phenotype of B cell subpopulations from the peripheral blood of active systemic lupus erythematosus (SLE) patients before & after treatment with humanized anti-CD154 mAb (5c8, BG9588) was examined and compared with normal controls. PBMC were analyzed by four color flow cytometry following staining immediately ex vivo with fluorochrome conjugated antibodies to CD19, IgD, CD38 & CD154 or CD69. Before treatment, SLE patients had the presence of circulating activated CD19high B cells that expressed CD154, CD69, and CD38. CD38 positive cells disappeared after two treatments and cells expressing CD69 & CD154 disappeared during the post-treatment period. In addition, before treatment, active SLE patients also had circulating CD38bright Ig secreting cells (ISC) that were not found in normals. These disappeared after 2 treatments with anti-CD154 mAb. Consistent with this finding, highly purified CD19high B cells from the periphery of active SLE nephritis patients cultured in vitro spontaneously proliferated and secreted Ig. Both functional outcomes were inhibited by anti-CD154 mAb (5c8). Finally, the GC founder, centroblast and centrocyte B cell subsets present in the periphery of active SLE patients disappear following treatment with humanized anti-CD154 (5c8). Together, these results indicate that active SLE patients exhibit abberancies in the peripheral B cell compartment that are driven via CD154-CD40 interactions and may reflect or contribute to the propensity of these patients to produce autoantibodies. Disclosure:

Introduction: LJP 394 is a novel B cell toleragen that has been shown to specifically reduce anti-dsDNA B cells in animals and circulating anti-dsDNA antibodies in SLE patients in 6 clinical trials. A double-blind, placebo-controlled trial (90-05 trial) showed that LJP 394 reduced time to renal flare and time to treatment with high dose corticosteroids and/or cyclophosphamide (HDCC) in patients with anti-dsDNA antibodies that have high affinity binding to the LJP 394 dsDNA epitope. Objective: To compare the effect of LJP 394 or HDCC administration in decreasing circulating anti-dsDNA antibodies in patients with a history of SLE renal disease. Methods: In the 90-05 trial, 230 SLE patients with a history of renal disease and anti-dsDNA antibodies ⱖ15 IU/ml by Farr assay were randomized to receive weekly infusions of 100 mg LJP 394 or placebo for 16 weeks, followed by intermittent dosing with 50 mg LJP 394 or PBO for 60 weeks. HDCC was administered at the investigator’s discretion and 1st exposure in a patient was defined as any exposure to cyclophosphamide or systemic prednisone (or prednisone equivalent) when increased ⱖ 15 mg per day over baseline to ⬎20 mg per day for more than 2 days, or when the daily prednisone dose exceeded 200 mg. Results: LJP 394 reduced anti-dsDNA levels from 100 ⫾ 15 IU/ml at baseline to 68 ⫾ 12 IU/ml (25 ⫾ 3% reduction) after 4 weekly treatments. In patients receiving placebo, levels increased from 106 ⫾ 16 IU/ml at baseline to 118 ⫾ 19 IU/ml (11 ⫾ 5% increase) at 4 weeks, representing a difference in anti-dsDNA of 36% between LJP 394 and placebo. Sixty two patients required HDCC (23 LJP 394 and 39 placebo) and 58/62 had at least one anti-dsDNA determination after the initial dose of HDCC. Mean and median dose of prednisone in the HDCC group were 151 ⫾ 42 mg/day and 50 mg/day, respectively. Mean anti-dsDNA levels prior to receiving HDCC were 163 ⫾ 47 IU/ml and 178 ⫾ 43 IU/ml in LJP 394 and placebo groups, respectively. The lowest anti-dsDNA levels within 4 weeks of HDCC treatment were 76 ⫾ 33 and 116 ⫾ 25 IU/ml for LJP 394 and placebo, respectively, representing decreases of 38 ⫾ 9% and 23 ⫾ 5% for LJP 394 and placebo, respectively. Conclusion: Weekly treatment with 100 mg LJP 394 effectively reduces anti-dsDNA antibody levels with no other known effects on the immune system. The magnitude of this reduction was similar to that observed within one month following treatment with high dose corticosteroids and/or cyclophosphamide.


Disclosure: Vibeke Strand and Robert Bagin are consultants to La Jolla Pharmaceutical Company. Work was supported by La Jolla Pharmaceutical Company.

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ANALYSIS OF CD5 EXPRESSION ON PERIPHERAL B CELLS FOLLOWING TREATMENT OF ACTIVE SLE PATIENTS WITH HUMANIZED ANTI-CD154 MAB (5C8, BG9588). Hanan Gur, Peter E Lipsky, Satoshi Shinohara, Eduardo Vazquez, Gabor Illei, Amrie C Grammer Tel Hashomer, Israel and Bethesda, MD

GENETIC VACCINATION USING VH-DERIVED CTL EPITOPES ABLATES AUTOANTIBODY-SECRETING B CELLS AND AMELIORATES LUPUS. R R Singh, H I Brunner, G- C Fan Cincinnati, OH

Systemic lupus erythematosus (SLE) patients are known to have increased numbers of circulating CD5⫹ B cells, but the nature of the pathophysiologic events leading to their expansion is not known. To address this, the phenotype of B cell subpopulations from the peripheral blood of active SLE patients before & after treatment with humanized anti-CD154 mAb (5c8, BG9588) was examined and compared with normal controls. PBMC were analyzed by flow cytometry following staining immediately ex vivo with fluorochrome-conjugated antibodies to CD19, IgD and CD5. Before treatment, SLE patients, but not normal controls, exhibited a markedly increased number of abnormal circulating B cells that expressed CD5 very brightly. These CD5bright B cells were observed exclusively in the IgD⫹, but not IgD-, subset of SLE B cells. The CD5bright B cells disappeared after two to four treatments with anti-CD154 mAb. Of note, SLE patients had a greater percentage of CD5⫹ B cells expressing CD5 at a density comparable to CD5⫹ B cells from normal individuals. However, this population was not consistently affected by anti-CD154 mAb treatment. Together, these results indicate that active SLE patients exhibit abberancies in CD5 expression in the peripheral B cell compartment. Moreover, the presence of increased numbers of CD5bright B cells in SLE patients appears to be affected by in vivo interactions between CD40 and CD154, suggesting that these CD5bright B cells are the products of germinal center reactions and may contribute to the propensity of thee patients to produce autoantibodies. Disclosure:

Current treatments for lupus cause non-specific immune suppression. Since organ damage in lupus is caused, at least in part, by autoantibodies such as anti-dsDNA Ab, depletion of dsDNA-specific B cells would inhibit lupus without causing non-specific immune suppression. Here, we applied bioinformatics to predict the MHC class I-binding epitopes: In the VH of an anti-DNA mAb (A6.1), 5 epitopes were predicted to have high proteasomal cleavage probability; all 5 had high dissociation half-time and binding to MHC class I, Kd or Ld. We confirmed these predictions in a cellular binding assay using a TAP-deficient cell line, T2. Conventional immunizations with synthetic peptides induced weak or no CD8⫹ T cell response in lupus-prone NZB/NZW F1 (BWF1) mice. We then cloned the cDNA encoding the VH epitopes into a plasmid DNA vector, pCD. Treatment of BWF1 mice with peptide-expressing pCD (’minigenes’) induced peptide-specific CD8⫹ T cells that exhibited strong CTL activity against a syngeneic, dsDNA-specific B cell hybridoma (A6.1), but not against a syngeneic non-autoreactive hybridoma (375-57) that expresses Ig that does not contain the VH epitope. Importantly, anti-VH T cells killed B cells from aged (6-9-mo-old) female BWF1 mice, but did not kill B cells from 1-2-mo-old BWF1 mice that did not have detectable anti-DNA-secreting B cells. The anti-VH T cells did not kill B cells from MHC-matched, normal CWF1 mice, unless the B cells were pulsed with the relevant VH epitope. The CTL activity was abrogated by an anti-CD8a Ab. The minigene-induced T cells inhibited anti-DNA production in vitro, enhanced TGF-␤ and IFN-␥, and decreased IL-4 and IL-13 (p⬍0.01). To determine the effect of VH minigenes on the development of lupus, we vaccinated BWF1 mice with a minigene (pCD.III), a null vector (pCD) or saline (PBS) (n⫽14/group). The pCD.III-treated mice had reduced serum IgG anti-DNA Ab (p⫽0.04-0.003, Student’s t-test) and delayed development of nephrotic-range proteinuria (p⬍0.0002, log-rank test) compared with pCD- or PBS-treated mice. Total serum IgG, however, was not decreased in the pCD.III-treated mice. The PAS and H&E-stained kidney sections showed markedly decreased glomerular proliferation and sclerosis, interstitial and perivascular infiltrates and tubular atrophy in the pCD.III-treated compared with the control mice. Finally, the survival was prolonged in the pCD.III-treated mice (p⬍0.001 vs. pCD or PBS treatment, log-rank test). In conclusion, minigene induction of anti-VH CTLs that ablate autoreactive B cells represents a novel approach to treat lupus. Disclosure:

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SUPPRESSION OF A LUPUS-LIKE SYNDROME IN MICE WITH REGULATORY T CELLS GENERATED EX-VIVO WITH TRANSFORMING GROWTH FACTOR-BETA. Song Guo Zheng, Michael N Koss, Francisco Quismorio, Jr, David A Horwitz Los Angeles, CA Although regulatory T cells (Treg) play a major role in controlling autoimmunity, how they are generated is poorly understood. The functional activity of Treg is decreased in SLE. We have previously reported that CD8⫹ cells and CD4⫹ T cells activated in the presence of TGF-␤ develop potent suppressive activity. Here we tested the hypothesis that Treg generated ex-vivo can alter the course of a lupus-like syndrome induced by transferring DBA/2 (D2) T cells into DBA/2 x C57BL/6 F1 mice. Eight week old female F1 mice received 80 million parental D2 cells. A second group received D2 cells with an additional 20 million alloactivated D2 CD4 or total T cells (Tcon). A third group received D2 cells ⫹ CD4 or total T cells alloactivated in the presence of TGF-␤ (Treg). By 2 weeks the transfer of parental D2 cells to F1 mice resulted in a marked increase in serum IgG and the appearance of anti-dsDNA antibodies. By 10 weeks these mice had developed lipemic serum and marked proteinuria. Similar findings were observed in mice that also received Tcon. By contrast, in mice that had received D2 cells with Treg, serum IgG was only minimally increased and anti-dsDNA autoantibody production was markedly suppressed. Moreover, urine protein was only minimally increased. Examination of the kidneys at 8 to 10 weeks revealed significant swelling and injury in mice that received D2 cells. The addition of the control alloactivated T cells to D2 cells further increased glomerular injury. These mice had a sclerosing glomerulonephritis. By contrast, transferring Treg with D2 cells markedly inhibited disease. Glomeruli in these mice were near normal or had only minimal changes. Immunofluorescence studies revealed marked deposition of IgG in the first two groups, but not in those that had received Treg. Examination of the mouse spleens revealed marked lymphocytosis at 2 weeks in all groups that had received D2 cells. By 8 weeks the total spleen cell number remained 3 fold increased in the first two groups, but had returned to near normal in those that received T reg. These studies have revealed that Treg generated ex-vivo with TGF-␤ can markedly suppress and in some cases prevent a lupus-like syndrome induced in normal mice. Although significant numbers of donor D2 T cells remain in the recipients, they are rendered tolerant to alloantigens. Further studies are in progress to learn the mechanism of action of this effect and to learn whether these Treg can suppress already established disease. Disclosure:

DENDRITIC CELL IMMUNIZATIONS INDUCE HIGH TITER LUPUS SPECIFIC AUTOANTIBODIES IN NORMAL MICE. Maria Georgiev, Lucila Agle, Keith B Elkon, Dalit Ashany New York, New York Most evidence indicates that autoantibodies result from antigen drive, yet the pathways responsible for antigen presentation remain to be defined. Specifically, immunization with self antigen plus powerful adjuvants fails to break tolerance to common lupus autoantigens in mice. To determine whether dendritic cells (DC) abrogate tolerance, we injected normal mice with DC, either intentionally loaded with apoptotic cells or not intentionally loaded (cells dying in the DC cultures may have provided a source of antigen). Two normal strains of mice (BALB/c and C57BL/6) received 3-4 i.v. injections of 0.5-1 x 106 syngeneic DC. Control groups received either 1x107 apoptotic cells alone, necrotic cells alone or PBS. The mice were evaluated serologically for ANA, anti-ssDNA, anti-cardiolipin, anti-ds DNA, anti-Sm. Mice were checked for proteinuria, and their kidneys evaluated for evidence of glomerulonephritis by H&E and for IgG deposition by immunofluorescence. Of DC injected mice, 17/20 developed a positive ANA with 2-4⫹ staining (rim pattern). Serologic analysis by ELISA demonstrated that 37/43 DC injected mice developed anti-ds DNA antibodies with 60% of sera at levels similar to those seen in 4-5 month old MRL/lpr mice. 23/25 DC injected mice developed anti-SmD antibodies; 7/21 developed anti-Sm B antibodies and 25/25 developed anti-cardiolipin antibodies. 20% of apoptotic cell injected mice developed autoantibodies at low titer but none of the control PBS injected mice did. H&E staining of kidneys revealed only a mild perivascular inflammatory infiltrate with segmental mesangial thickening in 2/8 kidneys from mice with high titer anti-ds DNA. Immunofluorescence staining revealed glomerular deposition of IgG in 14/15 DC immunized mice with only low level deposition in 2/15 control mice. None of the mice developed proteinuria or hematuria. These data suggest that DC are competent to break tolerance to lupus antigens. The lack of clinical manifestations of renal disease in the face of high titer anti-ds DNA antibodies suggests that the antibodies generated may be of a different subclass and/or different affinity that those seen in MRL/lpr mice. These data also have important cautionary implications for DC based vaccine strategies. Disclosure:

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ALTERED PPAR␥ EXPRESSION PARALLELS DISEASE DEVELOPMENT IN MRL/lpr MICE. Michelle B Crosby, Gary S Gilkeson Charleston, SC

SHORT TERM COSTIMULATORY BLOCKADE INDUCES LONG TERM MODULATION OF AUTOREACTIVE LYMPHOCYTES WITHOUT AFFECTING IMMUNE RESPONSES TO FOREIGN ANTIGEN. Weiqing Huang, Xiaobo Wang, Jayashree Sinha, Anne Davidson

Patients with systemic lupus erythematosus (SLE) produce more nitric oxide (NO) than controls. Furthermore, NO production correlates with disease activity measures. NO mediates inflammation through direct tissue damage and via peroxynitrite-mediated nitrosylation of key enzymes. The production of NO occurs in regions of tissue injury such as the kidney in both SLE patients and a murine lupus model: MRL/lpr mice. Spontaneous NO production increases with age in MRL/lpr mice in parallel with disease activity. Blocking NO production pharmacologically ameliorates renal disease in MRL/lpr mice. We postulate that one mechanism for the excess NO production in both lupus patients and the MRL/lpr mouse is a defect in the downregulation of the iNOS inflammatory pathway. One potential mediator of downregulation of NO is the nuclear hormone receptor Peroxisome Proliferation Activated Receptor gamma (PPAR␥). We and others have shown that PPAR␥ agonists inhibit iNOS expression and NO production by macrophages and mesangial cells. PPAR␥ is induced by IL-4, a component of Th2 immune response. However, MRL/lpr mice are Th1 aligned which may affect PPAR␥ expression and thus increase iNOS production. We investigated PPAR␥ protein and mRNA expression in the kidneys of MRL/lpr, BALB/c, B6, B6/lpr and MRL ⫹/⫹ mice over time. We found dramatic increases in iNOS expression in the kidneys of MRL/lpr mice compared to controls. However, there was no difference in mRNA expression for PPAR␥ between MRL/lpr mice and control strains. In contrast, MRL/lpr kidney cortices had altered expression of PPAR␥ protein as they aged compared with control mice. Western blot analysis shows increased expression of a larger PPAR␥ protein in the kidney as MRL/lpr mice age with loss of the “standard size” PPAR␥ protein seen in control strains. The similar levels of PPAR␥ mRNA in MRL/lpr mice compared to controls contrasted with the alterations in PPAR␥ protein expression suggest either alternative splicing or post-translational modification of PPAR␥ protein in MRL/lpr mice as they age. These findings likely impact the enhanced renal iNOS expression and NO production by these mice.

We have previously reported that treatment of SLE prone NZB/W F1 mice with short term combination CTLA4Ig/anti-CD40L results in a long delay in emergence of IgG anti-DNA antibodies and SLE onset that is due to decreased activation, class switching and somatic mutation of autoreactive B cells. To determine whether this effect is associated with induction of generalized B cell unresponsiveness, we immunized 10 treated and 10 control NZB/W F1 mice with the hapten oxazolone 8 weeks after a 2 week course of combination treatment and boosted 4 weeks later. IgG anti-Ox responses measured by ELISA were the same in both groups. ELISpots were performed on spleen cells 3 days after boosting for total Ig secreting cells and anti-Ox and anti-DNA secreting cells. The frequency of IgG anti-Ox secreting cells expressed as the ratio of anti-Ox IgG/total IgG secreting cells was slightly decreased in the treated mice (1/42 vs 1/18), but the frequency of IgG anti-DNA secreting cells was markedly diminished (1/491 vs 1/72). Hybridomas were generated from the spleens and tested for binding to DNA and to Ox. The frequency of Ox binding hybridomas was the same in treated and control mice (4.5% vs 4.0%) but the frequency of DNA binding hybridomas was markedly diminished in the treated mice (0.3% vs 3.2%). In control mice 30% of the anti-Ox hybridomas were cross-reactive with DNA compared with 7% in the treated mice. IgG cDNA libraries were generated from spleen cells for the VHOx heavy chain gene and for the VHBW-16 anti-DNA antibody associated VH gene and sequences from the libraries were analyzed for mutation frequency and pattern. Treated and control mice had similar frequencies of mutation of the VHOx gene and analysis of replacement to silent ratios in the CDR and FR regions demonstrated evidence of antigen selection in both groups. Treated mice had fewer mutations of the VHBW16 gene than controls (2.1 vs. 4.1 per sequence) and there was evidence of strong selection against mutations to arginine, particularly in the CDR2 region. These data show that following combination costimulatory blockade, treated mice whose spontaneous production of anti-DNA antibodies has been blocked are still able to respond to exogenous antigen and generate an effective class switched and mutated antibody response with concomitant regulation of cross-reactive autoantibody producing cells. Since these B cells have developed in a microenvironment devoid of costimulatory blockade, this regulation is most likely due to long term modulation of autoreactive T and/or B cells by combination treatment. Disclosure: NIAID NY Arthritis Foundation

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GENETIC DELETION OF CD1 IN LUPUS: EVIDENCE OF A REGULATORY ROLE. R R Singh, J Yang, A Singh, T Chun, S Hong, M Satoh, H Liu, C- R Wang, L van Kaer Cincinnati, OH; Nashville, TN; Chicago, IL and Gainesville, FL

IMMUNOGLOBULIN HEAVY CHAIN REVISION IN LUPUS-PRONE MRL MICE. Kimberly D Klonowski, Marc Monestier Philadelphia, PA

Objectives: To determine the role of CD1, a non-polymorphic antigen presenting molecule, in the development of lupus in 6 spontaneous or induced models. Methods: B6/129 CD1d1 knockout (KO) mice were backcrossed onto the NZB, NZW, NZM.2410, MRL-fas/fas and BALB/c for 8-10 generations; and CD1d1⫹d2 KO mice were backcrossed onto the MRL-lpr/lpr. A total of ⬎200 mice - CD1-/- (KO), CD1⫹/- (HTZ) and CD1⫹/⫹ (WT) littermates - were evaluated for renal disease, enlargement of lymphoid organs, anti-DNA and anti-erythrocyte antibodies (Ab), and rheumatoid factor (RF). Results: Among the genetically lupus-prone strains, namely NZM.2410, MRL-fas/fas, NZB and NZB/W F1, proteinuria, renal histological changes, anti-DNA and anti-erythrocyte Ab levels were slightly increased or unffected in KO compared to WT and HTZ mice; RF was increased in KO NZB/WF1 mice (p⫽0.04). To rule out the possibility that the CD1d1 gene deficiency is compensated by the second murine CD1 gene, CD1d2, we analyzed CD1d1⫹d2-/-MRL-lpr/ lpr mice. The latter KO mice had increased CD4-CD8-(DN)TCR␣␤⫹B220⫹ cells in lymph nodes (p⬍0.05). The weight and cellularity of lymphoid organs, anti-DNA Ab, and renal histology were similar between the KO and WT mice. We then considered the possibility that the effect of the genetic deficiency of CD1 may be compensated in genetically lupus-prone mice that have multiple immune abnormalities since early age. To obviate the latter possibility, we tested the effect of CD1 deficiency in an induced model of lupus: adult, pristane-injected CD1-/-BALB/c mice had increased proteinuria (p⬍0.05) and serum IgG anti-DNA Ab levels by ELISA (p⬍0.05, n ⫽ 22 WT and 28 KO mice); anti-dsDNA Ab by Crithidia assay were not significantly increased in the KO mice. Renal histology was evaluated in 18 ten-mo-old mice: Pristane-injected WT mice had mild to moderate mesangial or focal proliferative lesions, while 50% of KO mice had diffuse proliferative lesions with fibrous crescents and severe tubulointerstitial lesions. Conclusions: A correlation of CD1 deficiency with accelerated nephritis in pristane-induced SLE, with increased RF in NZB/WF1 mice, and with an expansion of DN, TCR␣␤⫹B220⫹ cells in MRL-lpr/lpr mice suggests a regulatory role of CD1 in at least some manifestations of lupus. A lack of major effect of CD1 deletion in genetically lupus-prone strains could be due to compensatory mechanisms that might arise in the congenital absence of CD1.

Antibodies to nucleosomes possess numerous positively charged amino acids, such as arginines, in the heavy chain CDR3. These cationic side chains are contact points with negatively charged sites on the DNA helix in chromatin. The presence of these arginine residues results in part from atypical VH-D-JH rearrangements such as D-D fusions. We have tested the hypothesis that newly emerging B cells from autoimmune mice possess more unusual rearrangements even before antigen selection. We have sequenced and analyzed libraries of MRL/⫹ and C3H/⫹ VH-D-JH junctions from newborn livers and adult pre-B cells. In the newborn, D-D fusions are significantly more frequent in the autoimmune MRL mice than in the control strain. Further, the heavy chain junctions in newborn MRL exhibit a preferred usage of VH-proximal D genes and distal JH genes suggestive of secondary D-JH gene rearrangements. Adult MRL pre-B cells, which have not yet undergone antigen selection, show similar rearrangement patterns in that they possess more atypical junctions than those of C3H mice. However, the MRL pre-B cell library does not display the biased usage of D and JH genes observed in unselected B cells. These results suggest that secondary gene rearrangements at the heavy chain locus MRL mice can explain the frequent occurrence of potentially self-reactive VH-D-JH junctions in lupus. The analysis of B cell development in the MRL strain indicates that most bone marrow B cell fractions are decreased in this autoimmune strain whereas the peripheral B cell subsets are normal. These results suggest that a larger proportion of MRL B cells are allowed to emigrate out of the bone marrow or that peripheral B cells are longer lived in this strain. These differences could account for the increased frequency of atypical rearrangements in MRL and their proclivity to yield autoreactive specificities. Disclosure:

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HISTONE DEACETYLASE INHIBITORS (HDI) DOWN-REGULATE TH1 AND TH2 CYTOKINE GENE EXPRESSION IN THE MRL/LPR/LPR MURINE MODEL OF LUPUS. Nilamadhab Mishra, Doris R Brown, Christopher M Riley, Gary S Gilkeson, Gary M Kammer Winston-Salem, NC and Charleston, SC

INTERACTIONS BETWEEN NZM-DERIVED Sle1 AND Sle3/5 LUPUS SUSCEPTIBILITY LOCI IN THE GENERATION OF ELEVATED CD4:CD8 RATIOS IN MIXED CHIMERAS. Eric S Sobel, Raquel Baert, Edward J Butfiloski, Laurence Morel Gainesville, Florida

The MRL/lpr/lpr murine model of lupus is characterized by autoantibody production and inflammation, including nephritis, vasculitis and arthritis. These mice exhibit markedly increased mRNA expression and protein secretion of Th1 (IFN-␥ and IL-12) and Th2 (IL-6 and IL-10) cytokines. Our laboratory recently demonstrated that the HDI, Trichostatin A (TSA), reverses the skewed expression of several genes implicated in the immunopathogenesis of human SLE. TSA significantly down-regulated CD154 (CD40-ligand) and IL-10 mRNA and protein while simultaneously upregulating IFN-␥ mRNA and protein expression in T cells (PNAS 98:2628, 2001). We asked whether TSA and Suberoylanilide Hydroxamic Acid (SAHA), a structurally related compound, could modify abnormal gene expression in the MRL/lpr/lpr model in vitro. Utilizing splenocytes from younger (10 wk) and older (24 wk) mice, TSA and SAHA suppressed IFN-␥, IL-12 p40, IL-12 p35, IL-6 and IL-10 mRNA levels in a dose-dependent manner by semi-quantitative PCR while having no effect on GAPDH transcript levels. These inhibitors also suppressed Con A-induced IFN-␥ transcript and protein secretion by ELISA. When LPS and IFN-␥ together were used to stimulate splenocytes, TSA and SAHA significantly inhibited IL-12 p40, IL-12 p35, IL-6 and IL-10 transcripts and protein secretion in vitro. This suppression of gene expression was associated with increased acetylation of histone 3(H3) and 4(H4) by immunoblotting, which may regulate gene expression via chromatin remodeling. In summary, HDIs down-regulate the expression of multiple cytokine genes implicated in lupus immunopathogenesis in MRL/lpr/lpr mice. Together with our data in human SLE, we propose that HDIs be investigated as candidates for treatment of SLE. Disclosure:

Two lupus susceptibility loci originally derived from NZM2410 have been identified and backcrossed onto the non-autoimmune C57BL/6 (B6) background. On this background, Sle1 on chromosome 1 conferred only attenuated autoimmunity characterized mainly by IgG antibodies to the H2A/H2B/DNA subnucleosome particle, with minimal antigenic spreading and no renal disease. Sle3/5 on chromosome 7, in contrast, conferred an elevated CD4:CD8 T cell ratio and a low-penetrant autoantibody production to both chromatin and dsDNA. Mice congenic for both intervals had an accentuated elevation of CD4:CD8 ratio above Sle3/5 alone and highly penetrant glomerulonephritis. Mixed chimera experiments have shown that Sle1 was expressed on B cells and T cells while Sle3/5 was expressed on non-T and non-B cells of hematopoietic origin. Because the two loci appear to be expressed in different cell populations, it was of interest to determine whether the phenotype expression of the double congenics could be fully reproduced with a mixture of bone marrow from the single congenics. To study this further, B6 mice were lethally irradiated and reconstituted with a combination of bone marrow as shown in the Table. After one year, the mice were sacrificed, and cells from the spleen characterized by flow cytometry. Mice receiving bone marrow from B6.Sle3/5 had an elevated CD4:CD8 ratio, as expected. Interestingly, mice receiving a combination of bone marrow from B6.Sle1 and B6.Sle3/5 had a greatly enhanced increase despite the fact that the presence of Sle1 alone had no effect. Because these mice had no allelic T cell marker, it was not possible to assess mixed chimerism at the T cell level nor to determine the relative contribution of each donor partner to the CD4:CD8 ratio. However, mixed chimerism was well-balanced by immunoglobulin heavy chain expression on splenic B cells. Donor Composition (n)

CD4:CD8 Ratio

%IgM(a)

B6.Igh(a) &B6 (10) B6.Sle1.Igh(a) & B6 (7) B6.Igh(a) & B6.Sle3/5 (11) B6.Sle1.Igh(a) & B6.Sle3/5 (8)

1.5 ⫾ 0.2 1.6 ⫾ 0.2 1.9 ⫾ 0.3 2.5 ⫾ 0.7

35 ⫾ 7 27 ⫾ 8 37 ⫾ 14 48 ⫾ 16

Overall, these data indicate that the epistatic interactions between Sle1 and Sle3/5 seen in the double congenics in the control of CD4/CD8 ratios can be separated into different cellular compartments. Disclosure: This work was supported by NIH grants AI-43454 and AI-039824.

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MECHANISM OF INHIBITION OF NITRIC OXIDE PRODUCTION IN MESANGIAL CELLS BY PPAR-␥ AGONISTS AS ASSESSED BY GENE ARRAY. Christopher M Reilly, Gary S Gilkeson Charleston, South Carolina

NEONATAL THYMECTOMY IN A MURINE MODEL OF SYSTEMIC LUPUS ERYTHEMATOSUS: ROLE OF REGULATORY T CELLS IN SYSTEMIC AUTOIMMUNE DISEASE. Harini Bagavant, Katsuhiro Ohno, Kenneth SK Tung Charlottesville, VA

Cytokine effects on inflammation depend on activation of signal transducers and activators of transcription (STATs), NF␬B, and AP-1. Thiazolidinediones are ligands for the nuclear receptor perixosome proliferator activated receptor (PPAR-␥). Once activated, PPAR-␥ inhibits many inflammatory mediators including nitric oxide (NO) and TNF-␣. Mesangial cells from MRL/lpr mice overproduce NO when stimulated with LPS/IFN␥ as compared to control mice. Previously we have demonstrated that PPAR-␥ activation blocks NO production in mesangial cells. To delineate the mechanism of how PPAR-␥ activation inhibits NO production, we pretreated MRL/lpr mesangial cells with ciglitazone two hours prior to stimulation with LPS/IFN␥. Using micro-array gene analysis, we found that stimulation with LPS/IFN-␥ increased inteferon regulatory factor-1 (IRF-1), Stat-1 and NF␬B expression leading to increased NO production. Two-hour pretreatment with ciglitazone did not alter the LPS/IFN␥ induced induction of Stat-1, NF␬B or IRF-1 gene activation. However, iNOS gene expression, protein synthesis and NO production were abrogated by ciglitazone pretreatment of LPS/IFN␥ stimulated mesangial cells at 8 and 24 hours. Although it is known that multiple signaling pathways induce iNOS gene activation these results suggest that PPAR-␥ activation specifically blocks iNOS induction via acting directly to either inhibit iNOS gene transcription or the co-activators responsible for iNOS gene induction without affecting upstream mediators. Disclosure:

The recently defined immunoregulation mechanism of peripheral tolerance mediated by CD4⫹CD25⫹ regulatory T cells is abolished by thymectomy between day 1-4 after birth (d3tx). D3tx results in organ specific autoimmune diseases such as gastritis, oophoritis, orchitis or thyroiditis in susceptible strains of mice. However, little is known on the role in systemic autoimmune diseases in general and SLE in particular. We hypothesized that depletion of regulatory T cells by d3tx may accelerate spontaneous lupus manifestations in mice. Thus, d3tx was done on NZM2328 mice, a lupus model with anti-dsDNA autoantibodies and chronic glomerulonephritis (GN) with a female gender bias for the development of renal disease. Control mice were sham thymectomized (sham tx). The treated and control mice were monitored for proteinuria, appearance of dsDNA autoantibodies, and sacrificed at 20 or 30 weeks for histological studies of renal disease. D3tx male and female mice showed an accelerated onset of dsDNA antibody by 9 weeks. At 20 weeks there was an increase in incidence of acute GN in d3tx females. The early onset of acute GN was associated with increased C3 and immune complex deposition in renal glomeruli. Moreover, in females, by 30 weeks the immune complex deposits extended into capillary loops with changes of chronic GN. Surprisingly, acute proliferative GN with immune deposits confining to the mesangia were found in the d3tx male by 20 weeks but not in the control males. Unlike the female mice, the renal disease in d3tx males did not progress to chronic GN at 30 weeks. D3tx in both male and female NZM2328 mice resulted in frequent and severe organ specific autoimmune disease, affecting the salivary gland, lacrimal gland, thyroid and prostate. These data suggest that 1) regulatory T cells are present and operative in mice programmed to develop spontaneous lupus; and their depletion by d3tx results in acceleration of autoantibody response and acute GN, 2) Spontaneous development of systemic autoimmunity in NZM2328 has not resulted from global deficiency of the immunoregulatory CD4⫹CD25⫹ T cells, and 3) Acute lupus GN is a process that may be dissociated from chronic lupus GN with renal failure, suggesting independent mechanism for their occurrences. The latter finding corroborated with the pathological and genetic data originally obtained in the initial characterization of NZM2328. These results have basic implication in GN pathogenesis and suggest novel therapeutic approaches.


Disclosure: Supported by NIH grants AR45222 and AI 41236

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ANTIGEN PRESENTING CELLS ACTIVATED BY PRISTANE INDUCE NAIVE MICE TO PRODUCE LUPUS AUTOANTIBODIES. Minoru Satoh, Akiei Mizutani, Hideo Yoshida, Krista M Behney, Westley H Reeves Gainesvilel, FL and Toyoake, Japan

SPONTANEOUS AUTOIMMUNITY IN BALB/c Fcg RIIB DEFICIENT MICE, SYNERGISTIC EFFECT OF PRISTANE. Nicola Calvani, Hanno B Richards, Minoru Satoh, Westley H Reeves, Raphael Clynes Gainesville, FL and New York, NY

Pristane induces lupus in normal mice along with a long-lived, proliferating subset of peritoneal CD11b⫹ macrophages and CD11c⫹ dendritic cells. The role of antigen presenting cells (APC) in pristane-induced lupus was examined in BALB/c mice. Unfractionated cells from the spleen, peritoneal cavity, and thymus/LN of pristane treated anti-nRNP/Sm (⫹) mice were adoptively transferred to BALB/c SCID recipients. Recipients of spleen, peritoneum, or thymus/LN cells produced high levels of anti-nRNP/Sm as early as 2 wks after transfer, indicating that cells required for autoantibody production are present in all 3 locations. Next, CD11c⫹, CD11c-/CD11b⫹, and CD11c-/CD11b- cells were isolated from Igh-a mice using biomagnetic beads and adoptively transferred to naive Igh-b (allotype congenic) recipients. Recipients of CD11b⫹ cells from the peritoneal cavity, spleen, or thymus/LN of pristane treated BALB/c mice (anti-nRNP/Sm⫹, anti-Su⫹) produced both specificities within 2-4 wks. The recipients’ B cells were responsible for these autoantibodies (rather than donor B cells contaminating the CD11b⫹ population), because the IgG2a anti-nRNP/Sm and Su autoantibodies all expressed the recipient (b) allotype. Since T cells are required for the induction of autoantibodies by pristane, it was possible that antigen-primed TH cells contaminating the CD11b⫹ population induced autoantibody production. However, ⬍2% of the transferred cells were CD3⫹ and the CD11b⫹ subset was far more efficient at inducing autoantibody production than the CD11b- (T cell-enriched) subset, arguing that autoantibodies were not induced by the transfer of primed T cells. Both macrophages and peritoneal B-1 cells are CD11b⫹. However, we have previously shown that pristane treatment depletes B-1 cells. In agreement, ⬎90% of the CD11b⫹ cells transferred were CD5-, CD19-, sIgM-, suggesting that the APC stimulating autoantibody formation may be macrophages rather than typical B-1 cells. Whether induction of autoimmunity occurred in the peritoneal cavity or elsewhere was unclear. Only ⬃10% of CFSE labeled CD11b⫹ cells injected i.p. were recovered at 48 hrs, suggesting that they migrate rapidly to other sites. We conclude that APC from pristane-treated mice can prime naive T-cells leading to anti-nRNP/Sm and anti-Su autoantibody production by naive B cells. The data provide evidence that a primary APC defect is sufficient to initiate an autoantibody response characteristic of SLE. Disclosure:

INTRODUCTION. Altered homeostasis in Fc␥ receptor (Fc␥R) expression has been implicated in the development and progression of autoimmune diseases. ITAM (activation motif) containing Fc␥RI and III are required for immune complexes to activate effector cells in mice, thereby triggering tissue damage. In contrast ITIM (inhibitory motif) containing Fc␥ RIIB functions to restrict effector cell responsiveness. Fc␥RII deficient mice have been reported to develop spontaneous autoimmunity in a strain dependent (B6) manner. This study was aimed at examining the role of ITAM vs. ITIM Fc␥ R pristane-induced lupus in BALB/c mice. METHODS. Fc␥RI/III (n ⫽ 17) and Fc␥RIIB (n ⫽ 24) deficient (-/-) and control (⫹/⫹) (n ⫽ 21) BALB/c mice were treated with either pristane or PBS. Serial sera were analyzed by ELISA for IgG anti-chromatin, anti-nRNP/Sm and total IgG levels. Anti-nRNP/Sm was determined by immunoprecipitation and anti-dsDNA by Crithidia assay. Proteinuria was measured 8 months after treatment. RESULTS. Absence of Fc␥RI/III or Fc␥RIIB had little effect on anti-DNA/chromatin, anti-nRNP/Sm and anti-Su induction by pristane, as the frequency of these autoantibodies was similar in pristane-treated Fc␥RI/III -/-, Fc␥ RIIB -/- and ⫹/⫹ mice. Spontaneous production of anti-chromatin and anti-dsDNA was seen in half of the Fc␥RIIB -/- mice. In contrast no spontaneous production of anti-nRNP/Sm or anti-Su was observed. Pristane induced hypergammaglobulinemia in both Fc␥RI/III and Fc␥RIIB -/- mice without a significant difference in total IgG1 and IgG2a levels compared to ⫹/⫹ mice. Pristane-induced nephritis as indicated by proteinuria was significantly accelerated in the absence of Fc␥RIIB whereas lack of Fc␥RI/III abolished renal disease. CONCLUSION. These results suggest that spontaneous autoimmunity develops in BALB/c Fc␥RIIB-/- mice. Pristane has a synergistic effect on antiDNA/chromatin production in these mice and the absence of Fc␥ RIIB dramatically accelerates nephritis. Absence of Fc␥RI/III prevents pristane-induced nephritis but has little or no influence on autoantibody induction. Finally, in contrast to anti-DNA/chromatin, Fc␥R mediated mechanisms are likely not involved in anti-nRNP/Sm and anti-Su production. ⫹/⫹ anti-dsDNA anti-chromatin anti-nRNP/Sm anti-Su Disclosure:

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pristane 67% 82% 73% 27%

RI/III -/PBS 0% 10% 0% 0%

pristane 90% 90% 50% 38%

RIIB -/PBS 0% 29% 0% 0%

pristane 91% 93% 54% 62%

PBS 60% 50% 0% 0%

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AN ALTERNATIVE ROLE FOR FACTOR B IN THE PATHOGENESIS OF DISEASE IN MRL/lpr MICE. Margaret K Elliott, Gary S Gilkeson Charleston, South Carolina

AN ANGIOTENSIN CONVERTING ENZYME-INHIBITOR, CAPTOPRIL MODULATES PRODUCTION OF IL-4, IL-10 AND TGF-␤ AND RETARDS PROGRESSION OF MURINE LUPUS NEPHRITIS. D A Albuquerque, S R Kakumanu, F D Finkelman, R R Singh Cincinnati, OH

Complement activation plays a role in the pathogenesis of end organ disease in Systemic Lupus Erythematosus (SLE). Activation of the alternative pathway (AP) of complement correlates with lupus disease activity. The involvement of the alternative pathway of complement was investigated in the mouse model of SLE, the MRL/lpr mouse. MRL/lpr mice deficient in AP activation were derived by genetic knock out of factor B, an essential protein for AP activation. The absence of factor B has a significant inhibitory affect on renal disease in MRL/lpr mice. One finding, which might contribute to this effect, is the profound depression of sera IgG3 and IgG3 cryoglobulin levels in MRL/lpr Bf-/- mice. IgG3 is implicated in immune complex deposition in the kidneys of MRL/lpr mice and is the major isotype represented in response to T-independent (TI) antigens type 1 and 2. These antigens can activate the AP as well as stimulate the production of IgG3. To determine the mechanism by which factor B affects IgG3 production, the immune response of Bf-/- MRL/lpr mice to TI antigens was evaluated in vivo. We found that the Bf-/- mice had significantly lower levels of anti-TNP IgG3 antibodies in response to immunization with TNP-ficoll (a prototypic TI-2 antigen) when compared to Bf⫹/- and Bf⫹/⫹ littermates. The Bf-/- IgG3 response to TNP-LPS (TI-1 antigen), however was not significantly lower than Bf⫹/- and Bf⫹/⫹ littermates. The mechanism by which IgG3 production is inhibited is currently under investigation. This data supports the hypothesis that factor B may be essential for the IgG3 response elicited by TI-2 antigens and that factor B may contribute to nephritis in MRL/lpr mice via alternative mechanisms other than its role in complement mediated damage.

Background: While patients with acute lupus nephritis respond well to therapy, management of chronic lupus nephritis remains dismal and the mechanisms that cause progression to end stage renal disease remain unclear. Angiotensin converting enzyme inhibitors (ACE-Is) such as captopril are used to control hypertension. In rodent models and patients with primary chronic nephropathies, these agents improve renal pathology and function more than would be expected from their control of hypertension. The beneficial effects of treatment may result from reduction of renal TGF-␤. It is not known, however, whether ACE-Is affect production of TGF-␤ or other cytokines in lymphoid tissues. We were concerned that systemic suppression of TGF-␤ production might enhance T-B cell activation, autoantibody production and disease. Methods: We addressed this issue by treating lupus-prone and normal strains with captopril, with a control antihypertensive, verapamil, or with no medication. Mice were evaluated for proteinuria, anti-DNA Ab and cytokine production and renal histology. Results: In 6 separate experiments (total of 30-40 mice/group), proteinuria progressed in untreated and verapamiltreated mice but failed to progress or decreased in captopril-treated NZB/W and MRL-lpr/lpr and MRL-fas/fas mice. There was no significant effect on anti-DNA Ab, except for elevation in one strain after a long-term treatment. The improvement in disease was associated with the modulation of cytokine production by spleen cells: decreased type 2 cytokines, namely interleukin 10 (IL-10; p⬍0.01, Student’s t-test) and IL-4 (p⬍0.05); IFN-␥ levels were slightly increased or unaffected (p⫽NS). Circulating and renal TGF-␤ expression were decreased after a long-term (6 weeks) treatment of most, but not all, strains; In MRL-fas/fas mice, 4-month treatment with captopril decreased serum TGF-␤1 to undetectable levels; these mice had severe inflammatory skin lesions. Spleen cell production of TGF-␤1 was also decreased or remained unaffected after a long-term treatment. Intriguingly, short-term (2 weeks) treatment, however, enhanced spleen cell production of TGF-␤1 (p⬍0.05), probably due to compensatory mechanisms or due to a differential effect of captopril on renal versus lymphoid tissues. Conclusions: Captopril may be a systemic immunomodulator that has a broad effect on autoimmunity, rather than simply an inhibitor of renal TGF-␤ production. Such immune modulation may be responsible for its beneficial effect in immune-mediated nephropathies. Disclosure: Supported by an NIH P30 P&F grant.

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B CELL DEVELOPMENTAL PATHWAYS CORRELATE WITH CLINICAL OUTCOMES IN THE NZM MURINE MODEL OF SYSTEMIC LUPUS ERYTHEMATOSUS. Laurence Morel, Byron P Croker Gainesville, FL

EXPRESSION OF DNA METHYLTRANSFERASES IN T CELLS: RAPID UPREGULATION OF DNMT3A AND DNMT3B WITH INVOLVEMENT OF THE RAS-MAP KINASE AND CALCINEURIN SIGNALING PATHWAYS. John T Attwood, Bruce C Richardson Ann Arbor, Michigan

This study is aimed at establishing a functional correlation between the aberrant development of various B cell compartments, B1 cells, marginal zone B cells, and plasma cells, and susceptibility to SLE in the NZM mouse model. We have compared two related strains with both opposite SLE-suceptibility and B cell development. B6.NZM.Sle1/2/3 is a triple congenic strain that combines 3 of the NZM2410 SLE susceptibility loci, Sle1, Sle2, and Sle3, on a C57BL/6 background. We have shown recently that this strain reconstitutes NZM2410 immunopathology, in which an early and intense activation of the B cells plays a critical role. TAN is a novel NZM strain with 70% of its genome derived from NZW and 30% from NZB. Autoimmune manifestations in the TAN mice are very mild, and do not result in nephritis as in most other NZM strains. Both B6.NZM.Sle1/2/3 and TAN show an elevated number of B1a cells in their peritoneal cavity, with the increase being significantly greater in the TAN. This shows that, while an increased B1a compartment may contribute to SLE pathogenesis, it is not sufficient for the disease development. SLE-prone B6.NZM.Sle1/2/3 spontaneously develops an abnormal splenic accumulation of plasma cells, leading to plasmacytomas (PCT) in 80% of the mice. In parallel to this plasma cell expansion, B6.NZMSle1/2/3 mice show a significantly reduced to absent MZ B cell compartment. In contrast, TAN mice develop a marked splenic MZ enlargement leading to MZ lymphomas with a high penetrance (92%) by 12 months of age. TAN serum immunoglobulin levels are comparable to that of B6, indicating a normal plasma cell development. TAN and B6.NZMSle1/2/3 share Sle1, Sle2, and Sle3 loci, but on different backgrounds (a mosaic of NZB and NZW, or C57BL/6, respectively). We propose that the 3 Sle loci on a permissive B6 background mediate an intense autoimmune activation and proliferation of the B cells, which favors plasma cell formation. We hypothesize that the TAN genome contains modifiers that divert B cells from an abnormal terminal differentiation into plasma cells to the marginal zone compartment, ultimately leading to a neoplastic process, while protecting the mice from autoimmunityinduced pathology. Functional analyses of the B cell development and their immunopathological correlates in the NZM may provide unique insights into the role of the various B cell compartments in systemic autoimmunity. Disclosure:

We have reported that the DNA 5-methylcytosine content in T cells from patients with lupus is reduced, and in murine models have implicated this hypomethylation in disease pathogenesis. T cell DNA hypomethylation in lupus is associated with reduced activity of DNA methyltransferase 1 (DNMT1) and impairment of the Ras-MAP kinase signaling pathway. Recently, two further methyltransferases with de novo activity have been identified, DNMT3a and DNMT3b. Nothing is known about their expression or regulation in T cells. We therefore utilized realtime RT-PCR to determine whether, in addition to Dnmt1 expression, stimulated murine T cells express Dnmt3a or Dnmt3b. Following T cell activation with immobilized anti-CD3 and soluble anti-CD28, we found that the mRNA for both Dnmt3a and Dnmt3b is rapidly upregulated. Transcripts are detectable within 2-3 hours of T cell activation and peak at about 6 hours. There is a gradual fall in transcript levels over the next 48 hours. The kinetics of Dnmt1 upregulation is quite different, with the first detectable rise at 8 to 10 hours and peak levels reached at about 20 to 24 hours. Examination of CD4⫹ and CD8⫹ T cell subsets, as well as naive and memory T cells, revealed that both Dnmt3a and Dnmt3b are expressed in all activating T cells. We used inhibitors of cell signaling pathways to determine regulation of Dnmt3a and Dnmt3b expression. Dnmt3a upregulation is completely suppressed by inhibitors of the Ras-MAP kinase, calcineurin and phosphatidylinositol-3 kinase pathways, suggestive of cooperative binding to NFAT and AP1 regulatory sites. In contrast, whilst upregulation of Dnmt3b is completely suppressed by inhibitors of the calcineurin and PI3 kinase pathways, it is only partially suppressed by inhibitors of the Ras-MAP kinase pathway. Finally, although we have previously demonstrated involvement of the Ras-MAP kinase pathway in regulating Dnmt1 expression, its late upregulation suggests additional factors controlling its expression. We hypothesize that the RAS-MAP kinase defect in lupus T cells may lead to DNA hypomethylation by affecting both DNMT1 and DNMT3a expression. Disclosure:

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A MURINE ICOS-IG FUSION PROTEIN IS BIOLOGICALLY ACTIVE AND CAN BE USED TO DEFINE THE ROLE OF ICOS IN AUTOIMMUNE DISEASE MODELS. Meera Ramanujam, Lena Schiffer, Xiaobo Wang, Weiqing Huang, Alla Ackerman, Anne Davidson Bronx, NY

ACCELERATED NEPHROTOXIC NEPHRITIS IN FCR GAMMA CHAIN AND FC GAMMA RECEPTOR III DEFICIENT MICE. Kevin A Davies, Ruth M Tarzi, Marina Botto, Mark J Walport, J Sjef Verbeek, H Terence Cook London, United Kingdom; Leiden, The Netherlands

Costimulatory blockade is an effective method for treating autoimmune disease mediated by activated T and B cells and, when used in combination with conventional or other biologic agents, may be safer and more effective than current therapies. New costimulatory molecules that are potential therapeutic targets have recently been discovered. ICOS is a CD28-like costimulatory molecule that is highly expressed on activated T cells in the apical zone of germinal centers where somatic mutation occurs. Ligation of ICOS results in costimulation of Th2 cytokines and upregulation of CD40L on T cells which induces B cell differentiation and immunoglobulin synthesis. ICOS also appears to predominate over CD28 as a costimulator of the secondary B cell response. We have generated a fully murine ICOS-IgG2a fusion protein (mICOS-Ig) that acts as a soluble antagonist of the binding of ICOS to its ligand. mICOS-Ig was expressed in adenovirus and the expressed protein found to be a dimer of 95KD. Ad-mICOS-Ig was administered to non-autoimmune CBA/J mice. After 3 days, the mice were challenged with the hapten oxazolone. Anti-oxazolone antibody responses of both the IgG1 and IgG2a isotypes measured at weekly intervals after treatment were markedly attenuated in the treated mice. Treated and control mice were splenectomized and ELISpots were performed for anti-oxazolone antibody producing B cells. There was no difference in the frequency of such cells between treated and control mice suggesting that the effect of ICOS-Ig is on affinity maturation of the B cell response. No anti-mICOS antibodies were detected in the sera of treated mice. However, since mICOS-Ig did not appear in the serum for 24-48 hours after adenovirus injection, anti-adenoviral antibodies measured by ELISA were not attenuated in the treated mice and mICOS-Ig levels were sustained for only 4 weeks. Ad-mICOS-Ig was administered to 20 week SLE prone NZB/W F1 mice together with four doses of anti-CD40L or CTLA4Ig (days 0,2,4,6). These mice will be followed for long term effects of treatment on disease outcome. Our studies show that mICOS-Ig is a biologically active and non-immunogenic agent that can be used to evaluate the role of ICOS and the effect of its blockade in murine autoimmunity disease models.

Fc␥ receptors link IgG immune complex deposition to innate inflammatory responses. In the mouse there are two activatory Fc␥ receptors, Fc␥ receptor I (Fc␥RI) and Fc␥ receptor III (Fc␥RIII) which both possess a common ␥ chain signalling subunit. We have investigated the role of Fc␥ receptors in accelerated nephrotoxic nephritis induced in pre-immunized mice with sheep nephrotoxic serum, using mice with gene targeted deletion of the FcR ␥ chain (lacking Fc␥RI and RIII) and mice with a knockout of Fc␥RIII alone. The FcR ␥ chain -/- mice (FcR␥-/-) were generated on a C57BL/6 background and the Fc␥RIII knockout mice were backcrossed to C57BL/6 for nine generations. Compared with wild type (WT) and Fc␥RIII-/- mice, FcR␥-/- animals were strongly protected from glomerular thrombosis - 0% vs 71⫾9% (WT) and 59⫾9% (Fc␥RIII-/-) at day 7 post nephrotoxic serum (mean⫾SEM, n⫽9 per group). They were also protected from crescent formation - 0% vs 27⫾6% (WT) and 16⫾3% (Fc␥RIII-/-) and uremia - serum creatinine 50⫾3mmol/l (FcR␥-/-) vs 152⫾17␮mol/l (WT) and 112⫾17␮mol/l (Fc␥RIII-/-). The difference between FcR␥-/and WT is strongly significant (p⬍0.001). Whilst there is a trend towards milder disease in the Fc␥RIII-/- mice compared with WT, this does not reach statistical significance. Both WT and Fc␥RIII-/- mice had proteinuria at day 7 (urinary mg albumin/mmol creatinine 690⫾123 (WT) and 1059⫾161 (Fc␥RIII-/-),NS). Some FcR␥-/- mice developed proteinuria and mild glomerular hypercellularity dependent on the dose of nephrotoxic serum. Serum anti-sheep antibody levels were the same in all groups. These results show that lack of Fc␥RIII alone cannot explain the marked protection of FcR␥-/- mice from accelerated nephrotoxic nephritis. Disclosure:

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DIFFERENT AUTOANTIGENS INITIATING ANTI-snRNP IMMUNE RESPONSE RESULTS IN DIVERSE B CELL EPITOPE SPREADING PATTERNS RESEMBLING THOSE OBSERVED IN LUPUS PATIENTS. Umesh S Deshmukh, Shu Man Fu Charlottesville, VA

REDUCED SERUM AUTOANTIBODY LEVELS IN MRL/lpr MICE BEARING H-2b/b HAPLOTYPE. Hideharu Sekine, Ivan D Molano, Christopher M Reilly, Gary S Gilkeson Charleston, SC

Autoantibodies reactive with multiple proteins within the small nuclear ribonucleoprotein (snRNP) complex are an important feature of systemic lupus erythematosus. In this investigation, the mechanisms for B cell epitope spreading were studied in an experimental system. A/J mice were immunized with either purified recombinant SmD, or U1RNP associated A protein. Sera at different time points were analyzed for anti-snRNP reactivities using Western blot and immunoprecipitations. In mice immunized with SmD, cross reactive antibodies to SmD and either SmB or the A protein were detected readily by day 30 after initial immunization. By day 60, antibodies specific for A protein and SmB were detected. In addition, the day 60 immune sera absorbed with SmD were capable of immunoprecipitating the whole snRNP complex. In mice immunized with the A protein, antibodies reactive with A protein, SmB, SmD and 70kDa protein were detected by Western blots 90 days after initial immunization. Absorption with the A protein completely depleted antibodies reactive with the A protein, SmB and SmD. The absorbed sera showed persistent antibodies to the 70kDa protein, indicating intermolecular B cell epitope spreading. Whether the absorbed immune sera precipitate the intact snRNP complex is being investigated. This result suggests that the initiating antigen dictates the epitope spreading patterns. Thus, in one case, specific antibodies to SmD and the A protein are generated with SmD as the immunogen, whereas specific antibodies against the A protein and 70kDa protein are generated with the A protein as the immunogen. These varied patterns resemble those observed in lupus patients, providing an insight into the pathogenesis of this disease.

Introduction: MRL/lpr mice (H-2k/k), a strain that spontaneously develops a lupus-like disease, were developed by interbreeding several mouse strains bearing either the H-2k, H-2b, or H-2d/f haplotype. Several gene loci are linked with disease susceptibility in MRL/lpr mice, but the role of MHC alleles, if any, in disease in this model remains unclear. To assess the effect of MHC alleles on disease in MRL/lpr mice, we determined serum autoantibody levels in H-2k/k, H-2b/k, and H-2b/b MRL/lpr mice. Methods: We backcrossed (C57BL6x129) F1 mice (H-2b/b) to MRL/lpr mice (H-2k/k) for nine generations, then derived H-2k/k, H-2b/k, and H-2b/b MRL/lpr mice by interbreeding F9 H-2b/k MRL/lpr mice. H-2 of each mouse was determined by PCR-RFLP method by monitoring MHC class II I-A locus. Serum autoantibody levels (anti-dsDNA and GBM) were measured by ELISA. Results: More than 12 mice of each haplotype were generated. At 16 and 20 weeks of age, there were significant reductions of serum autoantibody levels in H-2b/b MRL/lpr mice compared to H-2k/k and H-2b/k MRL/lpr mice. There were no statistically significant differences in autoantibody levels between H-2k/k, and H-2b/k MRL/lpr mice. Conclusion: These results suggest that genes encoded within or closely linked to the MHC region regulate serum autoantibody production and may affect the development of the disease in MRL/lpr mice.

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A MAJOR MECHANISM OF IMMUNE TOLERANCE INDUCED BY A PEPTIDE THAT DELAYS CLINICAL SLE IN MICE MAY BE IMPAIRMENT OF IFN-␥ RESPONSES. George A Karpouzas, Fanny M Ebling, Bevra H Hahn Los Angeles, CA

A CO-REGULATED PATHWAY RESPONSIBLE FOR THE IMMUNE RESPONSE TO ATHEROSCLEROSIS-ASSOCIATED ANTIGENS, APOPTOTIC CELLS AND MICROBIAL PATHOGENS: A POSSIBLE ROLE IN ATHEROGENESIS IN AUTOIMMUNE DISEASES. Gregg J Silverman, Christoph J Binder, Sohvi Horkko, Denise Dwyer, Asheesh Dewan, Mikyung Chang, Peter X Shaw, Wulf Palinski, Joseph L Witztum La Jolla, CA

IFN-␥ plays a major role in SLE in several strains of lupus-prone mice. In some strains, including NZB/NZW F1 (BWF1), inhibition of IFN-␥ or Knockout of IFN-␥ receptors delays autoantibody formation and disease. PConsensus (pCONS) is an artificial immunomodulatory peptide, restricted by I-Ed, based on T-cell stimulatory peptides from IgG anti-dsDNA from BWF1 mice. Similar Ig-derived peptides are stimulatory for PBL lymphocytes of SLE patients. In vitro, pCONS activates T cell help for anti-DNA production. In vivo, given in tolerizing doses to BWF1 females, it induces peptide-non-specific tolerance in T cell help, thus delaying appearance of multiple autoAb and prolonging survival of BWF1 mice, begun before or after disease appears. Furthermore, treatment delays the dramatic increases in serum levels of IFN-␥ and IL-4 that herald disease onset (Hahn BH et al, Arthritis Rheum 2001). Experiments reported here test whether pCONS-induced immune tolerance alters cytokine responses in lymphoid tissues. PCONS was used as both tolerogen and immunogen. Five-wk-old female BWF1 mice received 1 mg of pCONS i.v. Seven days later they were immunized with 100 ug pCONS in CFA. Draining lymph node cells were collected 10 days later. Control BWF1 mice were immunized but not tolerized. 5x105 cells/well in 96 well plates from each group were activated in vitro by culture with anti-CD3 and anti-CD28 monoclonal antibodies (1ug/ml), with or without pCONS and other stimulatory and non-stimulatory peptides. IFN-␥, IL-2 and IL-4 were quantitated by ELISA in culture supernatants collected at 24 or 48 hours. Production of IFN-␥ was significantly decreased in the tolerized group- from means of 157.2 pg/ml in the non-tolerized to 110.4 pg/ml in the tolerized mice (p⬍0.0001, Mann-Whitney test). In contrast, IL-2 production was not significantly different (p⫽0.4); IL-4 elaboration was modest and similar in both groups. Conclusions: In BWF1 mice, in vivo tolerization with pCONS significantly decreases IFN-␥ elaboration (but not IL-2) from lymphoid cells studied ex vivo, supporting earlier observations of decreased plasma levels of IFN-␥ in vivo. An important mechanism by which pCONS protects from disease may be its ability to impair production of IFN-␥ . Studies are in progress to determine the molecular basis of this effect.

Despite improvements in the treatment of RA and SLE, these patients, who are generally devoid of cardiovascular risk factors, increasingly succumb to accelerated atherosclerosis. The pathogenetic basis is unknown, but mounting evidence indicates that the immune system plays an important modulating role in atherogenesis. Pathologic atherosclerotic plaques contain oxidatively modified LDL (OxLDL), a major target of the associated immune response. The recognition of OxLDL by B cells and T cells leads to specific antibody responses, and autoantibody titers correlate with occurrence and severity of atherosclerosis-associated diseases. Significantly, LDL immunization has been shown to induce a specific response that ameliorates disease in rabbits and mice. To investigate the origin of autoimmune responses occurring during atherogenesis, we have cloned splenic B-cell lines from naive atherosclerosis-prone, apolipoprotein E-/- (ApoE) mice, and these monoclonal IgM bound to oxidized phospholipids on OxLDL and apoptotic cells and blocked their uptake by macrophages, the major contributors to vascular lesions. Unexpectedly, these IgM were found to share complete gene sequence identity with the classic anti-phosphorylcholine (PC) clone, T15, the dominant source of protective antibodies to several common infectious pathogens, and these B cells arise from the B-1 lymphocyte pool. In ApoE-/- and LDL-receptor-/- mice with advanced disease, there are greatly enhanced spontaneous in vivo expansions of the T15 clone compared to control mice. In LDL-receptor-/- mice, immunization with pneumococcal extract (R36A) induced expansions of OxLDL-specific and T15 IgM-secreting B-cells are cross-reactive between PC and OxLDL, and the induced high titer antibody responses also intensely stain human atherosclerotic lesions. Finally, by flow cytometry these post-immune sera specifically stained aortic endothelial cells undergoing apoptotic death, and this reactivity was specifically inhibited by PC-BSA. These findings suggest that host autoimmune responses to conserved oxidative neo-self-determinants in LDL, vascular lesions, and apoptotic cells can contribute to the clonal expansion of the same autoreactive B-cell clone. These findings may characterize features of the autoimmune response associated with RA and SLE that contribute to accelerated atherogenesis.

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LUPUS LIKE DISEASE IN SEVERE COMBINED IMMUNODEFICIENT (SCID) MICE AND IN HUMAN/ MOUSE RADIATION CHIMERA. Zev Sthoeger, Heidy Zinger, Benjamin Dekel, Fabian Arditti, Yair Reisner, Edna Mozes Rehovot, Israel

AN IMMUNOGLOBULIN-RELATED ARTIFICIAL PEPTIDE TOLEROGEN (pCONSENSUS-pCONS) IS RESTRICTED BY I-Ed AND LOSES IMMUNOSTIMULATORY CAPACITY IF A SINGLE AMINO ACID IS ALANINE-SUBSTITUTED, SUGGESTING THE RELATIVE UNIQUENESS OF THE SEQUENCE. Fanny M Ebling, Jatinderpal Kalsi, Akio Ametani, Eli Sercarz, Ram R Singh, Bevra H Hahn Los Angels and La Jolla, CA and Cincinnati, OH

The objective of the present study has been to develop a human SLE model for a better understanding of the mechanisms underlying the development and progression of lupus. To this end we have established and compared two models: 1) SCID mice reconstituted with peripheral blood lymphocytes (PBL) of either SLE patients or healthy controls and 2) lethally irradiated BALB/c mice radioprotected with bone marrow of SCID mice, to which human PBL were transferred intraperitoneally (human/mouse chimera). Mice were tested for levels of human immunoglobulin (Ig), for the production of antibodies and autoantibodies and for Ig deposits in the kidneys. Engraftment was successful in most recipient mice as determined by the levels of human IgG measured (3-5mg/ml). In about 50% of either the SCID or human/mouse chimera mice that were successfully engrafted with PBL of SLE patients significant anti-dsDNA autoantibodies as well as serum human ANA titers were determined. Kidneys of 78% of the SCID mice with human DNA specific antibodies had human Ig deposits in their kidneys. The prevalence of immune complex deposits in the human /mouse chimera was lower. Interestingly in a significant number of recipients of PBL of the healthy controls, of both models, the above manifestations could be observed as well suggesting that PBL of some healthy controls have the potential to develop SLE associated autoantibodies under the appropriate stimulatory conditions. Nevertheless, the anti-DNA titers were lower in the recipients of PBL of healthy donors than in recipients of PBL of SLE patients. Further, high antibody titers to the recall antigen, tetanus toxoid, were detected in sera of both the SCID and human/mouse chimera models reconstituted with control PBL whereas the antibody responses of recipients of patients, PBL appeared to be more specific to SLE associated autoantibodies. Thus, SLE serology and glomerular pathology were reproducibly demonstrated in two models of human SLE. These models should allow the evaluation of potential therapies for the treatment of lupus patients.

pCONS is an immunomodulatory artificial peptide based on T cell determinants in the FRl/CDR1 region of VH of human and murine IgG antibodies to dsDNA. When administered to BWF1 females as a tolerogen, it delays the apperance of autoAb and dramatically prolongs life. T cells from SLE patients release cytokines when incubated with peptides from the same region of human autoAb and similar sequences. Understanding the requirements of pCONS to engage MHC molecules and then the TCR will help move this strategy from laboratory to bedside. MHC Class II restriction was measured by using pCONS to immunize mice with I-Ad and I-Ed , which responded with T cell proliferation. Immunized mice expressing I-Au and I-Eu did not respond. Therefore, restriction was I-Ad or I-Ed . Binding of pCONS to I-Ed was confirmed in inhibition assays. pCONS bound I-Ed with moderate affinity. In contrast, pNEG which does not cause T cell proliferation or affect disease in BWF1 mice, bound with high affinity. This was similar to the wild Ig-peptide p34, which stimulates Th1 and Th2 responses from BWF1 lymphocytes but does not alter clinical disease. HyHEL34, from a VH region similar to that of p34 and pCONS, bound I-Ed weakly. We conclude high-affinity binding of I-Ed does not correlate with clinical efficacy, since one high binder (pNEG)either cannot engage TCR nor activate T cells, and another (p34) engages TCR, produces proliferation and T cell help but does not alter disease. The moderate-binding pCONS is more effective. Alanine substitutions of each amino acid in pCONS examined requirements for full MHC binding and TCR engagement. Peptides were presented on a murine lymphoma expressing I-Ad, I-Ed, to peptide-specific T cell hybridoma, #422, and IL-2 production measured by proliferation of an IL-2 dependent T cell line, HT-2. The sequence of pCONS is FIEWNKLRFRQGLEW. Mean stimulatory index for the unmodified pCONS was 14. No substitutions were tolerated except K6 (S.I. 7), R10 (S.I. 7), and E14(S.I. 9.5). Intact pCONS was repeatedly the most stimulatory sequence; stimulation was still measurable if one charged amino acid was substituted. pCONS is presented by I-Ed and requires each of its amino acids for full immunostimulatory capacity. Therefore the entire sequence of pCONS is probably important in its powerful effect on murine lupus. Disclosure: Supported by NIH P60 AR 36834, R37 AI46776, the Arthritis Foundation, and the Paxson Foundation.

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LPR B CELLS STILL SHOW AN INTRINSIC DEFECT ESSENTIAL FOR AUTOANTIBODY PRODUCTION IN THE ABSENCE OF FASL OVEREXPRESSION. Michael A Maldonado, Hong Yuan Luo, Robert A Eisenberg Philadelphia, PA

IS CAROTID VASCULAR DISEASE SIMILAR IN SYSTEMIC LUPUS AND RHEUMATOID ARTHRITIS: COMPARISONS OF PLAQUE, INTIMA-MEDIA THICKNESS AND RISK FACTORS. MC Wasko, F Selzer, K Sutton-Tyrrell, A Cunningham, A Kao, S Manzi Pittsburgh, PA

One of the hallmarks of Systemic Lupus Erythematosus is the production of autoAb directed primarily at nuclear antigens. Experimental work designed to elucidate the cellular and molecular controls that normally prevent the production of autoAb suggest that there are both developmental and functional controls both intrinsic and extrinsic to the B cell population. This work is designed to further clarify the relative role of intrinsic B cell control in the production of autoAb. Lpr mice, which are deficient of Fas, spontaneously produce SLE autoAb in a strain-dependent pattern. Mixed bone marrow (BM) chimeras derived from Fas-sufficient (wildtype) and Fas-insufficient (lpr) derived BM still produce autoAb. The use of Ig heavy chain allotypic markers in these chimeras has shown that only the B cells derived from the lpr BM produce autoAb. The Fas-sufficient B cells are presents, as indicated by FACS analysis of spleen cells and allotype-specific measurement of serum IgM. These results have suggested two hypotheses: (1) The absence of Fas expression on lpr B cells allows for these cells to escape normal immunoregulatory mechanisms delivered through FasL; or (2) the overexpression of FasL on lpr derived T cells (J Exp Med, 1995, 181;393) may downregulate potential autoAb production by Fas-sufficient B cells through a non-physiological mechanism. These alternatives could be distinguished by creating mixed chimeras in which the overexpression of FasL in lpr T cells is prevented by making them simultaneous deficient in FasL (gld). We therefore utilized lethally irradiated B6lpr/gld (double homozygote) recipients and reconstituted them with mixed BM of normal (Igh “a” allotype) and Fas- and FasL-deficient (lpr/gld) (Igh “b” allotype). Analysis of sera at 4 month post BMT by allotype-specific ELISAs revealed that all the autoantibody derived from the lpr/gld donor, although immunoglobulin was present from both BM. These results suggest that the intrinsic expression of the lpr defect in autoantibody producing B cells reflects the failure of an important physiological mechanism of B-cell tolerance through interaction with FasL. Allotype

IgG2a (mg/ml)

IgM (mg/ml)

anti-ssDNA (normalized OD)

anti-chromatin (normalized OD)

“a” “b”

1.3 3.9

350 500

⬍0.14 0.27

⬍0.26 154

Disclosure:

Cardiovascular (CV) disease is a leading cause of death in systemic lupus (SLE) and rheumatoid arthritis (RA). AIM:We compared the prevalence and severity of carotid atherosclerosis in SLE and RA women with comparable CV risk factors. METHODS: Focal carotid plaque and intima-media wall thickness (IMT) were measured by B-mode ultrasound in 74 SLE and 37 RA women of similar age and race. Plaque was defined as a focal protrusion into the arterial lumen. Risk factor data were collected on the day of ultrasound testing. RESULTS: SLE and RA women were similar in age, education, disease duration (10.3 vs 13 yr), menopausal status, body-mass index, blood pressure, prevalent self-reported coronary heart disease (CHD) (4.1% SLE vs 8.1% RA, p⫽0.37) and total, LDL and HDL cholesterol. While the proportion of women currently on prednisone was comparable in the two groups (36.5% SLE vs 43.2% RA, n.s.), daily prednisone dose was higher in the lupus women (7.0⫹/-3.9 mg vs 4.6⫹/-1.8 mg, p⫽0.01). Of the SLE patients, 6.8% had renal disease,16.2% were anti-DNA positive, 13.7% anticardiolipin positive, mean SLAM score 6.4, SLICC damage score 1.3 (⫹/-2.0); 64.9% RA patients had had nodules, 80.9% were RF positive, 64% had taken ⬎/⫽3 DMARDs. Mean IMT was greater in lupus women than those with RA (0.76 vs 0.68 mm, p⬍0.01). Focal carotid plaque was present in 41.9% of SLE women vs 29.7% RA (p⫽0.21). There was a trend toward more severe carotid atherosclerosis in the SLE women. In the multlivariate analysis, after adjusting for other significant CV risk factors, increasing carotid IMT values were strongly associated with SLE (vs RA) (p⫽0.001)). The odds of having carotid plaque were 2.3 times higher in SLE compared to RA (p⫽0.10), and the odds of having more severe carotid disease were 2.5 times higher in SLE (p⫽0.06). CONCLUSIONS: In a sample of SLE and RA women of comparable age, race, menopausal status, and traditional CHD risk factors, focal carotid plaque was found in roughly one third of both groups. However,the overall prevalence and severity of carotid disease were more striking in lupus women, and those taking prednisone were on a greater daily dose than the RA prednisone users. Disclosure:

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CORONARY ARTERY ANGIOGRAPHY IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)PATIENTS WITH ABNORMAL MYOCARDIAL PERFUSION IMAGING. Emilia Inoue Sato, Elaine MC Sella, Wagner Leite, Antonio Barbieri, Japy A Oliveira Filho Sao Paulo, Sao Paulo, Brazil

RISK FACTORS FOR CORONARY-ARTERY CALCIFICATION IN FEMALES WITH SYSTEMIC LUPUS EYTHEMATOSUS BELOW THE AGE OF 50. K Manger, M Kusus, C Forster, D Ropers, J R Kalden, S Achenbach, B Manger Germany

Introduction: In the literature, myocardial perfusion abnormalities were reported in 16 to 64% of SLE patients. Usually patients with coronary artery disease risk factor (CAD-RF) and myocardial perfusion abnormalities are submitted to coronary angiography, but no study has evaluated the coronary artery by angiography in SLE patients with these abnormalities. Objective: The aim of this study was to evaluate coronary artery angiography in SLE patients with myocardial perfusion abnormalities. Patients and Methods: After signed consent term, approved by Ethics Committee, 90 consecutive SLE women (mean age 38⫾10 y.o, 54 white) with disease duration higher than 5 years and no previous myocardial infarction (MI), angioplasty or coronary bypass grafting were studied. All patients were submitted to myocardial scintigraphy with 99m Tc-sestamibi with dipyridamole stress. Thirty patients (mean age 40⫾9y.o) had abnormal scintigraphy. Fifty-eight percent of abnormalities were reversible defects. Twenty-one patients agreed to undergo coronary angiography. Eight of these patients (38%) with mean age 47 ⫾ 4 y.o. and mean disease duration 142 ⫾ 74 months presented atherosclerotic plaques. These patients had at least 4 CAD-RF. All had arterial hypertension and four patients complained of angina pectoris. At evolution, one patient presented MI, 3 were submitted to percutaneous transluminal coronary angioplasty, one patient performed myocardial perfusion revascularization and 3 were treated with vasodilator drugs. Conclusions: The coronary artery angiography was not able to demonstrate atherosclerotic plaques or significant narrowing of coronary artery in 62% of SLE patients with abnormal myocardial scintigraphy. This suggests that abnormal scintigraphy may be due to endothelial dysfunction with reduced vasodilator reserve or microvascular disease in SLE patients. Our results cannot exclude that myocardial perfusion abnormalities may represent an early step of atherogenic process.

The survival rate in SLE has improved dramatically over the past decades to 96.7% (5 years) in the Erlangen cohort of 338 patients. Therefore comorbidity and long-term damage such as accelerated atherosclerosis become increasingly important. In the Erlangen cohort, 6.5% of the patients had a myocardial infarction and 17.5% have angina, but asymptomatic coronary heart disease (CHD) is probably much more frequent. Material and methods: To identify the prevalence of coronary artery calcifications (CAC) in asymptomatic patients, we performed electron beam tomography (EBT) in 68 female SLE patients according to ACR criteria below the age of 50. The results were correlated with traditional risk factors and SLE -related risk factors. Statistical analysis was performed with uni- and multivariate logistic regression, mean values were compared with Student’s t-test. Results: In total 18 out of 68 pts. had evidence of CAC, 2/19 younger than 35 had CAC. Mean disease activity measured by ECLAM score was identical in both groups, SDI after five and ten yrs of disease was 1.7 vs. 1.2 (with vs. without CAC) and 2.8 vs. 2.1, and age at disease onset was 29 vs. 26 yrs. Looking for independent risk factors for CAC we could identify hyperlipidemia (p⫽0.06, OR⫽2.7, 39% vs. 16%) and smoking (p⫽0.02, OR⫽5.7, 72 vs. 48%,). In our cohort, traditional risk factors such as elevated CRP, a family history of CHD, body mass index or hypertension could not be identified as independent risk factors. However we were able to identify SLE related factors like proteinuria (p⫽0.05, OR⫽3.6, mean proteinuria 2032 vs. 511mg/d, p⫽0.001) and reduced renal function (p⫽0.1, OR⫽4.3, mean maximum creatinine 1.9 vs. 1.2mg%, p⫽0.06). Interestingly, the frequency of nRNP antibodies is lower in patients with CAC (33 vs. 52%). Therefore nRNP antibodies are protective in this model (p⫽0.06, OR⫽0.1). There was no correlation to phospholipid antibodies (APL). This analysis was adjusted for age at disease onset and disease duration until EBT. Conclusion: We could identify a subgroup of patients with CAC by EBT, which provides a useful, non-invasive technique to assess atherosclerosis in this population at risk. In addition to traditional risk factors, especially proteinuria and reduced renal function contribute to the high prevalence of CAC in women with SLE. Apparently SLE disease activity by itself or APL are not associated with CAC. Disclosure:

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LUPUS DISEASE ACTIVITY AND CORONARY ARTERY DISEASE. Christian A Pineau, Dafna D Gladman, Dominique Ibanez, Murray B Urowitz Toronto, Ontario, Canada

CARDIOVASCULAR RISK FACTORS AND CORONARY CALCIFICATION (CC) IN SYSTEMIC LUPUS ERYTHEMATOSUS. M Petri Baltimore, MD

AIM: To determine whether lupus disease activity, as measured by the Adjusted Mean SLEDAI (AMS) in the first three years of disease, predicts future CAD in SLE patients. METHOD: An inception cohort of patients seen at the Toronto lupus clinic within one year of diagnosis, and at least once a year for the first 3 years of disease was identified. Patients were followed for a minimum of 10 years. This group included 134 patients, 18 of whom had a CAD event (angina or myocardial infarction). To evaluate the average lupus disease activity of patients in their first 3 years of disease, we measured the area under the curve of the SLEDAI over that period and divided this by the time elapsed. This measure is called the Adjusted Mean SLEDAI (AMS). The variability of the SLEDAI over time (variability measure of SLEDAI ⫽ VMS) was estimated by the average change in SLEDAI from visit to visit. The SLEDAI at presentation and adjusted mean ESR in the first 3 years were also compared. Finally, the mean cholesterol level, mean dose of corticosteroids and use of antimalarials, immunosuppressants, ASA and NSAID’s was examined. RESULTS:

Noninvasive detection of coronary atherosclerosis is possible using helical CT, which measures coronary calcification (CC). We assessed associates of CC in a series of 52 SLE patients, selected because 50% had normal and 50% abnormal carotid duplexes. Results: Of those with coronary calcification, 29 were abnormal, 23 normal. Age was associated with CC (p⫽0.01). Sex, race, and age-of-onset were not associated with CC.

No CAD (n⫽116)

CAD (n⫽18)

P value

6.32 ⫾ 3.73 1.20 ⫾ 0.76 5.47 ⫾ 0.76 6.50 ⫾ 6.30

8.61 ⫾ 2.83 1.61 ⫾ 0.59 6.15 ⫾ 1.26 12.59 ⫾ 12.07

0.0139 0.0333 0.0849 0.0569

The SLEDAI at presentation and the Adjusted mean ESR were not associated with CAD. There was no statistical difference between the two groups with regards to the use of antimalarials, immunosuppressants, ASA or NSAID’s. Finally, in a stepwise logistic regression analysis, the AMS (OR ⫽ 1.156, 95% CI ⫽ 1.02 - 1.31, p ⫽ 0.026) and the mean dose of steroids (OR ⫽ 1.092, 95% CI ⫽ 1.03 - 1.31, p ⫽ 0.0063) were both associated with CAD when studied separately. The correlation between AMS and mean steroid dose was r ⫽ 0.52. CONCLUSION: SLE patients with CAD had a higher disease activity and variability of their disease activity in their first 3 years of disease. Higher disease activity (AMS) and mean dose of steroids were the two main predictors of future CAD.

Normal

Abnormal

p-value

190.9 144.5 48.5 113.4 92.36 48% 17% 1.92 8.74 9.37 334.2 12.4 162.8

202.6 118.6 58.8 120.5 90.97 48% 55% 1.03 10.5 9.28 342.9 27.6 178.7

NS NS 0.01 NS NS NS 0.0092 NS 0.08 NS NS 0.03 NS

Conclusion: 56% of the helical CTs showed CC. In general, lipids were not predictive of CC. Paradoxically, a HIGHER HDL-cholesterol was a strong predictor. We speculate that this reflects prednisone increasing HDL. Hypertension and lipoprotein(a) were also strong predictors of CC. Homocysteine had suggestive p-values. These results suggest that some traditional cardiovascular risk factors (but not lipids) play a major role in coronary atherosclerosis. Thus, there exist multiple possible pathways for intervention in prevention studies. Disclosure:

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AMS VMS Mean Chol. (mmol/l) Mean steroid dose (mg/d)

Cholesterol Triglycerides HDL-cholesterol LDL-cholesterol Glucose Smoking Hypertension x 6 mo CRP Homocysteine PAI-1 Fibrinogen Lipoprotein(a) Weight

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MRI EVALUATION OF MYOCARDIUM IN PATIENTS WITH SLE: A PROSPECTIVE STUDY. Jasvinder Singh, Pamela K Woodard, Victor G Davila-Roman, Alan D Waggoner, Jei Zheng, Seth A Eisen St. Louis, MO

RISK FACTORS FOR CARDIOVASCULAR DISEASE IN SLE. Elisabet Svenungsson, Kerstin JensenUrstad, Mikael Heimburger, Fei Gouzhong, Anders Hamsten, Ulf deFaire, Joseph Witztum, Johan Frostegard Stockholm, Sweden and San Diego, California

Objective: To determine whether cardiac MRI imaging demonstrates disease dependent myocardial abnormalities in SLE patients. Methods: 11 consecutive female patients (ages 30⫾10 years), 6 with active SLE and 5 with inactive SLE, were studied prospectively. All subjects underwent a detailed history and physical examination, laboratory evaluation, ECG, chest x-ray, echocardiogram (including parameters of systolic and diastolic function), and cardiac MRI. Five age, gender, and race matched healthy volunteers served as controls. MRI T2-weighted relaxation parameters were measured as an indication of tissue edema and cellular infiltration in 4 myocardial segments from mid-left ventricular short-axis images. Segmental measurements were averaged to derive global T2 relaxation times. Results: Patients with active SLE had significantly higher myocardial global T2 relaxation times by comparison to patients without active disease (84⫾15 ms vs. 66⫾8 ms respectively, p⫽0.036) and healthy controls (62⫾10 ms, p⫽0.02). Patients with active SLE demonstrated no differences in heart rate, systolic and diastolic BP, or echo derived measurements of ejection fraction, fractional shortening, left ventricular mass index, and Doppler mitral valve flow velocity E/A ratio compared to inactive SLE patients and healthy controls. End-diastolic volume index (ml/m2) was higher in active SLE patients compared to inactive patients (109⫾23 vs. 74⫾10 respectively, p⫽0.01) and healthy controls (50⫾7, p⬍0.01). Conclusions: Higher myocardial T2 relaxation times in patients with active SLE may be indicative of myocardial edema and/or cellular infiltration. These findings were detected even in absence of echocadiographically detectable changes in hemodynamic or functional ventricular parameters. This suggests that cardiac MRI may be a more sensitive tool than echocardiography for detection of myocardial abnormalities in patients with active SLE.

Objective: Cardiovascular disease (CVD) is overrepresented in patients with systemic lupus erythematosus (SLE). We determined the prevalence of traditional and non-traditional risk factors for CVD in SLE patients with and without CVD as compared to controls. Methods: Twenty-six women (52⫾8.2 yrs) with SLE and a history of CVD (SLE cases) were compared with 26 age matched women with SLE but without manifest CVD (SLE controls) and 26 age matched population-based control women (population controls). Common carotid intimamedia thickness (IMT) was measured by B-mode ultrasound as a surrogate measure of atherosclerosis. Results: SLE cases had increased IMT as compared to population controls (IMT 0.66mm ⫾ 0.15 vs 0.59 ⫾ 0.12, p⫽0.04) while the IMT of SLE controls (0.60mm ⫾ 0.14) did not differ from that of population controls(0.59mm ⫾0.12). As compared to SLE controls, SLE cases were characterized by raised plasma concentrations of circulating Oxidized LDL (Ox)LDL (p⫽0.03), measured by the monoclonal antibody E06, and autoantibodies to epitopes of OxLDL (aOxLDL; p⬍0.001); dyslipidemia with raised triglycerides (p⬍0.001) and lipoprotein (a) (p⫽0.002), and decreased HDLcholesterol concentrations (p⫽0.03); inflammation with raised levels of soluble Tnf-a (p⬍0.01) and alfa-1-antitrypsin (p⫽0.002); lupus anticoagulant (p⫽0.007) and high levels of homocysteine (p⫽0.03). Tnf-a levels correlated with dyslipidemia. SLE cases had received higher cumulative prednisolone dose (p⫽0.04) compared to SLE controls. Disease duration, smoking, blood pressure, BMI or diabetes mellitus did not differ significantly between the groups but osteoporosis was more frequent in SLE cases as compared to SLE controls (p⫽0.03). In conclusion, our data indicate that a set of distinct CVD risk factors separate SLE cases from SLE controls and population controls. If these observations can be confirmed in a prospective study, they could be used to identify SLE patients at high risk for CVD in order to optimize treatment.

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MAXIMUM OXYGEN CONSUMPTION, ANAEROBIC THRESHOLD AND HEART RATE IN SLE PATIENTS AND SEDENTARY HEALTHY CONTROLS. Maria RP Carvalho, Emilia I Sato, Antonio S Tebexreni, Vera L Tambeiro, Raquel TC Heidecher, Turibio L Barros Sao Paulo, Saõ Paulo, Brazil

VALVULAR ABNORMALITIES ARE STRONGLY ASSOCIATED WITH ARTERIAL DISEASE IN SLE. Elisabet Svenungsson, Kerstin Jensen-Urstad, Anders Hamsten, Ulf deFaire, Johan Frostegard Stockholm, Sweden

Oxygen consumption (VO2 max) in SLE patients is poorly studied and, in the literature there is no study evaluating the physical conditioning in these patients using the American Heart Association (AHA) classification. Objective: to evaluate the oxygen uptake (VO2max), anaerobic threshold (AT), maximum heart rate (max HR) and physical conditioning level according to AHA classification in SLE patients. Patients and Methods: Fifty SLE women (ACR criteria), and 102 sex-, age-, body weight-, and body mass index-matched healthy sedentary controls (HSC) were evaluated after signing the consent form. The Beck Depression Inventory and the Heath Assessment Questionnaire (HAQ) were applied to all patients. Patients and controls were submitted to a protocol of incremental load in treadmill (SensorMedics 2.000 treadmil-USA) with gas computed metabolic analysis, breath by breath with mean at each 20 seconds. The VO2max and VO2AT were calculated using Vmax29C analyzer SensorMedics (VISION-USA). The heart rate (HR) was measured by electrocardiogram (Cardiosoft program, Corina-USA). The AHA criteria of VO2max were used to classify the physical conditioning in patients and HSC. Statistic analysis included Student t test, Leven’s test, and Pearson‘s correlation coefficient. P values ⬍0.05 were considered statistically significant. Results: SLE patients showed significantly lower values of VO2max (24.64 ⫾ 4.91 vs 32.93 ⫾ 5.84 ml/kg/min, p⬍0,001), VO2AT (18.01 ⫾ 3.95 vs 20.20 ⫾ 4.72 ml/kg/min, p⬍0.05 ), and max HR (169.60 ⫾18.36 vs 180.82 ⫾ 12.16 beats-1, p⬍0.001) when compared to HSC. According to the AHA criteria, 62% of SLE patients and 15% of HSC had mean VO2max bellow of the mean (p⬍0.001). Physical conditioning was considered weak in 42% vs 16% and very weak in 20% vs 0% of patients and HSC, respectively (p⬍0.001). Twenty-four percent of patients had mild depression and 14% had mild/moderate to severe depression. The Beck inventory questionnaire showed correlation with HAQ (0.438, p⬍ 0.05) and max HR (-0.292, p⬍ 0.01). Conclusions: This study showed low oxygen uptake in SLE patients. It is possible that VO2max and VO2AT were lower due to fatigue, depression, decreased functional capacity, physic inactivity with involvement of peripheral muscles, all of these contributing to limit max HR as a physiological response. This is the first study classifying SLE patients according the AHA physical conditioning criteria.

Objective: to study cardiac valve morphology and function and ventricular function in patients with SLE with and without cardiovascular disease (CVD) and in population controls. Methods. Twenty-six women (52⫾8.2 years) with SLE and a history of CVD (SLE cases) were compared with 26 age-matched women with SLE but without manifest CVD (SLE controls) and 26 age matched population-based control women (population controls). CVD was defined as a history of objectively verified angina pectoris, myocardial infarction, cerebral infarction or intermittent claudication. Echocardiography was performed to assess valvular abnormalities and manifestations of ischaemic heart disease. B-mode ultrasound of carotids was used to estimate degree of atherosclerosis. Results. 13 / 26 (50%) of the SLE cases but only one of the SLE controls and one of the population controls had cardiac valvular abnormality. Three of the SLE cases had undergone valve replacement and one had a significant aortic insufficiency, the other nine had thickening of usually mitral leaflets without hemodynamic significance. Among SLE cases, patients with valvular abnormalities had higher homocysteine (p⬍0.001) and triglyceride (p⫽ 0.02) levels than patients without valvular disease. Among SLE cases there was no association between valvular abnormalities and degree of atherosclerosis as determined by intima media thickness and plaque occurrence Antibodies to phospholipids and double stranded DNA, disease duration and acute phase reactants did not differ between SLE cases with or without valvular abnormalities. Valvular abnormalities were not more common in SLE cases with stroke as compared to those with myocardial infarction, angina or claudication. Conclusion: Valvular abnormalities are strongly associated with CVD in SLE. Raised levels of homocysteine and triglycerides correlate with cardiac valve abnormalities.

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DIMINISHED AEROBIC CAPACITY IS A MECHANISM OF FATIGUE IN WOMEN WITH MILD SYSTEMIC LUPUS ERYTHEMATOSUS. Randall E Keyser, Violeta Rus, William T Cade, Neeta Kalappa, Raymond H Flores, Jamal A Mikdashi, Barry S Handwerger

FACTORS ASSOCIATED WITH DIABETES MELLITUS IN SYSTEMIC LUPUS ERYTHEMATOSUS. Abraham ZonanaNacach, Favio E Martinez, Antonio Fraga Mexico City, Mexico

Eighty percent of people with systemic lupus erythematosus (SLE) have life-limiting fatigue. Low aerobic capacity is known to cause fatigue and limit one’s ability to engage in physical activity. The degree to which low aerobic capacity limits the ability to participate in physical activity and relationships among fatigue scale scores and quantitative measures of diminished aerobic capacity have not been established in patients with SLE. The aim of this study was to determine if fatigue in patients with mild SLE is associated with low aerobic capacity. Methods: 18 women (age⫽35⫹9 yrs, wt⫽72.5⫹30.5 kg) with mild SLE (SLAM⫽3.1⫹2.1) and no other condition known to impair aerobic capacity and 16 healthy but sedentary women Measures of oxygen consumption (VO2) were recorded during the treadmill tests.Results: Peak VO2 was lower (p⬍.0006 vs. controls and p⬍.0001 vs expected) in subjects with SLE (19.2⫹4.4 ml/kg/min) compared to controls (27.4⫹4.7 ml/kg/min) and expected values (30.7⫹3.1 ml/kg/min). Functional aerobic impairment was observed in 14 of the 18 subjects with SLE. In subjects with SLE, ventilatory threshold, a marker for the onset of lactic acidemia, was observed at the lowest energy requirement for activities of daily living. Peak VO2, in the subjects with SLE, was similar to the energy requirements for the most vigorous activities of daily living, but below level needed for more intense occupational and recreational activities. In contrast, peak VO2 was significantly higher (p⬍.0000) than the energy requirements for all normal activitiers of daily living in controls, providing a substantial energy reserve. FSS was 44.9⫹12.8 in subjects with SLE, with 14 of the 18 subjects having scores above 36, a score at which fatigue would be expected to limit physical activity: of the 14, 11 (79%) had functional aerobic impairment. An FSS score of greater than 36 was not observed in controls (mean ⫽22.7⫹6.1). Conclusion: Women with SLE have diminished aerobic capacity, to a level where one would expect limited ability to perform activities of daily living and negligible energy reserve for engaging in occupational and recreational activities. Functional aerobic impairment was observed in conjunction with FSS scores that indicated the presence of clinically relevant perceived fatigue. These data provided physiological evidence for life-limiting fatigue and elucidated the validity of the FSS as an assessment tool in patients with SLE. Disclosure:

Objective. Assess risk factors associated with development of diabetes mellitus (DM) after SLE onset. Methods: Case-control study. During January-July 1999, 300 clinical records of patients with SLE seen at the Rheumatology outpatient clinic were reviewed. Those patients who developed DM after SLE diagnosis where paired by gender, age, and disease duration with SLE patients who did not develop DM. Factors for DM assessed at DM onset were family history of DM in first degree relatives, history of alcoholism, systemic hypertension, body mass index (BMI), total cholesterol, HDL, triglycerides, previous use of diabetogenic drugs, cumulative and high doses of prednisone (60 mg/day for 2 months). Statistical analysis was done using ␹2, Student t test, and logistic regression. Results:Thirty patients developed DM after SLE onset.Univariate analysis shows: Variable

LSLE-DM, nⴝ30

SLE-noDM, nⴝ27

p

Age, yrs SLE duration,mo History of DM, n History of alcoholism, n Systemic hypertension,n BMI Glucose, mg/dl Triglycerides, mg/100 ml HDL, mg/100 ml Pred cumulative dose, mg, ␮ Pred high dose, mg, ␮

44⫾13 110⫾85 20 7 16 30⫾6 232⫾122 311⫾194 73⫾69 19,300 47

42⫾12 101⫾83 5 2 7 28⫾6 84⫾5 163⫾69 53⫾19 17,175 50

0.52 0.70 0.001 0.09 0.03 0.75 0.001 0.01 0.003 0.76 0.76

The only variable retained in the logistic regression analysis was history of DM in first-grade relatives,(p⫽ 0.008). Conclusion: This study shows that while there are well known factors related with DM (obesity, corticosteroids), the positive history of DM in first degree relatives of patients with SLE was the most significant factor associated with DM after SLE onset Disclosure:

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IS DISEASE ACTIVITY DIFFERENT IN PREGNANT AND NON-PREGNANT WOMEN WITH ESTABLISHED LUPUS? Elizabeth M Chang, Eli G Shamiyeh, Susan Manzi, Winston Sequeira, Rosalind Ramsey-Goldman, Participating Physicians in the Lupus Registry Chicago, IL; Pittsburgh, PA

PREGNANCY-INDUCED HYPERFILTRATION IN WOMEN WITH LUPUS NEPHRITIS. Carl A Laskin, Jamie N Nadler, Christine A Clark, Karen A Spitzer Toronto, Canada

Although controversial, pregnancy may be a risk factor for the development of active disease in women with established lupus (SLE). To further evaluate this question, 173 women with SLE from Chicago (21 pregnant; 42 non-pregnant) and Pittsburgh (42 pregnant; 68 non-pregnant), were prospectively followed from February 1991 to May 1997 using an identical protocol for 9-15 mths. Study subjects were seen at 3 mth intervals for a minimum of 3 visits (if pregnant, in each trimester and at 3 and 6 mths postpartum). Maternal disease activity was assessed using the modified systemic lupus activity measure (mSLAM which excludes weight loss and ESR. Active disease was defined as mSLAM ⱖ 7 at any study visit. Data from both sites were combined as there were no site differences except that most of the African-American patients were from Chicago. Pregnant subjects compared with non-pregnant patients had a slightly shorter disease duration (4.6 vs. 5.0 median yrs, NS) and were more likely to be Caucasian than African-American (82% vs. 62%, p⫽0.04) but were younger at age of diagnosis (30 vs. 34, median yrs, p⫽0.0001). At first study visit, they were also younger (28.5 vs. 32.7 mean yrs, p⫽0.0001); on lower prednisone (5.6 vs. 8.8; mean mg, p⫽0.02); and less likely to be on plaquenil (7% vs. 60%, p⫽0.0001). When stratified by disease activity at first study visit, 10 (16%) pregnant and 38 (35%) non-pregnant subjects were classified with active disease and 53 (84%) pregnant and 72 (65%) non-pregnant patients had inactive disease. When disease was active at study visit 1, 8 (80%) of pregnant patients and 35 (92%) non-pregnant patients had at least one subsequent study visit rated with active disease (NS). Of these initially active patients, 1 (5%) pregnant subject and 4 (10%) control subjects continued to have active SLE throughout the duration of the study. In contrast, when disease status was inactive at the first study visit, 21 (40%) of the pregnant subjects and 42 (58%) of non-pregnant subjects had a change in status with at least 1 subsequent study visit rated with active disease (p⫽0.04). Of the subjects with inactive disease at visit 1, 32 (61%) of pregnant and 30 (42%) of non-pregnant subjects remained inactive for the duration of the entire study (p⫽0.04). Overall SLE disease activity was not significantly increased during pregnancy or postpartum.

Objective: To monitor the effects of pregnancy on kidney function in women with lupus nephritis. Methods: Twenty-four hour urine collections were obtained every 1 to 2 months during pregnancy (including pre- and post-pregnancy) in 12 women with lupus nephritis. Proteinuria levels were then compared with disease activity (using SLEDAI), medications and weeks gestation at the time of collection. Results: Six of the 12 women had pre-pregnancy proteinuria below 1.0 gm/day, and remained stable throughout their pregnancies. Four of the remaining 6 women who had pre-pregnancy proteinuria above 1.0 gm/day showed an average increase of 4.26 gm/day between 18 and 36 weeks gestation followed by a sharp decrease postpartum. The increased proteinuria showed no correlation with changing levels of clinical or serological disease activity or changes in medication. Conclusion: Women with lupus nephritis who are spilling over 1 gram of protein/day pre-pregnancy are at greater risk for developing deteriorating renal function during pregnancy, demonstrated by increased proteinuria beginning around 23 weeks gestation. This condition appears to be pregnancy related hyperfiltration and not a manifestation of disease activity. This condition also appears to be temporary, as all the women showed a sharp decrease in proteinuria immediately postpartum. Disclosure:

Disclosure: NIH/NIAMS P60 AR30692,K24 AR02138; MO1 RR00048; AF-IL and PA Chapters; AF-Clinical Science Grant; LFA-IL and PA Chapters

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PREGNANCY OUTCOME IN PATIENTS WITH LUPUS NEPHRITIS: ANALYSIS OF 23 PREGNANCIES. J Font, F Carmona, M Ramos-Casals, R Cervera, M Garcia-Carrasco, M Cayuela, S Jimenez, V Cararach, M Ingelmo Barcelona, Spain

PREDICTORS OF FETAL LOSS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS. Laura Gregg, Wilmer L Sibbitt Jr, Samir Patel, Marcos E Maldonado, Arthur D Bankhurst Albuquerque, New Mexico

OBJECTIVE. To study the outcome of pregnancy in women with systemic lupus erythematosus (SLE) and lupus nephritis (LN). PATIENTS. We prospectively evaluated 23 pregnancies in 22 SLE patients with LN (65% class IV) and 46 pregnancies in 37 SLE patients without nephropathy, using the same protocol (planned pregnancy during inactive SLE). If there were autoimmune complications (mainly antiphospholipid antibodies or thrombocytopenia), aspirin and/or prednisone at low-medium dose was used. Patients were monitored as having high-risk pregnancies. RESULTS. No statistical differences were found between pregnant women with and without LN concerning mean age at the time of delivery (28.6 years vs 30.4 years), clinical manifestations or immunological features of SLE. Analysis of the obstetric complications showed a lower mean gestational age at delivery (36,5 weeks vs 37,2 weeks, p ⬍ 0.05) and a higher frequency of preeclampsia (21% vs 5%, p⬍0.005), but perinatal mortality (5.2% vs 6.5%) and preterm delivery (26% vs 19%) were similar in both groups. Additionally, caesarean sections were performed more frequently in the LN group (47% vs 19%, p ⬍ 0.05). SLE relapses were similar in both groups (26% vs 37%), and no patient presented renal impairment during or after pregnancy. Active disease at conception and significant pregestational proteinuria were associated with a poor outcome (hypertension, birth weight). CONCLUSION. Pregnancy in SLE women with nephropathy is associated with a lower mean gestational age. Perinatal mortality and maternal SLE relapses were not higher in these patients than in pregnant SLE women without nephropathy. Nowadays, pregnancy in patients with SLE and LN should not be regarded as an unacceptable high-risk condition for the mother or her baby provided that conception is accurately planned and patients are managed according to a careful multidisciplinary treatment schedule.

BACKGROUND: Patients with systemic lupus erythematosus (SLE) have an increased risk of fetal loss (spontaneous abortion, miscarriage, or stillbirth). This has been attributed to several factors, including elevated levels of antiphospholipid antibodies (aPL). Based on our experience, we hypothesized that elevated levels of aPL would not correlate with fetal loss in patients with SLE, and that other clinical markers of lupus activity and vascular injury would be more predictive. OBJECTIVE: The aim of this study was to determine which clinical and serologic markers would be most predictive of fetal loss in patients with SLE. METHODS: We retrospectively evaluated 358 women with SLE with 726 pregnancies, examined fetal outcomes, and correlated these with clinical and serologic parameters of lupus activity. In addition to total fetal losses, we evaluated 1st trimester miscarriages as one cohort, and 2nd and 3rd trimester losses as a second cohort. RESULTS: The overall fetal loss rate was 31.7%. The presence of elevated aPL IgG or lupus anticoagulant (LA) was not associated with fetal loss. 1st trimester losses were most strongly associated with neurologic disease; specifically transient ichemic attacks (TIA) (p ⫽ 0.004), cerebral vascular accidents (CVA) (p ⬍ 0.0001), seizures (p ⫽ 0.0004), and elevated neuro SLEDAI (p ⫽ 0.01) and neuro SLICC (p ⬍ 0.0001) scores. In addition, immunologic disease (p ⫽ 0.0045) and hematologic disease (p ⫽ 0.03) were more common in women with 1st trimester losses than in women with successful pregnancies. 2nd and 3rd trimester losses were associated with the presence of renal disease (p ⫽ 0.007). CONCLUSIONS: The presence of elevated aPL and LA is not associated with fetal loss in patients with SLE. Its association to fetal loss appears to be subordinate to other markers of lupus activity and vascular injury. These markers are manifested most prominently by CNS disease for 1st trimester losses and renal disease for 2nd and 3rd trimester losses.

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PREGNANCY OUTCOME IN MOTHERS WITH ANTI-SSA/RO ANTIBODIES: A MONOCENTRIC STUDY OF 103 PREGNANCIES. Nathalie Costedoat, Zahir Amoura, Du Le Thi Hong, Bertrand Wechsler, Daniele Vauthier, Marie Elisabeth Dermer, Jean-Marc Lupoglazoff, Isabelle Denjoy, Marie Claude Diemert, Yves Darbois, Jean Charles Piette Paris, France

IL-10, sTNFR p55 and p75 DURING PREGNANCY IN SLE. Andrea Doria, Maurizio Cutolo, Anna Ghirardello, Francesca Vescovi, Sandra Zampieri, Leonardo Punzi, Pier Franca Gambari Padova and Genova, Italy Aim. To evaluate serum level variations of some soluble markers of immune activation considered suitable indicators of disease activity out of gestation, during SLE pregnancy. Methods. 17 consecutive pregnancies in 17 patients (mean age 30.5, range 21-38) with SLE (ACR criteria) and 8 pregnancies in 8 matched healthy subjects, as controls, were prospectively studied. All subjects were monthly monitored for clinical and laboratory parameters. Disease activity was evaluated by ECLAM score modified for pregnancy. Sera obtained within the last 3 months prior to pregnancy (in 12 programmed pregnancies) and at 9, 17 and 29 weeks of pregnancy, and 1 month after delivery, were stored at -20°C until analysis. Serum IL-10 and sTNFR p55 (I) and p75 (II) were detected by ELISA. Statistics were performed by PMDP package using the variance analysis for repeated measures, linear regression analysis and Student’s t test. Results. IL-10 serum levels were found significantly and persistently higher in SLE patients than in healthy women, either before(⬍0.0001) or during (⬍0.001) pregnancy. IL-10 levels did not vary throughout pregnancy in SLE patients, whereas in controls they were higher in the third trimester than before conception (p⫽0.029) or during the first trimester (p⫽0.038). sTNFRI serum levels were found higher in patients than in controls before conception (p⫽0.043) and in postpartum (p⫽0.040), but they did not significantly differ during pregnancy, even though significant differences in the serum level profile were observed (FT 22.4, p⬍0.0001, FGXT 3.9 p⫽0.02). sTNFRI was found increased during gestation both in SLE and in controls, however, the increment was lower in SLE. No differences were noted in sTNFRII serum concentration. Disease activity changed during pregnancy and postpartum (F⫽2.88, p⬍0.05). No correlation was observed between IL-10 as well as sTNFR levels and ECLAM score within any distinct pregnancy period. Conclusions. IL10 or sTNFR do not seem to be suitable indicators of SLE activity during pregnancy. Moreover, the abnormally and persistently high serum levels of IL-10 in SLE patients, either out of or during pregnancy seems to support the view that IL-10 overproduction in SLE is constitutive. The lower than expected sTNFRI level increase during gestation could suggest that in such condition the TNF/sTNFR balance might be influenced by specific factors (hormones?) related to SLE pregnancy itself rather than to SLE activity.

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Introduction : Maternal anti-SSA/Ro antibodies (ab) may lead to neonatal lupus erythematosus syndrome (NLES) characterized by a transient lupus dermatitis, systemic and hematologic abnormalities, and/or congenital heart block (CHB). Moreover, questions have been recently raised concerning QT interval prolongation without CHB in asymptomatic infants born from mothers with anti-SSA/Ro ab, potentially leading to increased life-threatening arrhythmias (A/R 2000,43:1049). Patients : Sixty women with anti-Ro/SSA ab were followed during 103 (including 2 twins) pregnancies. Connective tissue diseases (CTD) were SLE (n⫽34), primary Sjogren syndrome (n⫽20), mixed (n⫽1) or miscellaneous/ unclassified CTD (n⫽4). One additional patient was asymptomatic but was known positive for anti-Ro/SSA antibodies after bearing a child with CHB. Anti-La/SSB were associated to anti-Ro/SSA ab in 22 women. During pregnancies women received either prednisone (n⫽76), aspirin (n⫽71), LMWH (n⫽9), antimalarials (n⫽33) or no treatment (n⫽11). Nine pregnancies occurred in 4 women who previously had a child with CHB. Results : Maternal outcome : Eighteen pregnancies were complicated by mild CTD flare, 6 by mild hypertension. Fetal outcome : pregnancies resulted in therapeutic abortion (n⫽3, including 2 performed for cardiac abnormalities not associated with CHB), spontaneous abortion (n⫽18), stillbirth (n⫽3), premature birth (n⫽28, including the 2 twin pregnancies) and full-term birth (n⫽51). Therefore, 81 newborns were delivered at a mean gestational age of 37.8 weeks [30-41] with a mean weight of 2,841g [1,250-4,250g]. One premature twin died from pulmonary hypoplasia. Six children presented transient lupus dermatitis. No CHB or other manifestation of NLES occurred. EKG was available in 35 children. PR interval was 88 ⫹/-12 msec (m ⫹/- SD) vs 90 ⫹/-12 msec for 30 control children born from mothers with CTD devoid of anti-Ro/SSA or anti-La/SSB ab (p⫽NS). QTc interval was 408 ⫹/-32 msec vs 410 ⫹/-36 msec for control (p⫽NS). Sudden infant death or symptomatic arrhythmias were not observed during follow-up. Conclusion : no significant QT prolongation was associated to maternal anti Ro/SSA ab. Neither sudden infant death nor CHB occurred, even in mothers previously bearing a child with CHB.

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CLINICAL, HORMONAL AND THERAPEUTIC ASSOCIATIONS OF MENSTRUAL DISTURBANCES IN SLE PATIENTS WITHOUT ALKYLATING THERAPY. Sandra G Pasoto, Berenice Mendonca, Eloisa Bonfa Sao Paulo, SP, Brazil

REGISTRY FOR THE ANTIPHOSPHOLIPID SYNDROME: ASSOCIATION OF ANTIPHOSPHOLIPID ANTIBODIES WITH POST-MENOPAUSAL ARTERIAL EVENTS. Joan T Merrill, Tatsuya Atsumi, Hong Wei Zhang, Ranit C Shriky, Jill P Buyon New York, NY; Sapporo, Japan and Oklahoma City, OK

The role of alkylating agents in the pathogenesis of ovarian dysfunction and menstrual alterations in SLE is well established, however less is known about other factors. We, therefore, have evaluated 36 consecutive female SLE patients, age 18-39 years, without current or previous alkylating therapy, in order to determine the prevalence of the menstrual disturbances and its association with ovarian and thyroid function, prolactin levels, disease activity, steroid and azathioprine therapy. Seventeen patients presented normal cycles (Group A), where as menstrual alterations, varying from increased flow to amenorrhea, were observed in 19 (53%) (Group B). The patients from groups A and B were comparable concerning the mean age (28 ⫾ 5 vs 30 ⫾ 5 years, p⫽ 0.2), race (p⫽ 0.8), menarche age (14 ⫾ 2 vs 13 ⫾ 2, p⫽ 0.6), and disease duration (median 5 vs 5 years, p⫽ 0.3). Ovarian function was preserved in both groups, with similar levels of estradiol (median 39 vs 32 pg/mL, p⫽ 0.3), FSH (5 vs 4 IU/L, p⫽ 0.5), and LH (5 vs 5 IU/L, p⫽ 0.6). Subclinical thyroid disease (normal free T4 and mild elevated TSH levels), and slightly increased prolactin levels were detected in 14% and 8% of the patients. However, the frequency of these hormonal alterations was similar in patients with and without menstrual disturbances (p⫽ 0.3 and p⫽ 1, respectively). Similarly, the frequency of current use and the mean dosage of azathioprine were not associated with the occurrence of menstrual disorders (p⫽ 0.6 and p⫽ 0.5, respectively). Percentages of current use of prednisone (11/17 vs 16/19), mean dose (10 ⫾ 12 vs 19 ⫾ 17 mg/day), and percentages of patients on high doses (ⱖ 30 mg/day) of this steroid drug (1/17 vs 6/19) were comparable in patients from groups A and B (p⫽ 0.3, p⫽ 0.062 and p⫽ 0.09, respectively). Remarkably, the mean SLEDAI values (2 ⫾ 2 vs 6 ⫾ 6, p⫽ 0.02) and the frequency of patients with severe disease activity (SLEDAI ⱖ 8) (0/17 vs 7/19, p⫽ 0.008) were significantly higher in patients with menstrual disturbances. Moreover, 5/7 (71%) patients (Group B) with SLEDAI ⱖ 8 were on low steroid dose or without this medication due to recent flare. Our data strongly support that disease activity, as measured by the SLEDAI, is the major factor involved in the pathogenesis of the menstrual disorders in SLE patients without previous or current alkylating therapy.

The issues surrounding cardiovascular changes of menopause are complicated in healthy women and compounded in SLE. We undertook a non-interventional cross-sectional study of 101 postmenopausal lupus patients, 22 of whom had no history of antiphospholipid antibodies (aPL) (Group 1), 39 of whom had documented aPL but no major thrombosis (Group 2), and 40 of whom met the Sapporo criteria for the Antiphospholipid Syndrome (Group 3). There was a high incidence of both major and minor arterial events in this population, increasing in the thrombotic-prone group. Group 1 had total combined arterial events of 4/21 patients, group 2 of 9/39 patients and Group 3 of 23/40 patients (p⫽0.08), which included angina, myocardial infarctions, TIAs and cerebrovascular accidents. There were no differences between the three groups with regards to smoking history, hypercholesterolemia, hypertension, or diabetes. Thrombocytopenia was increased in the thrombotic group, being found in Group 1 in 1/22 patients, Group 2 in 6/39 patients,and Group 3 in 10/40 patients. In a univariate regression analysis, thrombocytopenia appeared to be associated with arterial events (p⬍0.003). Since multiple confounding variables could affect these data, a range of potentially independent, but confounding cardiac risk factors and medical therapies were considered. The variables most consistently impacting on arterial events in linear regression models were thrombocytopenia (p⬍0.001), use of cholesterol lowering agents (p⬍0.001), hydroxychloroquine (p⫽0.069), as well as hormone replacement therapy (p⫽0.038). We conclude that patients with antiphospholipid antibodies, particularly those with the antiphospholipid syndrome, may be at increased risk for post-menopausal arterial events and require intervention. Disclosure: The Registry for the Antiphospholipid Syndrome is partially supported by grants from Bio-Rad Laboratories and LaJolla Pharmaceutical Company.

Disclosure: Supported by FAPESP 95/0116-9

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IS ENDOMETRIOSIS ASSOCIATED WITH SYSTEMIC LUPUS ERYTHEMATHOSUS? Sandra G Pasoto, Mauricio S Abrao, Vilma ST Viana, Cleonice Bueno, Eloisa Bonfa Sao Paulo, SP, Brazil

DEHYDROEPIANDROSTRONE SULPHATE LEVELS IN PREMENOPAUSAL WOMEN WITH SYSTEMIC LUPUS ERYTHEMATOSUS. Sung Won Lee, Jin Hyuk Yun, Won Tae Chung Busan, Korea

Endometriosis is a major cause of pelvic pain and infertility in women in reproductive years. Recent evidence has implicated immunologic abnormalities in its pathogenesis and a few restrospective studies have suggested a possible association of endometriosis with systemic lupus erythematosus (SLE). The aim of this study is to determine whether women with histologically confirmed endometriosis are more likely to have rheumatic clinical and laboratory manifestations. The studied population consisted of 39 patients, age 18-40 years old, with pelvic endometriosis diagnosed prospectively by laparoscopy (10 in stages I / II and 29 in stages III / IV, as determined by APS criteria) (Group A), and 18 age-matched normal women without endometriosis, as confirmed by recent laparoscopy performed to reverse bilateral tube ligation (Group B). Patients and controls were clinically evaluated previous to the laporoscopic procedure and blood samples were obtained on days 1-3 of menstrual cycle for hematological and immunological evaluation. Urine analysis was performed during the same menstrual cycle. Antinuclear (ANA) and anti-dsDNA antibodies were detected by immunofluorescence on Hep-2 cells and Chritidia luciliae, respectively. Importantly, none of the patients with endometriosis fulfilled the ACR criteria for SLE. However, musculoskeletal complaints were more frequently observed in these patients compared with healthy women, such as poliarthralgia (ⱖ 5 peripheral joints)- 14/39 vs 0/18 (p⫽ 0.03), arthritis- 9/39 vs 0/18 (p⫽ 0.05), and generalized muscle pain- 8/39 vs 0/18 (p⫽ 0.05). Similarly, tender points were more commonly detected in group A patients- 16/39 vs 1/18 (p⫽ 0.01). The presence of these clinical manifestations was not associated with the laporoscopic severity of endometriosis, whereas all 6 sera with speckled ANA pattern with antibody titer varying from 1:80 to 1:1280 were from endometriosis patients classified as stage IV. Antibodies to the frequent targets in SLE (dsDNA, Sm, RNP and Ro/La) were uniformly negative in these patients and controls, and complement levels were within the normal range. Our prospective evaluation do not support the association of SLE and endometriosis. The high prevalence of musculosketal complaints, particularly poliarthralgia and fibromyalgia symptoms, in patients with endometriosis justify a multidisciplinary approach in the management of this chronic algic disorder.

Background/Aims : Dehydroepiandrosterone sulphate (DHEAS), the major steroidal product of the human adrenal, is abnormally low in patients with SLE. Moreover, a recent study confirms a positive effect of the precursor DHEA on the disease course in SLE, which supports an etiologically important role of the hormone on SLE. The aim of this study is to search for an interrelation between clinical manifestations, laboratory findings, and disease activities and DHEAS in patients with SLE in Korea. Method : DHEAS were measured by radioimmunoassay kit using 125I-labeled DHEA-SO4 antibody-coated tube in the serum of 49 patients with SLE and in 46 control subjects. Laboratory findings, clinical symptoms, signs and SLE disease activity index (SLEDAI) in SLE patients were evaluated by retrospective clinical chart review. RESULT : DHEAS was lower in patients with SLE compared to controls (52.06⫾61.79␮g/dl vs 101.55⫾56.54␮g/dl, p⬍0.001). The serum DHEAS levels were significantly negative correlation with systemic lupus erythematosus disease activity index (SLEDAI). There was no significant correlation between steroid dose and DHEAS (r⫽-0.041, p⫽0.795). CONCLUSION : Patients with SLE have low levels of DHEAS and significant negative correlation between DHEAS and SLEDAI. Disclosure:


Disclosure: Supported by FAPESP 99/04520-0 and 99/07483-8

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EFFECT OF MENOPAUSAL STATUS ON DAMAGE IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). Chi Chiu Mok, Carmen TK Ho, Ming Leung Szeto, Chak Sing Lau Hong Kong

PREDICTORS OF SELF-REPORTED PHYSICAL AND MENTAL FUNCTIONING IN SYSTEMIC LUPUS ERYTHEMATOSUS. Alan W Friedman, John D Reveille, Holly M Bastian, Barri J Fessler, Bruce A Baethge, Gerald McGwin, Jr, Seung-Ho Kang, Graciela S Alarcon Houston, TX; Birmingham, AL and Galveston, TX

Objectives: To study the effect of menopause on damage in an inception cohort of SLE patients. Patients and method: Consecutive female patients who fulfilled ⱖ4 ACR criteria for SLE were recruited as an inception cohort in 1998. The ACR/SLICC damage indices were obtained at entry and then scored yearly. Disease flares, defined by criteria modified from the SLEDAI-SELENA instrument, were also analyzed. At the third year, the change in damage scores was compared between those who were and were not postmenopausal at entry. The effect of menopause on damage was studied by regression method. Results: 216 patients were studied. The mean age at entry was 35.3⫾10.6 yrs. 36 patients (17%) were postmenopausal (22 cyclophosphamide[CYC]-induced and 14 physiological) and had a significantly older age (49.7⫾10.3 vs 32.4⫾8.0yrs, p⬍0.001), longer disease duration (119⫾106 vs 65⫾69mths, p⫽0.005), and higher baseline damage scores (1.56⫾1.65 vs 0.54⫾1.1, p⫽0.003) than the premenopausal patients. At the third year, 11 patients died (7 and 4 from the premenopausal and postmenopausal groups, respectively) and had greatest damage. The increment in damage scores was higher in the postmenopausal patients (0.85⫾0.96 vs 0.50⫾1.1, p⫽0.06), but the trend was insignificant. The no. of major flares was similar between the two groups (0.50⫾0.71 per patient vs 0.46⫾0.75, p⫽0.76) but the no. of mild/moderate flares in the postmenopausal patients was significantly lower (0.41⫾0.70 vs 0.91⫾1.1, p⫽0.001). Univariate analysis revealed that menopause at entry (RR 3.87 [1.84-8.14], p⬍0.001), increasing age (RR 1.04[1.01-1.07], p⫽0.006), baseline damage scores (RR 1.29[1.03-1.62], p⫽0.003), no. of major flares (RR 2.64[1.73-4.05], p⬍0.001), CNS disease (RR 2.30[1.30-4.10],p⫽0.004), and use of CYC (RR 3.13[1.72-5.70],p⬍0.001) or azathioprine (RR 2.14[1.21-3.80], p⫽0.01) were predictors for damage increase. The no. of major flares, however, was the only multivariate predictor for further damage (RR 2.46[1.36-4.44], p⫽0.003). Menopause at entry was not a significant risk factor for damage, after adjustment for other confounding variables (RR 1.90[0.64-5.65], p⫽0.25). Conclusions: In our inception cohort of SLE patients, those who were postmenopausal at study entry had more serious disease and baseline damage, and accrued more damage than those premenopausal over a 3-year period. However, after multivariate adjustment, the number of major disease flares, rather than the menopausal status, was the independent predictor for further damage.

PURPOSE: To determine the predictors of self-reported physical and mental functioning in patients with SLE from the 3 ethnic groups comprising the LUMINA cohort. METHODS: SLE patients of Hispanic (H, n⫽87), African-American (A, n⫽125) and Caucasian (C, n⫽ 110) ethnicity with disease duration of ⱕ 5 years were enrolled in a longitudinal study of outcomes. Sociodemographic, clinical (i.e. organ manifestations, disease activity, damage), immunogenetic, immunologic and behavioral variables were ascertained at baseline, and yearly thereafter. Self-reported physical and mental functioning were assessed using the physical and mental component summary scores (PCS and MCS, respectively– higher scores reflect better function) of the Medical Outcomes Study Short Form-36. Independent predictors of self-reported function over time were identified using Generalized Estimating Equation (GEE) multivariable regression analyses, which permit the use of all available data points for the cohort (mean disease duration ⫽ 55 months, mean followup ⫽ 33 months). RESULTS:

Disclosure:

outcome PCS

MCS

predictor

t-statistic

p-value

pain fatigue helplessness age SLAM score (disease activity) musculoskeletal involvement (per the SLAM) hospitalization for SLE in past year non-Hispanic ethnicity constitutional symptoms (per SLAM) male gender abnormal illness behaviors fatigue helplessness poor social support constitutional symptoms (per SLAM)

-9.9 -8.8 -7.6 -5.8 -3.8 -3.5 -3.5 -2.5 -2.3 -2.0 -10.2 -4.3 -3.6 -3.5 -2.9

⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.0001 0.0005 0.0005 0.0126 0.0229 0.0425 ⬍0.0001 ⬍0.0001 0.0003 0.0004 0.0038

CONCLUSIONS: Pain, fatigue, helplessness and abnormal illness behaviors are stronger predictors of self-reported function over time than specific disease manifestations, autoantibodies, disease activity or damage. Some of these may be amenable to change via behavioral interventions in addition to standard medical therapies. Disclosure:

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LONGTERM FUNCTIONAL AND SOCIAL OUTCOME IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AND RHEUMATOID ARTHRITIS (RA) - RESULTS FROM THE GERMAN RHEUMATOLOGICAL DATABASE. Rebecca Fischer, Joachim Listing, Christof Specker, Matthias Schneider, Angela Zink Duesseldorf and Berlin, Germany

COMBINATION THERAPY WITH METHOTREXATE AND CYCLOSPORINE A IN JUVENILE IDIOPATHIC ARTHRITIS. Angelo Ravelli, Chiara Moretti, Francesca Temporini, Federica Rossi, Silvia Magni-Manzoni, Stefania Viola, Gabriele Rossi, Angela Pistorio, Alberto Martini Pavia, Italy

OBJECTIVE: To describe the effects of SLE on long-term functional and social outcome in comparison to RA. METHODS: Data from the German rheumatological database on all 3,106 patients (p) with SLE seen at 21 collaborative arthritis centers and 3,106 p with RA, matched for age, sex, age at manifestation and disease duration, were analyzed. Physician global assessments of activity and severity, functional disability, pain, global assessment of health status, educational level and employment status were compared. RESULTS: Drawn from about 35,000 documented RA patients, there was a perfect match indicated by 89.1% female p with mean age of 42.5 y in SLE (RA 42.6), age at manifestation 32.1 y (RA 31.8y) and mean duration of disease of 9.2 y in both groups. Physicians‘ assessments of severity of the disease was “severe” or “very severe” in 18.4 vs 15.2 % (RA vs SLE) and of high activity 17.3 vs. 12.1 % . Patients’ assessed functional status was dependent on age but generally worse for p with RA (e.g. 20.1 % severe disability in RA vs 10.1 % in SLE). Severe pain was stated by 26.5 % in RA and 16.8 % in SLE. The global assessment of health status was similar in both patient groups (SLE: mean 3.06 vs. RA mean 3.36 on a numerical rating scale from 1-10) as also the marital status. With 41,2% the employment rate was slightly lower in SLE than in RA (44.1 %). Educational status did not differ significantly, but no vocational training was more frequent in SLE (15.5 % vs. 12.6 %). Early retirement was given in 18.4 % (SLE) vs.16.1 %; duration of work disability during the last year was longer in SLE (mean 9.3 vs. 6.2 weeks, median 4 weeks in both groups). CONCLUSION: There is an obvious misbalance between the judgement of activity and severity in long-term surviving lupus p and their functional and social impairment in comparison to matched RA p. Possible explanations comprise the different physician‘s judgement of severity and activitiy in SLE and RA. In addition, early retirement of lupus p may be due to initial severe disease followed by stable conditions in the longterm follow up.

Background. Little information exists on the use of combination therapies in juvenile idiopathic arthritis (JIA). Objective. To evaluate the efficacy and safety of the combination of methotrexate (MTX) and cyclosporine A (CyA) in patients with JIA who were refractory to MTX as a single agent. Methods. Seventeen consecutive patients with JIA, 6 boys and 11 girls, who did not improve or relapsed after 10 to 77 months (median 30.5 months) of MTX therapy at the dose of 15 to 25 mg/m2/week and were continued with MTX with the addition of CyA (4 mg/kg/day) for 6 to 30 months (median 10 months) were analyzed. The disease onset subtype was systemic in 9 pts, polyarticular in 5 pts and oligoarticular (with polyarticular course) in 3 patients. The clinical response to therapy was assessed through the preliminary definition of improvement (PDI) in JIA (A&R 1997;40:1202-9) and the radiographic progression by measuring the carpal length (Radiology 1978;129;661-8). The interobserver and intraobserver correlation coefficients for radiographic assessments were 0.97-0.99 and 0.98-099, respectively. Results. At the end of the treatment, as compared to the time when CyA was added to MTX, 8 patients (47%) demonstrated a clinical response according to the 30% PDI criteria; five of them (29%) also showed improvement with the 70% PDI criteria and 2 (12%) had achieved the clinical remission. In the whole cohort, the median number of active joints was decreased from 17 (range 6-42) to 8 (range 0-52) (p⫽0.027) and the median global articular severity score from 68 (range 18-162) to 27 points (range 0-242) (p⫽0.024). As compared to the treatment period with MTX as a single agent, during combined administration of MTX and CyA the median radiographic progression rate improved from -0.78 to ⫹0.35 in the better wrist (p⫽0.028) and from -0.66 to ⫹0.09 in the worse wrist (p⫽0.099). Seven patients (41%) experienced side effects during treatment with MTX ⫹ CyA (4 gastrointestinal discomfort, 2 raised creatinine level and 1 liver transaminase increase), which never required treatment discontinuation. The frequency of adverse events was similar to that observed during therapy with MTX alone (47%). Conclusions. In our JIA patients who were refractory to MTX as a single agent, the addition of CyA led to a significant clinical improvement in a consistent proportion of cases and seemed to reduce the rate of radiographic progression, without increasing the risk of side effects. Disclosure:

Disclosure: supported by German Ministers of Health and Science

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STEADY-STATE PLASMA CONCENTRATIONS OF ROFECOXIB IN CHILDREN (AGES 2-5 YEARS) WITH JUVENILE RHEUMATOID ARTHRITIS (JRA). Earl St. Rose, Manuel Ferrandiz, Maria Kiss, Oystein Forre, Richard Vehe, Gloria Higgins, Richard Rennenbohm, Arturo Porras, Peggy Wong, Lisa M DeTora, Kenneth E Truitt Rahway, NJ; Vieja La Molina, Peru; Sao Paulo, Brazil; Oslo, Norway; Minneapolis, MN; Colombus, OH; West Point, PA

THERAPEUTIC EFFICACY OF HUMANIZED ANTI-IL-6-RECEPTOR ANTIBODY IN SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS. Shumpei Yokota, Takako Miyamae, Tomoyuki Imagawa, Norihiro Nishimoto, Kazuyuki Yoshizaki, Raphael Hirsch, Masaaki Mori Yokohama, Kanagawa and Osaka, Osaka, Japan and Cincinnati, OH

OBJECTIVE: To compare steady-state AUC(0-24 hour) of rofecoxib in children aged 2 to 5 years vs adult historical controls (adultHCs) who received a single 25-mg tablet daily (mean weight of 77.7 kg; corresponding to ⬃0.32 mg/kg/day). 25 mg rofecoxib q.d. is anticipated as the dose for RA in adults. STUDY DESIGN: A steady-state pharmacokinetic study in 2-5 year-olds (n⫽11) with JRA used ⬃0.32 mg/kg/day; results indicated that 0.32 mg/kg/day approximated the exposure of 12.5 mg in adults. A confirmatory study in additional 2-5 year-olds (n⫽10)used 0.7 mg/kg/day. Patients received daily rofecoxib for 8 days then had trough and post-dose blood samples drawn for up to 24 hours. To minimize blood draws 2 separate sampling schedules with different time points were used. Comparison to adults HCs was made by calculating the AUC(0-24hr) geometric mean ratio (GMR) with 90% confidence intervals. Safety was assessed by investigator-reported adverse experiences. Global efficacy was assessed on an exploratory basis. RESULTS:The systemic exposure of rofecoxib in 2-5 year-olds was dose-proportional. The 0.7 mg/kg/day dose slightly exceeds the exposure of 25 mg in adults. The calculated dose to approximate the 25-mg adult exposure is 0.6 mg/kg/day. Efficacy measures were consistent with clinical improvement. Rofecoxib was generally well-tolerated. CONCLUSION: Rofecoxib 0.6 mg/kg/day in children aged 2 to 5 should approximate exposure to 25 mg in adults. TABLE: PEDIATRIC GROUPS VERSUS ADULT HISTORICAL CONTROLS

AUC(0-24hr) ng hr/mL

2 - 5 year-olds 0.32 mg/kg/day, n⫽11 2 - 5 year-olds 0.7 mg/kg/day, n⫽10 Adult Controls, n⫽26

Geometric Mean 2581 5649 4543

Cmax ng/mL GMR (90%CI) 0.57 (0.48, 0.73) 1.24 (1.02, 1.51) -

Geometric Mean 204 382 361

GMR (90%CI) 0.58 (0.45, 0.74) 1.06 (0.83, 1.35) -

Disclosure: Supported by a grant from Merck & Co., Inc

Background: Systemic juvenile idiopathic arthritis (S-JIA) is a severe disease frequently refractory to steroids and/or immunosuppressive drugs, and sometimes progresses to macrophage activation syndrome. Pathogenic role of serum IL-6 and IL-6/sIL-6R complexes is suggested in S-JIA. Increased IL-6 levels induce production of sIL-6R to make IL-6/sIL-6R complexes, which bind to gp 130, referred to as signal transducing protein, on cell surface. Two children with severe S-JIA were effectively treated by humanized anti-IL-6R antibody (rhMRA). Case Report: Two boys with S-JIA (N.S. 5 year old, and N.R. 6 years old) have been treated for 1.5 year and 1 year, respectively, with prednisolone and cyclosporin A, being complicated with short of stature, obesity and destruction of vertebrae (Th 5 and Th7). Their general condition became worse with spiking fever, rash, and arthritis, thus they were not able to leave their beds at all. High levels of CRP were persistently detected. Increased levels of IL-6 in their sera prompted us that administration of rhMRA might be effective to relieve their symptoms and to minimize laboratory abnormalities. With the informed consent of their parents and permission of the Ministry of Health and Welfare, 4 mg/kg of rhMRA was administered once a week. Spiking fever and arthritis were subsided, and CRP decreased from 12.9 mg/dl to 1.3 mg/dl in N.S., and from 10.2 mg/dl to 0.4 mg/dl in N.R. in one week. Accumulation of rhMRA in sera was achieved within 4 weeks accompanying with gradual decreases of IL-6 levels, and CRP levels were persistently below 0.5 mg/dl as well as the decreases of ESR and SAA . Both boys became better, getting up on beds and walking around in the room after couple of weeks. HAQ scores were remarkably improved. Conclusion: rhMRA effectively neutralize sIL-6R to inhibit the formation IL-6/sIL-6R complexes, and presumably block signal transduction via gp130. Thus, inflammatory markers were subsided, and clinical symptoms disappeared. Disclosure:

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EVALUATION OF NABUMETONE (RELAFEN®) IN PATIENTS WITH JUVENILE RHEUMATOID ARTHRITIS. Steven I Goodman, Paul F Howard, Brian R MacDonald, Ann E Haig, Bela R Patel Delray Beach, Florida; Scottsdale, Arizona and Collegeville, Pennsylvania

COMPARISON BETWEEN INTARARTICULAR TRIAMCINOLONE HEXACETONIDE AND ACETONIDE IN OLIGOARTICULAR JUVENILE IDIOPATHIC ARTHITIS. Giorgia Martini, Daniela Gobber, Caterina Agosto, Andrea Vianello, Francesco Zulian Padua, Italy

Purpose: Once-a-day dosing with nabumetone has been shown to be effective in adults with rheumatoid arthritis. The purpose of this study was to establish dosing recommendations for nabumetone in children with juvenile rheumatoid arthritis (JRA). Methods: An open-label, multicenter study was conducted in children with JRA between the ages of 2-16 yrs and weighing ⬎14kg (30lbs) requiring nonsteroidal anti-inflammatory drugs (NSAIDs) for control of symptoms. All NSAIDs were discontinued 1 day prior to study initiation in order to minimize disease flare. Patients received NAB approximately 30mg/kg once daily (as a tablet or a slurry with 5-10 mL warm water) for 12 weeks. Blood sampling for pharmacokinetic (PK) analysis, efficacy assessments, and safety evaluations were performed at weeks 1,3,6, & 12. Blood samples were obtained for determination of unbound 6-MNA (the active metabolite of nabumetone) concentrations. Efficacy evaluations were based on 7 standard rheumatology variables. Since flare was not required, true change from baseline could not be ascertained. Instead, the number and percent of patients who did not experience a flare of disease activity during the treatment period was determined. Since this was an open-label study, only descriptive statistics were obtained for efficacy variables. Routine safety assessments were completed for all patients. Results: 99 patients with JRA were enrolled and 89 completed the study; mean age was 9.2 yrs (range 2-16 yrs). Population PK analysis indicated that JRA patients with lower body weight (⬍40kg) had higher unbound 6-MNA concentrations compared to JRA patients weighing ⱖ40kg. The proportion of nabumetone-treated patients with no flare in disease activity was 92/99 (93%). Improvement was noted in each of the 7 efficacy assessments. Abdominal pain (5%), dyspepsia (4%), and headache (2%) were the most frequently reported adverse events of probable relationship to study medication. Conclusion: Nabumetone dosed at 30mg/kg/day (up to 2000mg/day) demonstrated a safe and effective profile in children with JRA. Dose individualization can be achieved by suspending tablets in a warm water slurry.

Aim : to compare safety and efficacy of intraarticular Triamcinolone Hexacetonide (THA) and Triamcinolone Acetonide (TA) in a cohort of children with oligoarticular JIA followed prospectively. Methods: patients undergoing intraarticular injections from January 1996 to January 2001 were selected to receive either THA or TA according to a random sampling code. Clinical assessment was performed at baseline and 1, 3, 6, 9, 12, 18 and 24 months afterwards using an articular score (Preliminary Core Set of Outcome Measures, Marco Island, 1994). Inflammatory markers (ESR, CRP) were tested at baseline, and disease duration, as well as NSAIDs and DMARDs treatments were noted. A good response was defined as a decrease of the articular score ⱖ 60%. The ␹2 and t-tests as well as the multivariate analysis stepwise and the Kaplan-Meier life tables were used to estimate the differences between the two study groups. Results: 84 patients entered the study, 41 in the THA group, 43 in the TA group. 127 injections were performed, 67 using THA, 60 with TA. The average dose used was 1.01 mg/kg of THA and 1.1 mg/kg of TA. There was no significant difference between the two groups in disease duration, baseline articular score, ESR and CRP values and NSAIDs and/or DMARTs. The average duration of improvement was 10 months with TA, 21 with THA. After 6 months the rate of response was significantly higher with THA than with TA (79% and 54% respectively, p⫽0.01) and no other variable, such as inflammatory markers, dose of drug or disease duration, affected the result except the drug used. The Log-Rank test showed that the probability to achieve the joint remission was significantly higher with THA than with TA (56% and 36% after 12 months respectively, p⫽0.0001). THA maintained good efficacy over time: with 40% of remission 24 months after the procedure while only 12% were observed with TA (p⫽0.0001). 2 patients in each group developed skin atrophy in the site of the injection, no other side effects such as calcifications, infections or chemical synovitis were observed. Conclusion: our results suggest that THA is much more effective than TA for the intraarticular use, both in the short and in the long term follow up, and this observation is not affected by disease duration, degree of local and systemic inflammation. Disclosure:

Disclosure: This study was supported by SmithKline Beecham.

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PROLONGED EFFICACY OF ETANERCEPT IN REFRACTORY JUVENILE ANKYLOSING SPONDYLITIS (JAS). Andreas Reiff, Michael Henrickson Los Angeles and Madeira, CA

LEUKOPENIA IN CHILDREN RECEIVING SULFASALAZINE AND ETANERCEPT IN COMBINATION. Kim H Striegel, Karen B Onel, Thomas J A Lehman

Objective: The short-term efficacy of etanercept (Enbrel) in 5 children with refractory juvenile ankylosing spondylitis has been previously reported. This study investigates whether Enbrel can sustain long term efficacy in a larger cohort of 8 children with JAS and prior DMARD failure. Patients: Eight children, 7 males and 1 female, average age 15.9 years (range 12-25) with JAS onset at mean age of 11.5 years (range 8-16) were enrolled in the study and followed for a mean of 15.4 months. Six of 8 children were HLA-B27 positive; 2/8 including the female were HLA-B27 negative. Average disease duration prior to etanercept therapy was 4.5 years (range 1.2-17.5). Prior to etanercept treatment all 8 children had persistent synovitis and an elevated ESR, 5 had radiographic changes, and 6 had anemia. Etanercept was administered at 0.2-0.8mg/kg subcutaneously twice weekly; 3 patients had a dose increase while on the study. Four of 8 children received concomitant MTX during the study. Results at 12 month (mean):

Sulfasalazine and etanercerpt are two drugs commonly used in the treatment of children with juvenile idiopathic arthritis (JIA/JRA). Both drugs are known to cause leukopenia, but there have been no clinical studies suggesting an additive or synergistic effect. We noted a significant incidence of leukopenia in children whose white blood cell counts (WBC) previously had been stable on long term sulfasalazine therapy. These episodes occurred within a few months of beginning etanercept prompting us to investigate the relationship. To evaluate the additive or synergistic effect of etanercept and sulfasalazine, we studied the WBC of 15 pediatric patients aged 2 - 19 years. Nine were on etanercept and sulfasalazine, six were on sulfasalazine alone. Absolute neutrophil counts (ANC) were charted for three months prior to initiation of etanercept and for three to six months afterwards. Exclusions were made for documented illness or changes in other medications known to cause leukopenia. Results: Paired t-tests indicated a significant decrease in ANC for the group as whole 4.9 ⫾0.96 versus 3.27 ⫾ 1.22(p⬍.021). However, the results were not significant for the 6 children who were begun on etanercept alone 4.07 ⫾ 0.83 versus 4.35⫾ 0.95(p NS). However, for those 9 on etanercept and sulfasalazine the difference was highly significant 4.15 ⫾0.67 versus 2.55 ⫾0.32 P⬍0.001. In children in whom we noted leukopenia with the use of both drugs, reduction in the dosage of sulfasalazine resulted in correction of the leukopenia in all cases. Conclusions: This data demonstrates a significant additive effect of sulfasalazine and etanercept in the induction of leukopenia in children with JIA. It is important to monitor the WBC carefully when utilizing these drugs together to minimize the incidence of complications associated with leukopenia. In our experience a reduction in the dosage of sulfasalazine corrected the problem.

Etanercept duration (months) AM Stiffness (minutes) Active joint count Hemoglobin (g/dl) ESR (Wintrobe mm/h)

0

2

6

9

12

p value

175 7.6 11.2 57.6

6.3 2.8 12.5 28.6

2.9 1.7 13.6 19.7

0 0.8 14.2 9.86

0 0.3 13.9 11.4

⬍0.103 ⬍0.001 ⬍0.001 ⬍0.0001

Conclusion: Etanercept treatment resulted in a very dramatic and rapid therapeutic response in all 8 children with respect to ESR,active joint count,increased hemoglobin and decreased morning stiffness. Anemia resolved entirely in all 6 affected patients. All patients tolerated etanercept without any side effects. Despite limited power, these results indicate that Enbrel provides sustained efficacy in refractory JAS.

Disclosure:

Disclosure: Andreas Reiff, MD is a clinical consultant for Wyeth-Ayerst Pharmaceuticals and Immunex Corporation

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SAFETY AND EFFICACY OF ETANERCEPT (ENBREL) IN CHILDREN WITH JRA LESS THAN 4 YEARS OF AGE. Deborah Rothman, Kristen Smith, Yukiko Kimura Springfield, MA and Hackensack, NJ

INFLIXIMAB TREATMENT IN THE REFRACTORY CHRONIC UVEITIS OF JUVENILE IDIOPATHIC ARTHRITIS (JIA). Visa Honkanen, Marjatta Lappi, Leena Koskinen, Paivi Lindahl, Pekka Lahdenne Helsinki, Finland

Introduction and Methods Etanercept has been approved by the FDA for the treatment of juvenile rheumatoid arthritis (JRA) in children older than age 4. However, many children with severe disease are younger than this. A questionnaire was sent to pediatric rheumatologists to determine if etanercept is being used in children less than age 4, whether there have been any adverse effects, and their clinical response. Results A total of 16 patients were reported, 12 with systemic-onset JRA (S-JRA) and 4 with polyarticular JRA (Poly-JRA). Detailed information is available on 14 of those. The average age (months ⫾ S.D.) that etanercept was started was 25.8 ⫾ 8.9 (range 6-40). All but one child were on methotrexate, all were on glucocorticoids and NSAIDs, and, in addition 2 of the children were on hydroxychloroquine, 2 were on cyclosporine, and one was on azathioprine. All children were started on etanercept because of poor clinical response to their medication regime and/or unacceptable steroid toxicity. The dose was .4 mg/kg/ sc twice weekly. The mean duration of etanercept therapy was 12.6 ⫾ 5.8 (range 6-20) months. No serious adverse events occurred. One child developed a presumed viral upper respiratory infection and subsequent disease flare, one child with a prior history of urinary tract infections developed one, and one child developed diarrhea but only after both cyclosporin and a homeopathic medication were started. There were no reports of any hematologic abnormalities. There was clinical improvement in 10 patients (8 with S-JRA and 2 with poly-JRA), based on ability to taper other medications, resuming or starting ambulation (3 children), and improvement of the joint exam. Prednisone was discontinued in 6 children. Three children are now on etanercept as their only medication. Four children, 3 with S-JRA and one with Poly-JRA, have shown little or no improvement with etanercept therapy. Conclusion Etanercept appears to be well tolerated, safe, and effective in some children with JRA less than age 4. This survey suggests that early introduction of etanercept in severe disease may be warranted and that long term safety of this medication in children less than 4 should be studied. Acknowledgements Harry Gewanter, Kathleen Haines, Gloria Higgins, Donna Hummell, Anna Huttenlocher, David Rawlings, Edward Sills, Chuck Spencer, Richard Vehe, Carol Wallace.

Background: severe chronic uveitis may impair the vision, and cause a life-long disability. Hypothesis: TNF inhibition can control the inflammation in the eye. Methods: 8 children (mean age 10.5 yr, mean duration of uveitis 7 yr) were treated with infliximab infusions (3-5 mgs/kg) at 4 to 6 weeks intervals for six months. All the patients had active disease, and were on topical steroids and methotrexate. Six patients were on systemic steroids and 6 also received cyclosporin A. At the start of infliximab, all the patients were on unacceptable high dose of topical steroids (times 3 or more/day). Four had undergone surgery for uveitis. During the study the patients continued on steady dose of methotrexate. Cyclosporin A was discontinued at one month. Local and systemic steroids were adjusted by the opthalmologist based on the ocular findings. The study was unblinded, non-randomized survey. Results: At 3 months, there was a clinical response in 5 patients (62.5%) defined as a 50% decrease in the cellularity (4 stage scale). At 6 months (while receiving infliximab at 6 weeks intervals) two patients flared defined as a 50% increase in the cellularity. At 6 months, the systemic steroid was discontinued in two patients, and tapered down at least 50% in 3. The frequency of the topical steroid application was down 50% or more in 4 patients. Conclusions: Infliximab may be beneficial in selected cases of refractory JIA uveitis. The present results suggest that infliximab should be given on monthly basis to control the inflammation. Disclosure:


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EFFECTIVENESS AND SAFETY OF ETANERCEPT FOR THE TREATMENT OF JUVENILE RHEUMATOID ARTHRITIS (JRA) IN CLINICAL PRACTICE. Hermine I Brunner, Anne L Tomasi, Tina M Sherrard, Janalee Taylor, Murray H Passo, Thomas B Graham, Daniel J Lovell Cincinnati, OH

SKEWED T-CELL RECEPTOR REPERTOIRE IN CHIDLREN WITH JUVENILE DERMATOMYOSTIS. Ann M Reed, Sherry Cragoe, Michael Strausbach, Lauren M Pachman, Peter Wettstein Rochester, MN; Chicago, IL

Background: A randomized controlled trial of etanercept in polyarticular JRA demonstrated significant clinical benefits and an excellent short-term safety profile (NEJM 2000;342:763-769). The effectiveness and safety of etanercept in JRA in clinical practice in combination with other treatments over an extended period have not been well described. Objectives: 1) To document the long-term effectiveness and 2) the side effects of etanercept as well as 3) its potential to allow for prednisone and methotrexate (MTX) taper in clinical practice. Methods: The number of joints with active arthritis (AJC) or with limited range in motion (LOM), number of swollen joints (SJC), erythrocyte sedimentation rate (ESR), parent assessment of patient overall well-being (visual analog scale) and medication regimens of all JRA patients (pts) treated with commercially available etanercept at the CHMC were reviewed at the beginning of etanercept therapy (month 0); then at month 1-2, month 3 and 6-monthly post-initiation until April 2001. Group medians were compared using Kruskal-Wallis and Wilcoxon rank sum tests. Results: The 55 pts on etanercept (⫾13% of all JRA pts at CHMC; 32 pts with polyarticular, 12 pts with systemic and 11 pts with pauciarticular JRA at onset) had a mean age and JRA duration of 14 and 7 yrs, respectively. The median time on etanercept was 12 months (range: 1-27 months). At month 1-2 there was already a significant decrease in the AJC, SJC, ESR by ⱖ 40% (p⬍0.02). By month 3 the median AJC further decreased to 4 (8 at month 0; p⬍0.0003), the medians of both SJC and LOM to 3 (7 and 8 at month 0, respectively; both p⬍0.002), the median ESR to 12 mm/hr (31.5mm/hr at month 0; p⬍0.002) and the overall well-being improved by 13% (p⬍0.002). There was no further important improvement in AJC, LOM, SJC, ESR or overall well-being beyond month 3 or an increase in patient growth velocity by month 12. While treated with etanercept, MTX was discontinued 17/41 pts (41%) and prednisone was discontinued in 7/18 (39%) and in 11 pts decreased by a mean of 0.12mg/kg. Seven JRA pts discontinued etanercept [lack of efficacy (1), noncompliance (2), pregnancy (2), remission (1) and infection (1)]. Hospitalizations occurred for infections (5 pts) and disease flare (1 pt). Etanercept was held in 3 pts during hospitalization and in 11 other pts (median hold: 1 week) for minor infections. Herpes zoster occurred in 2 pts, viral meningitis in 1 pt and an abscess in a rheumatoid nodule in another pt. Side effects included injection site reactions in 11%, upper respiratory infections in 30%, rashes in 5% of the pts. Conclusions: In clinical practice etanercept is frequently combined with prednisone and/or MTX. Etanercept is associated with significant andsustained clinical benefits in JRA. Patient improvement mostly occurs within 3 months of therapy and often allows for tapering or discontinuation of prednisone and MTX. Even when combined with other medications, etanercept is well tolerated and safe. Disclosure: Daniell Lovell, MD serves as a consultant to Immunex Corportation, Lederle Labs, Merck Pharmaceuticals, Boehringer Ingleheim Pharmaceuticals Inc., Centrocor Inc., Barr Pharmaceuticals, and Amgen.

T-cell receptor (TcR) beta and alpha-chain variable (V), joining (J), and constant (C) gene segments are disproportionally represented in varied immunologic states. CDR3 length diversity is hypothesized to represent differences within the T cell receptor repertoire and to show skewing in specific disease states. Skewing is thought to be multifactorial and suggests a response to homeostatic cues. In juvenile dermatomyositis (JDM) reports of TCR V␤ expression is increased with the specific repertoire restriction unknown. The over-representation or restriction of TcR alpha and beta CDR3 lengths suggests specific antigens drive the clonal response. We hypothesize that TCR repertoire differences exist in JDM resulting in an altered immune response. To answer this question, PCR based spectrotyping was performed using 25 human V␤ and 35 V␣ families gene primers, amplifying CDR3 lengths in immunomagnetically isolated T cell population including CD4 and CD8 positive cells. Samples were analyzed using capillary electrophoreses DNA analysis. Seven healthy children were used to establish normal controls and the CDR3 lengths within TCR families were seen in a Gaussian distribution in CD4 and CD8 cells. Ten new onset JDM subjects CD4⫹ and CD8⫹ cells from the peripheral blood and the inflammatory infiltrate from muscle biopsy samples at onset were analyzed. CRD3 lengths determined using RT-PCR demonstrated significant restriction of the peripheral blood CD4⫹ and CD8⫹ CDR3 lengths in 8/10 JDM subjects compared to 1/10 siblings as healthy controls. CD4⫹ cells V␣ and V␤ CDR3 lengths were restricted to either single or double peaks in 50% of the JDM vs 15% of the siblings TCR families. CD8⫹ V␣ and V␤ lengths were restricted in 55% of the JDM vs 10% of the siblings TCR families. Muscle biopsy samples demonstrated restriction of 65% of the V␣ and 60% V␤ of the TCR families. DNA sequencing was performed on the single and double peaks showing clonality in 60%. JDM samples were significantly altered from both their siblings and the healthy control children, suggesting clonality of TCRs and altered repertoire. This could be a result of undefined antigen stimulation or an alteration of the immune response, which could play a role in the pathogenesis of disease. Disclosure:

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THE ASSOCIATION OF THE DQA1*0501 ALLELE AND ANTIBODY TO A 56 kD NUCLEAR PROTEIN IN SERA FROM PATIENTS WITH JUVENILE DERMATOMYOSITIS (JDM). Jennica L Lutz, Tamara O Fedczyna, David Isenberg, Ruth Sperling, Lauren M Pachman Chicago, IL; London, UK and Jerusalem, Israel JDM,the most common pediatric idiopathic inflammatory myopathy, afflicts 3.2/million children/ year. JDM susceptibility is associated with the presence of the DQA1*0501 allele. 85% of JDM children possess the DQA1*0501 allele and data suggests that children lacking this allele have a strikingly different genetic, immunologic, and clinical profile. Genetic profiling of untreated muscle from DQA1*0501 JDM children shows upregulation of many interferon inducible genes, including an interferon inducible 56kD protein, suggesting facilitation of an anti-viral response (Neurology 56: A210,2001). Over 60% of children with JDM reported contracting an infectious type illness, possibly viral in nature, prior to the first definite symptom of JDM. Circulating antibodies that recognize a 56kD protein,a component of large nuclear ribonuclear particles, are found in JDM (Clin. Exp. Rheumatol. 12:451-7, 1994). During viral replication, cell components could be released bound to a foreign viral product, overcoming self-tolerance and starting an autoimmune process (Nature 304:177, 1983). Purpose To ascertain that children with JDM, positive for the DQA1*0501 allele, have a higher incidence of circulating 56kD protein, indicating presumed exposure to an antecedent illness, compared with JDM children without this allele. Methods The 56kD protein was determined by Western Blot in sera from 29 JDM patients and 1 healthy control (previous publications indicated total absence of the 56kD protein in controls). The patient population consisted of 11 children with JDM lacking the DQA1*0501 allele (determined by Southern Blot) and 18 JDM patients positive for this allele. Results The 56kD protein was identified in 62% of the JDM patients tested regardless of DQA1*0501 status. Sera from 14/18 JDM patients with the DQA1*0501 allele contained the 56kD protein, significantly more than the 4/11 seen in the DQA1*0501 negative patients (p⬍0.03). Other variables that were not associated with the 56kD protein: The TNF␣-308A allele, ANA titers, disease duration prior to treatment, disease activity, or age at the first symptom of disease. Summary The 56kD protein is increased in sera from JDM children who carry the DQA1*0501 allele, suggesting a response to a viral stimulus. Conclusion In JDM, the DQA1*0501 antigen is associated with antibody to a 56kD protein, which is supportive of the concept of an anti-viral response. Disclosure: Supported in part by the Greater Illinois Chapter of the Arthritis Foundation.

WHAT MORE CAN WE LEARN FROM MUSCLE PATHOLOGY IN CHILDREN WITH DERMATOMYOSITIS/POLYMYOSITIS? Jennifer C Wargula, Daniel J Lovell, Murray H Passo, Joseph D Santangelo, Kevin E Bove, Joseph E Levinson OBJECTIVE:To correlate disease course and complications in children with juvenile dermatomyositis (JDM) or polymyositis (PMS) with specific features of muscle pathology. METHODS:This is a retrospective cohort analysis of 58 children diagnosed with JDM or PMS between 1965 and 1998 and followed at the Children’s Hospital Medical Center (CHMC) in Cincinnati, OH for a mean duration of 88 months (range 13-294). Disease course was defined as limited, chronic non-ulcerative or chronic ulcerative (Crowe et al., Arthritis & Rheumatism 25:126,1982) and determined by reviewing patients’ medical records. All children in the cohort had muscle biopsies performed at CHMC and had disease for at least two years in order to classify their disease course as limited or chronic. Children without a primary diagnosis of JDM or PMS were excluded. Features of muscle pathology that were evaluated included loss of capillary bed, infarct, perifascicular myopathy, direct immunofluorescence (DIF) of vessels on biopsy and specifically, the locale of DIF, i.e., arteries or capillaries. Specific disease complications that were assessed included death, gastrointestinal ulceration, calcinosis, contractures, muscle atrophy and lipodystrophy. Data analysis was completed using Fischer’s exact test. RESULTS:Twenty-two children had limited disease (37.9%), 24 had chronic non-ulcerative disease, and 12 had ulcerative disease (20.7%). Neither capillary bed loss nor perifascicular myopathy on biopsy significantly correlated with subsequent disease course. Eight of the 12 children with chronic ulcerative disease had DIF arterial staining (66.7%), significantly greater than DIF arterial staining in children with chronic non-ulcerative disease{8.3%DIF⫹}(p⫽0.0001)and in the limited and chronic non-ulcerative groups combined{19.6%DIF⫹}(p⫽0.004). Infarct on muscle biopsy was significantly associated with the development of chronic ulcerative disease (p⫽0.01), with a positive predictive value (PPV) of 75%. Infarct also correlated with death (p⫽0.01) and GI ulceration (p⫽0.02), but the PPV of infarct for each of these outcomes was 50%. DIF arterial staining was significantly correlated with death (p⫽0.03) and GI ulceration (p⫽0.007). CONCLUSIONS:Findings of infarct and DIF arterial staining on muscle biopsy are significantly associated with the development of chronic ulcerative disease in JDM or PMS. Their presence should suggest early consideration of second-line therapeutic agents to more quickly bring disease activity under control. Disclosure:

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HIGH DOSE CORTICOSTEROIDS MAY AGGRAVATE THE VASCULOPATHY THAT UNDERLIES ULCERATIVE JUVENILE DERMATOMYOSITIS (JDM). Hulya Bukulmez, Jennifer C Wargula, Kevin Bove, Daniel J Lovell, Thomas A Griffin

VOICE ABNORMALITIES ARE FREQUENT IN JUVENILE IDIOPATHIC INFLAMMATORY MYOPATHIES (JIIM) AND CORRELATE WITH MUSCLE STRENGTH AND FUNCTION. L Rider, B Sonies, B Solomon, B Feldman, M Perez, L Zemel, R Rennebohm, C Wallace, C Lindsley, S Bowyer, S Ballinger, A Reed, P White, I Katona, J Hicks, P Lachenbruch, F Miller Bethesda, MD Dysphonia is a recognized feature of JIIM, but a systematic examination of voice symptoms has not been performed. Parents of JIIM patients (n⫽97, mean age 9.4 yrs, 66 F/31 M, mean duration 23.4 mos) and 124 healthy childhood controls completed a 27-item questionnaire rating frequency of abnormalities in voice quality, resonance, speech rate and articulation on a 4 point Likert scale (1⫽never, 4⫽usually or always symptomatic). Strained, too soft, hoarse, low-pitched, silences, wet/gurgly, and too loud (all related to voice quality) were more frequently symptomatic in JIIM patients (4%-27% parents of JIIM reported symptoms vs. 0-15% controls, P⬍0.038). Voice quality symptoms were infrequent (mean score 8.2⫾1.8 in JIIM for sum of 7 items vs. 0.8⫾1.1 in controls, P⬍0.0001). Parents were good reporters of voice symptoms, as the inter-rater reliability between 70 parents and a speech pathologist was moderate (ICC⫽0.57;r⫽0.63, P⬍0.001). The 7-item voice quality score correlated with some measures of myositis activity, including parent global disease activity, skin global activity, distal MMT, and CMAS, a measure of muscle strength and function (rs⫽ 0.21-0.43, P⬍0.043). Voice scores correlated inversely with duration of active disease (rs⫽-0.21, P⫽0.05) and patients with a polycyclic course were more frequently symptomatic than those with monocyclic or chronic continuous courses (P⫽0.013). Voice scores were moderately responsive to change (SRM⫽0.62 over a 9 month interval in patients symptomatic at baseline). All JIIM patients and controls at one center (n⫽54) were examined by a speech pathologist: JIIM patients had greater degree of abnormalities in voice quality (mean score 1.6 in JIIM vs. 1.1 in controls, P⫽0.004) and diadochokinesis of speech (mean score 1.5 in JIIM vs. 1.1 in controls, P⫽0.007), but not in articulation. Voice quality and diadochokinesis together correlated with global disease damage, CMAS, total and distal MMT (rs⫽0.41-0.51, P⬍0.04). Videostroboscopy performed on 4 patients with hoarseness revealed vocal cord nodules, edema and erythema. We conclude that voice quality and diadochokinesis, not just hoarseness, are often abnormal in JIIM and correlate with muscle strength and muscle function. The presence of persistent hoarseness should signal additional evaluation, as vocal cord nodules require voice therapy.

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CLINICAL COURSE AND OUTCOMES IN CHILDREN WITH DERMATOMYOSITIS (JDM) AND POLYMYOSITIS (PMS). Jennifer C Wargula, Daniel J Lovell, Murray H Passo, Kevin E Bove, Joseph E Levinson

WHAT CRITERIA DO PEDIATRIC RHEUMATOLOGISTS USE TO MAKE THE DIAGNOSIS OF JUVENILE DERMATOMYOSITIS (JD)? Kirsten Moenkemoeller, Ross E Petty, Peter N Malleson, David A Cabral, Lori B Tucker Cologne, Germany and Vancouver, BC, Canada

OBJECTIVE: To review the clinical course and outcomes of 58 children with JDM, PMS or sclerodermatomyositis (SDM). METHODS:This is a retrospective cohort analysis of all children diagnosed with JDM, PMS or SDM between 1965-1998, with disease duration of at least 24 months (mos). Disease course was categorized as monocyclic, polycyclic or chronic continuous (Spencer et al., J Pediatr 105: 399,1984). Disease activity (ACR scale) and Steinbrocker Functional Class (SFC) were recorded at baseline, 24 mos and last visit (LV). Data analysis used Fischer’s exact and student’s t-tests. RESULTS: Fifty-one children had JDM (87.9%), 3 had PMS, and 4 had SDM. Thirty-eight children were girls (65.6%), and 25.9% were non-Caucasian (12 African-American, 2 Hispanic, 1 Asian). Age at diagnosis (mos), mean ⴞ 1SD (median), was 96.6⫾46.9 (87.5). Time to diagnosis (mos) was 5.3⫾5.3 (4.0) and was not significantly longer for non-Caucasian children (5.8 vs. 5.1 mos in Caucasians, p⫽0.69). Duration of follow-up (mos) was 88⫾61.2 (61.5). Disease course was monocyclic in 38%, polycyclic in 19% and continuous in 43%. Steroid therapy was used in 57 patients (98%). Commonly used second-line agents included methotrexate (41.4%), Plaquenil (24.1%), and IVIG (10.3%). At diagnosis, 100% had active disease, 50% had mild pain or limitation of motion with daily activities, 34.5% could not perform all ADL’s independently, and 6.9% were bed-/wheelchair-bound. Disease remained active in 12% at 24 mos and in 14% at LV. Total remission was seen in 22.4% of patients at 24 mos and in 58.6% at LV. SFC at 24 mos and LV were similar: 63% and 69% functioning without pain or limited motion, 24% and 31% having mild pain or limited motion, and 6.9% and 6.9% bed-/wheelchair-bound, respectively. By LV, 31% had developed calcinosis, 34.5% had contractures, 13.8% had chronic arthritis, and 10.3% had developed osteonecrosis; vertebral compression fractures and cataracts each occurred in 8.6%. Three patients died from disease complications, 4 had GI ulceration and 3 had removal of normal appendices due to RLQ pain (1 developed necrotizing fasciitis of the abdominal wall and 1 had unsuspected duodenal perforation). CONCLUSIONS: Time to diagnosis was similiar for Caucasians and non-Caucasians in our cohort. Two-thirds of patients had chronic disease, but by 24 mos, more than 80% had achieved partial or total remission. Death occurred in 5%, and many had complications of chronic disease or therapy. Lower abdominal pain in JDM patients should be fully evaluated with imaging studies prior to undergoing abdominal surgery.

There are no validated criteria for the diagnosis of JD. The Bohan and Peter criteria:symmetric muscle weakness, characteristic skin changes, elevation of serum levels of skeletal muscle enzymes, characteristic changes on electromyography(EMG)and muscle biopsy(MB), are frequently quoted in publications, but have never been validated for children, and we suspect are inconsistently applied. The purpose of this study is to determine what criteria pediatric rheumatologists(PR) use to make the diagnosis of JD. Methods: A questionnaire was mailed to 175 PR in North America and Europe. Physicians were asked to rate the importance of clinical findings, laboratory tests, MB ,EMG ,and MRI in establishing a confident diagnosis of JD using a 5 point Likert scale. Results: The response rate was 59%. Experience in the treatment of JD varied:19% of PR had treated 10 or fewer patients, and 81% had treated more than 11. More than 85% of respondents rated the classic skin rash and proximal muscle weakness, and 69% elevation of muscle enzymes, as very or extremely important in making the diagnosis of JD. Muscle biopsy and EMG were rated by 54% as only somewhat important or not important at all. Investigations routinely used to diagnose JD included MRI(39%), EMG(26%) and MB(25%). 41% of PR used none of these investigations routinely. MB and EMG were never used by 8% and 19% of PR, respectively. Muscle biopsies were reported as frequently normal in many cases where the classic JD triad(muscle weakness, skin rash and elevated muscle enzymes)was present; 55% of PR reported that a normal MB in this situation did not influence diagnostic decisions. Conversely, in the absence of the classic triad, MB was felt to be diagnostic by 73% of respondents. Conclusions: These data suggest that PR do not routinely use the Bohan and Peter criteria for the diagnosis of JD. MB or EMG are being used in only one quarter of PR. Based on current practice, JD could be diagnosed in the presence of muscle weakness, elevated muscle enzymes, and classic skin rash. Only in the absence of all three criteria would MB and/or EMG be indicated for diagnostic purposes. The role of MRI in the diagnosis of JD requires further evaluation. To facilitate consistency of diagnosis, new criteria specific for JD should be proposed and validated.

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Objective: To determine if high dose corticosteroid therapy (HDCT) contributes to ulcer development in JDM and to correlate this effect with underlying vasculopathy. Methods: In 21 JDM patients with arterial immunofluorescence (AI) on muscle biopsy, the date of onset of the following parameters was determined: rash, skin ulcers (SU), gastrointestinal perforations (GIP), and HDCT (30 mg/kg intravenous methylprednisolone pulses or ⬎1 mg/kg/day oral prednisone). A blinded pathologist assigned a muscle biopsy score (MBS) to each patient on a 0 to 6 scale: 2 points for any AI, 1 point for multiple AI, 1 point for muscle infarct, 0.5 points for any capillary immunofluorescence (CI), 0.5 points for multiple CI, 0.5 points for peripheral fascicular myopathy, and 0.5 points for capillary loss. Comparisons were performed using Fishers Exact Test. Results: 13/21 patients developed SU, and 5 of these 13 patients also developed GIP. In 7/13 patients, the onset or worsening of SU or GIP occurred within 1 month of initiating HDCT, whereas the mean duration of rash before initiation of HDCT in these 7 patients was 5.4 months (standard error ⫽ 1.5 months). Patients treated with HDCT who also had MBSⱖ4.5 were more likely to develop SU (p⫽0.057), and even more likely to develop GIP (p⫽0.01) compared to patients with MBS⬍4.5. In one patient with GIP, arterial lipid deposition in GI tissue was proven by fat staining of a frozen section. In paraffin sections of the same GI tissue, the lipid-laden appearance of arteries was similar to that seen in GI tissue from other patients with GIP. In one patient with a low MBS (1.5), who developed SU shortly after HDCT, arteriopathy and associated tissue necrosis was seen in a Gottron’s papule biopsy, even though the pathology of the skin overlying the muscle biopsy was normal. Conclusions: HDCT appears to contribute to the development of ulcers in JDM, particularly in patients with MBSⱖ4.5. Identification of arterial lipid deposition in GI tissue suggests a pathogenic role for corticosteroids by enhancing lipid deposition, which could aggravate the underlying vasculopathy. Higher MBS are associated with ulcerative JDM, but pathological examination of active skin lesions could provide even greater predictive power for ulcerative JDM. We suggest that alternatives to HDCT should be prospectively investigated in patients with underlying vasculopathy that are at risk for ulcerative JDM.

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INTERNATIONAL CONSENSUS FOR CORE SETS OF OUTCOME MEASURES FOR DISEASE ACTIVITY (DA) AND DISEASE DAMAGE (DD) ASSESSMENT. PART II: JUVENILE DERMATOMYOSITIS (JDMS). N Ruperto, Murray, Ravelli, Lovell, Giannini, Tsitsami, Rider, Pachman, Machado, Garay, Bae, Balogh, Burgos-Vargas, De Inocencio, Harjacek, Hofer, Huemer, Kanakoudi, Mihaylova, Nielsen, Nikishina, Pagava, Reed, Rennebohn, Romicka, Rooney, Martini Pavia, Italy and Cincinnati, OH

GENE TRANSFER OF TIMP-1 AND TIMP-3 INHIBITS PROLIFERATION OF RA SYNOVIAL FIBROBLASTS AND REDUCES CARTILAGE INVASION IN THE SCID MOUSE MODEL OF RA. Willemijn H van der Laan, Christian A Seemayer, Paul HA Quax, Jan H Verheijen, Tom WJ Huizinga, Ferdinand C Breedveld, Beat A Michel, Renate E Gay, Steffen Gay, Thomas Pap Leiden, The Netherlands; Zurich, Switzerland and Magdeburg, Germany

Objective: To identify core sets of outcome variables for JDMS DA and DD assessment. Methods: 2 consensus methodologies were used: Delphi Technique, a set of well defined mail surveys, and Nominal Group Technique, a structured meeting designed to reach consensus. The 1st survey was designed to select variables used in current clinical practice to assess if a child has responded to therapy. The 2nd survey was to rank the top 10 variables listed by ⱖ 10 physicians in the 1st survey. Next 40 pediatric rheumatologists from 34 countries met to select the domains (broad concepts that groups variables) and the list of variable(s) to measure each domain of both core sets. Results: Ist survey: 174/267 (65%) physicians responded with 37 variables listed by ⱖ 10 responders. II survey: 221/277 (80%) responded with the same top 17 variables selected by both PRINTO and PRCSG members. Consensus conference results: for each core set domains are listed with variables to measure each domain reported in parenthesis. JDMS DA core set: 1) physician’s global DA assessment (10 cm visual analogue scale-VAS); 2) muscle strength (Childhood Myositis Assessment Scale-CMAS, Manual Muscle Testing-MMT); 3) muscle enzymes (CPK, LDH, aldolase, SGOT, SGPT); 4) functional ability (FA) (Childhood Health Assessment Questionnaire-CHAQ); 5) parent/patient’s global assessment of overall well being (10 cm VAS); 6) health related quality of life (HRQOL) (Child Health Questionnaire-CHQ); 7) global JDMS DA tool (Disease Activity Score-DAS, Myositis Disease Activity Assessment-MDAA). Domains (and variables in parenthesis) included in the JDMS DD core set were: 1) physician’s global DD assessment (10 cm VAS); 2) FA assessment (CHAQ); 3) HRQOL (CHQ); 4) muscle strength (CMAS); 5) global JDMS DD tool (Myositis Damage Index-MDI); 6) growth and development (height, weight, menses, Tanner Stage). Conclusion: We propose DA and DD core sets as a minimum list of domains (and variables to measure those domains) for future short and long term JDMS clinical trials, and for current clinical practice to decide if a child has responded to treatment. A prospective large-scale international data collection effort is in progress to validate the DA/DD core sets. A second consensus conference will establish a definition of improvement that may be used in future JDMS clinical trials.

Objective: Joint destruction in rheumatoid arthritis (RA) is caused by the invasion of synovial cells into the articular cartilage. Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in mediating this process. We investigated the effects of overexpression of tissue inhibitors of MMPs (TIMPs) by the synovial cells on proliferation and cartilage invasion. Materials and Methods: RA synovial fibroblasts (RA-SF) were transduced with adenoviral vectors (Ad) encoding TIMP-1 or TIMP-3. Untransduced and mock transduced (empty vector) RA-SF were used as controls. Transduction efficacy was assessed by LacZ staining of ␤-galactosidase transduced RA-SF. To assess duration of gene expression in AdTIMP-1 transduced RA-SF, TIMP-1 was measured by ELISA in the culture supernatants every 10 days until 60 days after transduction. Proliferation was measured by 3H-thymidine incorporation. The AdTIMP-1 and AdTIMP-3 transduced RA-SF and the control RA-SF were co-implanted with human articular cartilage under the renal capsule of SCID mice for 60 days. The invasiveness was evaluated on paraffin sections using a semiquantitative score. Results: A transduction efficacy of 67% was achieved. TIMP-1 levels in the supernatants of AdTIMP-1 transduced cells were 51.5⫾6.5 ␮g/ml as compared to 8.7⫾3.4 ␮g/ml in the mock transduced cells and were maintained for at least 60 days after transduction. AdTIMP-1 and AdTIMP-3 gene transfer resulted in an inhibition of proliferation as compared to the mock transduced RA-SF (35% and 40% respectively; p⬍0.05). In the SCID mouse model, the untransduced and mock transduced RA-SF deeply invaded the cartilage. The invasion scores were 2.5⫾0.2 (n⫽3) and 3.2 (n⫽1) respectively. Inhibition of invasion was observed in the AdTIMP-1 and AdTIMP-3 transduced RA-SF, 0.9⫾0.4 (n⫽2) and 1.2⫾0.2 (n⫽3), respectively. Conclusion: Both AdTIMP-1 and AdTIMP-3 gene transfer inhibit proliferation of RA-SF and reduce cartilage invasion. These results indicate that gene transfer of TIMPs may be a useful approach to inhibit joint destruction in RA. Disclosure: T. Pap was supported by a grant of the Deutsche Forschungsgemeinschaft (PA-689-2/1). C.A. Seemayer was supported by the Swiss National Science Foundation (4037-55152). All others were supported by their institutions.

Disclosure: European Union grant no. QLG1-CT-2000-00514

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DEVELOPMENT OF DISEASE ACTIVITY AND DAMAGE INDICES FOR MYOSITIS: INITIAL TESTING OF FOUR TOOLS IN JUVENILE DERMATOMYOSITIS. C Pilkington, K Murray, D Isenberg, V Farewell, J Davidson, B Feldman, A Ravelli, R Rennebohm, C Ryder, E Allen, F Miller, L Rider London, United Kingdom

HIVVIRUS-MEDIATED GENE TRANSFER OF ANGIOSTATIN INHIBITS INFLAMMATION OF COLLAGEN INDUCED MOUSE MODEL. Ko Kato, Kouichi Miyake, Noriko Suzuki, Tsutomu Igarashi, Masakazu Nagashima, Shinichi Yoshino, Takashi Shimada Tokyo, Japan

Juvenile myositis is rare and standardized assessment tools are needed to further collaborative research and clinical trials. To address this, a series of meetings and surveys using the Delphi approach that involved 70 myositis experts around the world, were utilized to develop 4 new tools: 2 to assess disease activity (the Myositis Intention to Treat Activity Index [MITAX], and the Myositis Activity Assessment [MYOACT] using 10 cm Visual Analogue Scales [VAS]) and 2 to assess the extent and severity of damage (the Myositis Damage Index [MDI], and the Myositis Damage Assessment [MYODAM]. Six JDM patients (3F, 3M, ages 4-11 yr, disease duration 8 mth-5yr, with a range of disease activity [MD global VAS 0.5-6.3] and damage [VAS 0-8.8]), were examined by 6 paediatric rheumatologists experienced in myositis, and were assessed using the CMAS as well as the 4 tools. Analysis of variance was used to assess variability in physician ratings vs. true patient variation; little of the variation in any measure was associated with the order of observations. Using the CMAS, physicians’ variation in ratings was minimal (0.1% of the variability was due to physicians, 99.9% due to patients). On the activity and damage tools, the median variation attributable to patients was 62% [min 38%, max 99%] for 9 activity organ systems, and 82%[38%, 98%] for 11 damage organ systems. The median physician variation was 31%[8%, 55%] for activity and 11% [1%, 38%] for damage. The major area of disagreement was the skeletal system (physician variation 55%), with less variation in ratings of global activity, cutaneous, GI and pulmonary disease activity (range 8-38%). Physicians had difficulty understanding the instructions for the MITAX with little training, so inter-rater reliability could not be assessed. Global damage, as well as muscle, GI, skeletal, cutaneous, and endocrine systems had excellent inter-rater variation (range 1-10% physician variability); pulmonary and ocular systems had acceptable physician variability (20-26%), using the MYODAM, and little variation using the MDI (5%). These data suggest that physicians expert in myositis demonstrate good inter-rater reliability for assessments of myositis activity and damage using the CMAS and 3 of the 4 activity and damage tools. These tools, or variants of them, should enhance the evaluation of disease activity and damage in myositis patients and bring much needed consistency to outcome assessments.

Rheumatoid arthritis (RA) is now considered to be an important target of gene therapy. Genes for immunomodulators and antiinflamatory cytokines have been used for gene therapy experiments. As an alternative approach, we are interested in regulation of angiogenesis. Increased angiogenic promoting factors and remarkable vascularization in the synovial tissue from RA patients were reported by synovial groups including our own. We constructed an HIV vector containing the gene for angiostatin, which is a proteolytic product of plasminogen. Collagen induced mouse models were generated by intra-dermal injection of collagen and adjuvant two times. The viral vector was injected into right knee joint at the same time when the collagen booster was given. Six weeks after collagen injection, significant synovial hyperplasia and pannus formation were detected in the control left joints transduced with the GFP vector. Destruction of cartlige was also observed. On the contrary, the joint treated with the angiostatin vector showed minimal proliferation of synovial lining cells. No pannus formation and no cartilage destruction were observed. These results indicate that angiostatin efficiently suppressed the development of collagen induced arthritis. In addition, the swelling of the the non-treated distal paws which is usually observed after immunization with collagen, was significantly suppressed after treatment of the knee joint.The distal effects may be due to trafficking of transduced cells or leaking of the vector and/or angiostatin. Angiostatic gene therapy may provide a new approach to the effective treatment for RA. Disclosure:


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GENE TRANSFER OF A FIBRONECTIN PEPTIDE MINIGENE SUPPRESSES INFLAMMATION IN MOUSE COLLAGEN-INDUCED ARTHRITIS. Tomoyuki Imagawa, Shohei Watanabe, Shigeki Katakura, Gregory P Boivin, Raphael Hirsch Cincinnati, Ohio

CONSTITUTIVE OVEREXPRESSION OF IL-17 IN THE KNEE JOINT OF NAI¨VE MICE INDUCES BONE DESTRUCTION BUT NO IRREVERSIBLE CARTILAGE DAMAGE. Erik Lubberts, Birgitte Oppers, Leo Joosten, Liduine van den Bersselaar, Paul Schwarzenberger, Jay Kolls, Wim van den Berg Nijmegen, Gld, Netherlands and New Orleans, LA

Background: Cell adhesion plays an essential role in arthritis by recruiting and retaining leukocytes in the joint. This process is regulated by the interaction of adhesion molecules present on the leukocytes and on vascular endotherium, synovial lining, and extracellular matrix. Fibronectin is one of the major extracellular matrix components in synovium, and it plays a central role in cell-cell and cell-matrix interactions through ligation of cell surface integrins. In the present study, a 15 amino acid peptide derived from the carboxy-terminal 33-kD cell and heparin-binding domain of fibronectin (FN-C/H-II) suppressed established arthritis in mice, associated with reduced infiltration of inflammatory cells into the joint. Methods: The FN-C/H-II minigene was generated by PCR and the gene was inserted into the pNGVL3 plasmid under control of the CMV promotor. The resultant plasmid DNA encoding the FN-C/H-II minigene was injected intravenously into mice with established collagen-induced arthritis. The clinical and histological findings of mice were evaluated. Results: Following injection, circulating FN-C/H-II could be detected for at least 5 days. Mice treated with the FN-C/H-II-encoding plasmid, but not mice treated with a control luciferase-encoding plasmid, demonstrated a marked reduction in progression of arthritis. Not only was disease progression halted, but a significant improvement was observed in joint swelling within 2 days of treatment. Leukocyte adhesion and recruitment were inhibited by FN-C/H-II, both in vitro and in vivo. Histologic evaluation demonstrated a marked reduction in infiltration of both neutrophils and lymphocytes into synovium, persisting for at least 10 days. Conclusion: These studies suggest that antogonism of cell adhesion by soluble fibronectin peptides may provide a therapeutic approach towards attenuating chronic arthritis. Disclosure:

IL-17 is expressed in the synovium of RA patients. It is secreted by a restricted set of cells, whereas its receptor is ubiquitously expressed on many cell types. Recent in vitro data indicate that IL-17 may promote both joint inflammation as well as tissue destruction. In this study, we investigated the effect of local IL-17 application in the knee joint of naive and IL-1b knockout (KO) mice, with emphasis on synovitis and joint destruction. Histological analysis revealed joint inflammation after a single injection of 10^7 PFU of AdIL-17 at days 2, 5, and 10, which gradually increased during time. Predominantly influx of polymorphonuclear cells were noted at day 2, and at days 5 and 10 also mononuclear cells were detected. A single injection with the same dose of a control adenoviral vecotor did not induce joint inflammation at these timepoints. Although marked proteoglycan depletion was found in cartilage, no surface erosion, collagen damage and irreversible cartilage damage was noted, at day 10. However, at this timepoint erosion of the cortical bone in patella and femur/tibia, in particular was observed. Marked expression of RANKL and IL-1 mRNA were found in the synovium. Intriguingly, 5 and 10 days after the viral injection of AdIL-17 in IL-1b KO mice, significantly more bone erosion was observed. RT-PCR revealed marked increase of RANK but suppression of OPG mRNA expression in the synovium of the IL-1 KOs. AdIL-17 did not enhance cartilage damage in IL-1 KO mice. In conclusion, these data show uncoupling of irreversible cartilage damage and bone destruction under IL-17 overexpression conditions. IL-17 induced bone erosion seems to be mediated by the induction of RANKL and may be IL-1 independent. Increase in RANK expression by IL-17 may play an important role in the enhanced bone erosion in the IL-1 KO mice. Disclosure: This work was supported by Dutch Arthritis Association Grant NR 00-1-302

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PROTEASOME INHIBITORS REGULATE TRANSGENE EXPRESSION IN HUMAN SYNOVIUM FOLLOWING TRANSDUCTION BY ADENO-ASSOCIATED VIRUS. Shigeki Katakura, Kristi Jennings, Guangping Gao, James Wilson, Raphael Hirsch Cincinnati, OH and Philadelphia, PA

IN VIVO VIRAL IL-10 GENE ELECTROTRANSFER PREVENTS EXPERIMENTAL ARTHRITIS. Florence Apparailly, Norma Perez, Virginie Millet, Caroline Minot, Daniele Noel, Jacques Sany, Olivier Danos, Christian Jorgensen Montpellier and Evry, France

AAV is a single-stranded DNA parvovirus that is non-pathogenic in humans and can mediate long-term transduction without provoking a significant immune response. These properties make AAV attractive for use in gene therapy of rheumatoid arthritis (RA). However,not all cell types are efficiently transduced by AAV. The barriers to efficient transduction could include some steps. To investigate the potential of AAV as a gene transfer vector for arthritis, the ability of AAV to infect synovium in vitro and in vivo was tested. Three human RA and 2 murine (one DBA/1J and one DBA1J x C3H F1) fibroblast-like synoviocyte cell lines were infected with 10^4 particles/cell of a murine IL-10 encoding vector (AAV-mIL-10). The supernatants were harvested at various time points and assayed for mIL-10 expression by ELISA. Human and murine synovial cell lines infected with AAV alone demonstrated only low-level transgene expression. Treatment of AAV-infected human cell lines with the proteasome inhibitor,carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal (zLLL), resulted in a dose-dependent increase in mIL-10 secretion of up to 10-fold. This increase in transgene expression peaked 2 days following exposure to zLLL and returned to baseline by day 5. To further explore this finding, synovial cells were re-exposed to zLLL following the loss of transgene expression.We found that transgene expression could be repeatedly induced by re-exposure to zLLL. In each case, expression peaked 2-3 days following exposure to zLLL and then dropped off. We were able to reinduce transgene expression as far out as 25 days after infection. In contrast, induction of second-strand synthesis by a single exposure of cells to adenovirus or to g-irradiation resulted in a sustained elevation of transgene expression. As opposed to these observations in human synovial cell lines, transgene expression could not be induced in murine synovial cell lines either with zLLL or with adenovirus or g-irradiation. These data suggest that proteasome inhibitors might be used to regulate transgene expression in human synovium, and possibly other tissues derived from mesenchyme,following AAV-mediated transduction. Furthermore, there is a species difference in the transducibility of human and murine fibroblast-like synoviocytes by AAV. Further studies may allow optimization of AAV for the treatment of human arthritides.

We previously showed the efficiency of viral IL-10 (vIL-10) gene transfer mediated by an adenoviral vector, in both collagen-induced arthritis and SCID/hu mice models. However, vector immunogenicity, and short duration of transgene expression limit the use of adenovirus. The recent development of intra-muscular DNA electrotransfer methods offers new perspectives for cytokine gene transfer in RA as they show performances comparable with viral vectors. The purpose of this study is to assess the potential gene expression regulation and therapeutic efficiency of vIL-10 cDNA electrotransfer in DBA1 mice with collagen-induced arthritis (CIA). Moreover, we propose to regulate vIL-10 expression with tetracycline derivative (tetON system) and to improve the regulation system by expressing the tetracycline controlled transcriptional silencer (tTS) that binds promoter in absence of doxycycline. The co-injection in the tibialis anterior of DBA1 mice with a plasmid encoding vIl-10 under the control of a doxycycline-inducible promoter and a plasmid expressing tTS, immediately followed by electric pulses (200V, 2Hz, 8 pulses de 20ms), resulted in a dose-dependent increase in the vIL-10 production (2.86 ⫾ 1.11 ng/mg protein on day 7 with 40␮g plasmid), detectable for at least 8 weeks. There was a basal expression of the protein in injected muscles, increased significantly 8.5 times by doxycycline at 4 weeks. The doxycycline treatment (200 ␮g/ml in drinking water) showed significant inhibitory effects on paw swelling compared to the control group without doxycycline (1.79 ⫾ 0.22 versus 2.13 ⫾ 0.84 mm on day 32 post-immunization). CIA was delayed in the doxycycline-treated group as arthritis onset was 32.62 ⫾ 4.50 days, versus 28.38 ⫾ 3.62 days in the control group. Thus, vIL-10 gene electrotransfer not only led to a long-lasting and dose-dependent increase in cytokine expression, but also to a delay of arthritis and a decrease in severity of CIA. Disclosure:

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GENE THERAPY OF ARTHRITIS : DESIGN AND FUNCTIONAL IN VITRO STUDIES OF A CHIMERIC PROMOTER INDUCED BY PRO-INFLAMMATORY MEDIATORS. Charbel Massaad, Veronique Meynier de Salinelles, Colette Salvat, Jean-Luc Olivier, Gilbert Bereziat, Claire Jacques, Michel Raymondjean, Francis Berenbaum Paris, France, Metropolitan

ELECTROTRANSFER OF MURINE IL-10cDNA IN COLLAGEN-INDUCED ARTHRITIS: EARLY AND LATE EFFICIENCY. Nathalie Saidenberg-Kermanach, Natacha Bessis, Virginie Deleuze, Michel Bureau, Daniel Scherman, Marie-Christophe Boissier Bobigny and Vitry/Seine, France

Viral promoters are commonly used in gene therapy because of their high transcriptional activity. Nevertheless, their main disadvantages are the uncontrolled expression of the therapeutical gene and the extinction of their activity due to the cell defenses. With the aim of treating arthritis by gene therapy approach, we have designed a novel chimeric promoter induced by the inflammatory context (Interleukin-1, prostaglandins) to enhance the expression of a therapeutic gene. This chimeric promoter consists of the minimal promoter of the secreted type IIA phospholipase A2, which is induced by IL-1 and glucocorticoids (Massaad et al., J Biol Chem 2000). Upstream of the sPLA2-IIA promoter we have added a duplicate of the PPAR Response Element (DR1) that is activated by eicosanoids and non-steroidal anti-inflammatory drugs. We have designed three PPRE units formed by two DR1 in tandem separated by 18, 21 or 30 bp center to center. Gel shift assays showed that either PPAR ␣ or ␥ isoforms bind most cooperatively the PPRE unit formed by two DR1 separated by 21 bp. Rabbit articular chondrocytes in primary culture were transfected with this promoter and the reporter gene activity was assessed in the presence of different mediators. The synthetic promoter has very low basal activity, less than 5% of the RSV or SV-40 promoter activities, which are commonly used in gene therapy. Its induced activity reached 30% to 40% those of viral promoter. This synthetic promoter is induced by various mediators [IL-1 (3-fold) and lipids (prostaglandin-J2 (3-fold), Wy-14643 (4-fold)].The combination of IL-1 and prostaglandin-J2 exhibited a synergistic effect on the transcriptional activity of the promoter (8- to 12-fold). We also show that this promoter is induced by high doses ibuprofen(3.5-fold)and indomethacin (5-fold). In conclusion, this new synthetic promoter is perfectly inducible in vitro by the inflammatory context. Vectorization and in vivo experiments are now needed to validate this novel approach in gene therapy of arthritis.

Background: Gene therapy of rheumatoid arthritis is a promising strategy. Naked DNA encoding for anti-inflammatory molecules is quite simple and safe to use, but its use is frequently limited by a weak efficiency when just injected i.m. IL-10 is a potent anti-inflammatory cytokine already proven as efficient in several models of arthritis. Electrotransfer (ET) is a recently developped method that was shown to be more efficient in vivo than simple intramuscular DNA injection. Objective: To demonstrate anti-inflammatory efficiency of ET in collagen-induced arthritis (CIA) using murine IL-10 cDNA. Methods: 200ng of pCOR plasmid encoding for IL-10 was injected i.m. in tibial cranial muscle. ET was performed just after by applying square-wave electric pulses (8 pulses of 200 V/cm, 20 ms duration at 2 Hz). DBA/1 mice were immunized with bovine type II collagen. Control groups consisted in ET performed with empty plasmid or with saline. Results: In early treatments (ET performed twice on days 11 and 24), CIA was significantly inhibited in treated group (vs control group treated with empty plasmid), as evaluated by disease onset, clinical arthritis scores, and histological evaluation. Late treatment (ET on days 24 and 40, at the onset of the clinical disease) was followed by a significant reduction of clinical and histological scores. Conclusion: We show for the first time that ET of IL-10 cDNA inhibits inflammation in a model of arthritis. Such non viral systemic strategy of cytokine delivery is efficient in the treatment of CIA, even in late administration. Disclosure:

Disclosure: This work has been funded by CNRS and AVENTIS PHARMA and patented by AVENTIS PHARMA.

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TARGETING CATHEPSIN L BY SPECIFIC RIBOZYMES DECREASES PROTEIN SYNTHESIS AND CARTILAGE DESTRUCTION IN RHEUMATOID ARTHRITIS. Jorg Schedel, Christian A Seemayer, Wolfgang Zacharias, Thomas Pap, Michel Neidhart, Stefan Kuchen, Beat A Michel, Renate E Gay, Steffen Gay Zurich, Switzerland and Birmingham, AL

POSITIVE AND NEGATIVE SELECTION MECHANISMS CONTRIBUTE TO THE REGULATION OF AUTOREACTIVE HUMAN B-CELLS. Aimee Pugh, Amedeo Cappione, Gregg Silverman, Dan Ryan, Richard Insel, Inaki Sanz Rochester, NY and La Jolla, CA We have established an experimental system that analyzes the fate of B-cells expressing surface Ig encoded by the intrinsically autoreactive VH4-34 gene (VH4.34 cells). Using tonsils obtained from normal donors we have established that VH4-34 cells make up to 10% of the naive compartment but less than 1% of the memory and plasma cell compartments. Thus, more than 90% of naive VH4-34 cells are eliminated in the transition to the germinal center (GC) and memory compartments. Interestingly however, naive VH4.34 cells first undergo antigen-mediated positive selection from the resting Bm1 into the activated Bm2 subsets suggesting that these potentially pathogenic cells may play a beneficial role in the primary repertoire. Immunocytochemistry studies in more than 100 germinal centers from different tonsils indicate that VH4-34 cells accumulate in the follicular mantle but are absent from the germinal center proper. Furthermore, VH4.34 cells are abundant in the spleen marginal zone in normal individuals. In active SLE VH4-34 cells are significantly expanded and represent up to 30% of the memory and plasma cell compartments. Based on these observations, we postulate that in normal subjects autoreactive VH4.34 cells are prevented from entering the germinal center and therefore excluded from the T-cell dependent memory repertoire. We believe that our results are consistent with positive selection of follicular naive VH4.34 cells into the marginal zone where they may play a beneficial role in host defense and where tolerance may be maintained by interaction with regulatory T/NK-T cells. Studies to understand the mechanism of follicular exclusion and of compartmentalization into the marginal zone are currently underway in our laboratory. Disclosure:

Disclosure: J. Schedel was supported by the German Academic Exchange Service (DAAD), C.A. Seemayer and T. Pap by the Swiss National Science Foundation, S. Kuchen by the EMBO foundation, all others by their respective institutions.

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Objective: We examined whether ribozymes cleaving cathepsin L mRNA are able to decrease the production of cathepsin L and the invasion of RA synovial fibroblasts (SF) into cartilage in vitro and in the SCID mouse model. Methods: Two ribozymes were used which cleave cathepsin L at positions 532 (Rz532) and 790 (Rz790). Using retroviral gene transfer RA-SF were transduced with the ribozyme constructs or the empty vector. To examine the effect of the ®ribozymes on the mRNA level, quantitative analysis was performed using real-time PCR (TaqMan ). For evaluation at the protein level, ELISA using anti-cathepsin L antibodies was performed. In addition, transduced RA-SF were examined in vitro in an three-dimensional destruction assay evaluating their ability to degrade extracellular matrix produced by human chondrocytes. Matrix destruction was monitored by the release of soluble glycosaminoglycans (sGAG). Using the in vivo SCID mouse model of cartilage invasion, Rz532 transduced RA-SF were co-implanted with human cartilage for 60 days (n⫽6; mock⫽5). After sacrifice, invasion of RA-SF into the cartilage was evaluated by using a semi-quantitative score. Results: The expression of cathepsin L mRNA in Rz532- and Rz790-transduced RA-SF was decreased to 44% (range 25-62%) and 20% (range 1-43%), respectively, of the level in mock transduced RA-SF. The concentration of cathepsin L in supernatants of transduced RA-SF was decreased to 4.05 and 8.22 ng/ml (mean) in comparison to 13.15 ng/ml (mean) in the mock constructs. Using the in vitro cartilage destruction assay, the release of sGAG decreased to 46% and 60% of the release by mock-transduced cells after 14 days. Examining the effect of Rz532 in the SCID mouse model, a decrease of the invasion score was observed (score 1.6, SEM⫾0.3), in contrast to the mock transduced cells (score 2.8, SEM⫾0.6). Conclusion: Using retroviral gene transfer, ribozymes cleaving cathepsin L mRNA appear to be a feasible approach to inhibit specifically the synthesis of this catabolic enzyme. Moreover, our study suggests that ribozymes against cathepsins might present a novel and efficient tool to inhibit cartilage destruction in RA.

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DYSREGULATION OF ACTIVATION MOLECULES, CD20 AND CD40 LIGAND, IN SYSTEMIC LUPUS ERYTHEMATOSUS B-CELLS. Jennifer H Anolik, R John Looney, Inaki Sanz, Fay Young Rochester, NY

TAMOXIFEN ABROGATES ESTROGEN INDUCED ANTI-DNA TITERS IN A TRANSGENIC MOUSE MODEL. Elena Peeva, Xian Chen, Betty Diamond Bronx, NY

CD20, a B-cell specific surface molecule, is restricted to precursor and mature B cells but lost upon terminal differentiation into plasma cells. Though its precise function is unknown, it has been proposed to play an important role in B-cell activation, proliferation, and differentiation. CD20 on malignant human B cells has become an effective target for cancer immunotherapy and, more recently, early clinical trials in autoimmune disease. During a clinical trial of the chimeric mouse/human monoclonal anti-CD20 antibody rituximab in the treatment of systemic lupus, we examined the expression of CD20 on human peripheral blood lymphocytes. We postulated that CD20 may be upregulated in SLE as B cell activation is associated with increases in CD20 expression, and SLE is known to be associated with B cell hyperactivity. Flow cytometric analysis of peripheral blood B cells from patients with systemic lupus revealed a significantly higher density of CD20 (10-200%) compared to normal controls. This hyper-expression was not clearly related to disease activity or steroid treatment. Ex vivo stimulation of peripheral lymphocytes with CD40L caused a rapid and specific down-regulation of CD20 cell surface expression in both normals and SLE. Additional experiments to delineate the mechanism of this effect suggest that CD20 may be internalized following B cell activation through CD40 and support either a functional link between these two molecules or their signaling pathways. Surprisingly, activation through CD40 dramatically up-regulated CD40L expression on B cells, notably at a lower stimulation threshold for lupus B cells. This is the first demonstration that engagement of CD40 can induce CD40L on lupus B cells and may contribute to B cell hyperactivity through CD40-CD40L homotypic interactions. Overall, these results support dysregulation of co-stimulatory molecules and the cell surface molecule CD20 in systemic lupus.

Estrogen (E) has been demonstrated to exacerbate disease activity in murine models of lupus, while Tamoxifen (TAM), a selective estrogen modulator (SERM), to alleviate disease activity. E has also been shown to induce anti-DNA antibodies (Ab) and lupus-like illness in R4A␥2b BALB/c mice transgenic (tg) for the heavy chain of an anti-DNA antibody, by rescuing a naive autoreactive B cell population that is normally deleted. Objective: to identify whether the estrogen effect on anti-DNA B cells can be abrogated by TAM in R4A ␥2b tg mice. Methods: Anti-DNA Ab serum titers in E, E/TAM, TAM and P treated ovariectomized female mice were determined by ELISA. Kidneys were examined for IgG deposition by immunohistochemistry. Spontaneously secreting and inducible DNA-reactive B cells were enumerated by ELIspot assay. B cell subsets and the number of tg expressing B cells were determined by flow cytometry. Expression of the anti-apoptotic protein Bcl-2 and costimulatory molecule B7.2 was determined by flow cytometry. Results: E treated mice developed significantly higher serum titers of anti-DNA Ab (at week 4 of treatment p⫽0.024) than E/TAM and P treated mice. Kidney histochemistry showed IgG deposits in E treated mice only. There was an expansion of the tg bearing ␥2b B cells (p⫽0.002 and 0.001 respectively) and increased expression of Bcl-2 (p⫽0.04 and 0.02 respectively) in both E and E/TAM treated mice compared to P treated mice. However, the expression of B 7.2 was higher in E than in E/TAM treated mice compared to P (p⫽0.048 and 0.057 respectively). A significantly higher number of spontaneously secreting anti-DNA B cells was present in E than in E/TAM or P treated mice (p⬍0.01 for all 3 groups). There was no difference in the number of LPS induced secreting B cells, but there was significantly increased responsiveness to anti-CD40m Ab and IL-4 in both E and E/TAM treated mice compared to P treated mice. Conclusions: E does not cause expansion of ␥2b tg bearing population and increasing titers of pathogenic anti-DNA Ab when TAM is simultaneously given, which demonstrates therapeutic effects of TAM in murine lupus-like illness induced by E. The similar numbers of ␥2b tg bearing B cells, as well as anti-DNA B cells responsive to anti-CD40/IL-4 in E and E/TAM treated mice, suggests that TAM suppresses the stimulatory effects of E on anti-DNA B cell activation, but does not delete DNA-reactive B cells.

Disclosure:

Disclosure: This study was supported by NIH & SLE Foundation.

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MONITORING OF CD27 HIGH/CD19⫹ PERIPHERAL B CELLS REFLECTS THE DISEASE ACTIVITY IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS. Annett Marita Jacobi, Marcus Odendahl, Arne Hansen, Karin Reiter, Anne Bruns, Falk Hiepe, Andreas Radbruch, Gerd- Rudiger Burmester, Thomas Dorner Berlin, Germany

EVIDENCE OF A NOVEL IMMUNOMODULATORY MECHANISM OF ACTION OF PROSORBA THERAPY: RELEASE OF STAPHYLOCOCCAL PROTEIN A INDUCES VH REGION TARGETED APOPTOTIC DEATH OF B LYMPHOCYTES. Carl S Goodyear, Gregg J Silverman La Jolla, CA

Disturbances of peripheral B lymphocyte homeostasis with a reduction of CD27- naive B cells, a relative predominance of CD27⫹ memory B cells and an increase of CD27high/CD20- plasmablasts have previously been identified as being characteristic of patients with SLE. To assess whether changes of the frequency and absolute number of CD27high/CD20- plasmablasts are related to disease activity, the current study compared this particular B cell population with a variety of SLE disease activity parameters. In detail, the frequency of CD27high/CD20- plasmablasts determined by flow cytometric analysis was compared with the disease activity of 60 patients (53 female, 7 male) with SLE. The patients’ disease activity was assessed by SLEDAI. Whereas CD27high/CD20- B cells were found only infrequently in normal individuals (1.4 ⫾ 0.8%), there was a remarkable expansion of these cells in the peripheral blood of patients with SLE (11.4 ⫾ 11.1%) both with regard to their frequency and absolute numbers. Most notably, the frequency of peripheral plasmablasts was closely related to the SLEDAI (rP⫽0.498, p⫽0.0001, Pearson) and the titer of anti-dsDNA autoantibodies (rS⫽0.405, p⫽0.0015) at a similar degree as identified for the correlation of the SLEDAI and the titer of anti-dsDNA antibodies (rS⫽0.494, p⫽0.0001). Strikingly, the frequency of CD27high/CD20- B cells correlated with the SLEDAI also in patients lacking the detection of anti-DNA antibodies determined by Crithidia lucilliae immunfluorescence (49/60) (rS⫽0.436, p⫽0.0017, Spearman). In conclusion, the frequency of peripheral CD27high/CD20- plasmablasts is a reliable parameter of disease activity in patients with SLE regardless of their autoantibody profile. Disclosure:

For more than a decade, patients with autoimmune diseases have had the therapeutic option of treatment with the Prosorba extracorporeal pheresis column, and this therapy was recently approved for the treatment of rheumatoid arthritis. The column is composed of staphylococcal protein A (SpA) on an inert matrix, and while the mechanism of action has not been defined, it is unlikely to be due to the removal of pathogenic autoantibodies as the binding capacity of the column is insufficient to affect in vivo antibody levels. However, during each weekly treatment SpA is released into the patient, and we have recently reported that in a murine model even a limited in vivo exposure to SpA can induce a long term or even permanent loss of VH targeted mature B cells that are sources of autoantibodies. Moreover, if SpA-susceptible B cells are the source of an antigen-specific response, then deletion of these B cells by SpA can induce immunologic tolerance. To evaluate the mechanism(s) of SpA induced B-cell deletion, we have used mice transgenic for the clanVHIII Ig/T15i gene that conveys reactivity for SpA akin to the VH3 genes expressed by half of the human B-cell repertoire. We have found that within 16 hr of in vivo exposure, VH targeted B cells display dramatically downregulated surface Ig, and upregulation of the activation markers, CD86, CD69, and MHC class II with additional markers expressed at later times. Within 48-72 hrs, these targeted B cells undergo apoptotic cellular deletion, documented by increased Annexin V expression, DNA fragmentation, and increased cleaved Caspase 3. This VH targeted cellular fate progresses in vitro, but can be reversed by culture with CD40L or IL-4, while induction of apoptotic death by SpA is not impaired in T-cell deficient or in lpr mice, indicating that Fas is not required. These studies indicate that the oligovalent structure of SpA, which enables high avidity VH targeted BcR crosslinking, induces apoptotic cell death that results in immunomodulation of B-cell responses. Cumulatively, these studies define the probable mechanism of action of Prosorba therapy, and these findings may provide the basis for the development of more efficacious SpA-based therapies for the rheumatic diseases.


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SPECIFIC BLOCKING OF BACTERIAL CpG-DNA-INDUCED IMMUNE ACTIVATION BY INHIBITORY OLIGONUCLEOTIDES. Petar S Lenert, Laura L Stunz, Arthur M Krieg, Robert F Ashman Iowa City, IA and Wellesley, MA

POST-TRANSCRIPTIONAL REGULATION OF CD40 LIGAND (CD40L) EXPRESSION BY MITOGEN ACTIVATED PROTEIN KINASES(MAPK). Kyriakos A Kirou, Sojeong Kim, Mary K Crow New York City, NY

Various bacterial products and synthetic CpG-oligonucleotides (ODN) through a TLR-dependent step trigger innate immune system causing a secretion of proinflammatory cytokines. They also directly activate dendritic cells, macrophages, B cells and NK cells, and promote antigen-specific TH-1 biased immune responses. There is circumstantial evidence that bacterial DNA-induced local production of IL-12, IL-6, TNF-␣ could be associated with joint inflammation. Repeated systemic administration of CpG-ODNs may lead to systemic toxic shock syndrome. Obviously, procedures to prevent and/or treat these undesirable consequences of the immune activation are needed. We approached this problem by designing ODNs which potently inhibited early receptor binding/signal transduction events in CpG-DNA-treated cells. Almost one hundred 15-mer ODN with different backbone structure all based on a following motif: G, non-C, A or C, GGG were synthesized and tested for the inhibitory activity. Three most potent ODNs: 2088 (GGCGGG), 2114 (GGAGGG) and 2426 (GTCGGG) at the nanomolar level specifically blocked all downstream CpG-induced B cell effects (cell cycle entry, apoptosis protection, Bcl-XL upregulation, c-myc expression and p27kip1 degradation) and IL-6 secretion. They also inhibited IL-6 and IL-12 production by non-B cells. A single nucleotide change at the second base (from G or T to C), or a change from G to non-G at the fourth or fifth base of the motif resulted in ODNs which were between 10-100 fold less potent inhibitors. Inhibition was specific for ODN, since LPS, BCR or anti-CD40 antibody-triggered activation of B cells was not affected. While partial competition for cellular uptake could be observed at micromolar level, obviously this effect does not account for their biological potency in B cells at the nanomolar level. The inhibition occurred at the pre-transcriptional level, since inhibitory ODNs prevented CpG-induced nuclear translocation, DNA-binding activity and transcriptional activity of NF-␬B (p50, p65, c-Rel), AP-1 (c-Jun, Jun-B, Jun-D, c-Fos and Fos-B) and NF-IL-6. Delayed addition of inhibitory ODNs for up to 4 h following CpG stimulation completely prevented IL-6 secretion, and partially prevented cell-cycle entry. It was accompanied with decreased de novo nuclear translocation of NF-␬B and AP-1. Thus, undesirable consequences of the immune activation by CpG-DNA can be prevented/ reversed in a sequence-specific way by certain inhibitory ODNs.

CD40L, has been implicated in the pathogenesis of many diseases, including SLE, where its expression is higher, and more prolonged. Expression of CD40L depends on both transcriptional and postranscriptional mechanisms. Interestingly, CD28 stimulation stabilizes CD40L mRNA, and the binding of certain, yet uncharacterized, proteins to its 3’ UTR correlates with its stability. Phosphorylation of 3’ UTR-binding proteins might be critical for their function and might be mediated by the p38 and JNK MAPK, as these have been implicated in the regulation of other proteins’ mRNA stability. To determine the effect of MAPK on CD40L mRNA and protein expression, we used both freshly isolated primary T cells from healthy donors, and the D1.1 T cells which constitutively overexpress CD40L. The assessment for CD40L and GAPDH (reference gene) mRNA was done by semi-quantitative and quantitative (“real-time”, using SYBR green and the BioRad iCycler) RT-PCR. To assess mRNA stability we treated the stimulated cells with actinomycin D (10ug/ml) for 0.5-2 hours. CD40L protein was assessed by direct immunofluorescence and flow cytometry. Primary T cell stimulation was performed by 15-min incubation on ice with mouse anti-human CD3 (with or without anti-CD28), followed by interaction with goat anti-mouse IgG. T cells stimulated with the combination of antibodies had substantially more stable CD40L mRNA than that of T cells stimulated with anti-CD3 alone. Interestingly, treatment of the cells with the specific p38 inhibitor SB202190 (6mM) for 2 hours markedly decreased CD40L mRNA stability compared to stimulation with anti-CD3⫹anti-CD28 alone. At the protein level, treatment of T cells with SB202190 for 22h decreased their cell surface CD40L expression by ⬃30% (compared to DMSO treatment alone), without compromising their viability. On the other hand, the MEK inhibitor U0126 (10-25mM), although it also decreased CD40L expression, was toxic to the cells. In the case of D1.1 cells, treatment with either SB202190, or U0126 for 5-6h had a moderate effect (5-15% decrease) on CD40L expression, without apparent toxicity. In conclusion, p38 MAPK appears to be important in regulating CD40L mRNA stability and protein expression. Presumably, this occurs via phosphorylation of other intermediate proteins that may directly interact with the 3’ UTR of CD40L mRNA. Further characterization of this mechanism might provide a novel mode for controlling CD40L in disease, in which expression is dampened (or “normalized”) rather than ablated.

Disclosure:

Disclosure: Merck’s Rheumatology Young Investigators’ Program (Year 2000)

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THE p38 MAP KINASE IS CRITICAL FOR TH2 DIFFERENTIATION FROM MEMORY BUT NOT FROM NAIVE CD4 T CELLS. Francis Dodeller, Alla Skapenko, Joachim R Kalden, Hendrik Schulze-Koops Erlangen, Germany

LEFLUNOMIDE REDUCES TRANSENDOTHELIAL MIGRATION. Johannes Grisar, Daniela Eselbock, Marcus D Koller, Martin Aringer, Georg H Stummvoll, Peter Pietschmann, Josef S Smolen Vienna, Austria

It has been suggested that impaired Th2 cell differentiation contributes to the chronic Th1 driven immune response characteristic of rheumatoid arthritis. To delineate the molecular mechanisms regulating effector T cell generation, we investigated Th2 cell differentiation from naive CD4 T cells isolated from cord blood and from memory CD4 T cells isolated from the peripheral blood of healthy individuals. An in vitro system was employed that permitted the differentiation of effector cells after 5 days of priming. The cytokine secretion profile of the cells was assessed by cytometric analysis of cytoplasmic cytokines. Th2 differentiation could be induced from naive precursors by priming with anti-CD3 and anti-CD28. The addition of exogenous IL-4 was not required for Th2 cell differentiation and increased Th2 cell priming efficiency only minimally if at all. In marked contrast, in memory T cells, priming with anti-CD28 was sufficient to induce Th2 differentiation, whereas engagement of the T cell receptor inhibited Th2 generation. As the p38 MAP kinase has been recently implicated in regulating IL-4 production and subsequently, Th2 differentiation, we investigated the role of p38 in Th2 generation from human naive and memory T cells. Stimulation of memory T cells with anti-CD28 induced phospho-activation of p38 MAP kinase. Interestingly, priming of memory T cells with anti-CD28 in the presence of a specific inhibitor to p38 MAP kinase, SB203580, completely prevented Th2 differentiation. In naive T cells, phosphorylation of p38 MAP kinase was induced by anti-CD3/anti-CD28 stimulation. However, priming of naive T cells in the presence of SB203580 did not affect Th2 differentiation. Moreover, prolonged exposure of primed naive T cells to SB203590 did not alter Th2 frequencies. The data suggest that p38 MAP kinase is critical for Th2 differentiation from memory but not from naive T cells, and thus, that Th2 differentiation is regulated by different molecular mechanisms in naive and memory T cells. This might have implications for future immunomodulatory therapies aimed to alter the Th1/Th2 balance in autoimmune disease.

Leflunomide is widely used as a DMARD in the therapy of rheumatoid arthritis. Its active metabolite, A771726 leads to inhibition of dihydroorotate dehydrogenase (DHODH). We analyzed if A771726 also affected the transendothelial migration (TEM) of peripheral mononuclear cells (PBMC). We investigated the in vitro transmigration of PBMC through endothelial cell (EC) monolayers. Human umbilical vein ECs were cultured to confluence on collagen gels and then incubated with human PBMC of healthy blood donors. PBMC were recollected in three groups, namely 1) cells that did not adhere to the endothelium, 2) cells that bound to the EC, 3) cells that had migrated through the EC layer, and then counted by microscopic analysis. Experiments in which cells were pretreated with A771726 (in the absence or presence of uridine) were compared to parallel experiments in the presence of medium alone. No increased toxicity on PBMC treated with the doses of A771726 used in our experiments, was observed. A771726 incubation of PBMC only lead to a significant reduction in TEM by 33 ⫾19 % (p ⬍ 0.05). A771726 preincubation of EC alone also induced a pronounced reduction in PBMC migrated (36 ⫾36 %, p ⬍ 0.005). Incubation of PBMC and EC with A771726 potentiated the effects to a reduction by a mean of 47 ⫾27 % (7 ⫾5 % of treated vs 17 ⫾12 % untreated migrated cells, p ⬍ 0.004). Co-incubation with A771726 and with uridine reverted the migration inhibition (9 ⫾7 % cells without vs 15 ⫾10 % with uridine) suggesting that the inhibitory effects observed were due to inhibition of DHODH. Our data reveal that the anti-inflammatory effect of leflunomide may in part be related to the inhibition of PBMC transendothelial migration, possibly via downregulating adhesion molecules on EC and/or mononuclear cells. Since the decrease in TEM is reversible by adding uridine, the inhibition of the DHODH appears to be responsible for this mechanism. Thus, DHODH inhibition besides its other immunologic effects, also reduces the recruitment of mononuclear cells to inflammatory sites.

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THE CD23b PROMOTER IS A TARGET FOR NF-AT TRANSCRIPTION FACTORS. Christian Kneitz, Martin Goller, Corin Stibbe, Andris Avots, Edgar Serfling, Hans-Peter Tony Wuerzburg, Germany

EVIDENCE FOR A ROLE OF ␣(1,3)-FUCOSYLTRANSFERASE-VII IN T CELL RECRUITMENT TO INFLAMED JOINTS. Lars Rogge, Fabrizio De Benedetti, Mauro Biffi, Elisa Bono, Patrizia Pignatti, Margherita Massa, Alberto Martini, Costantino Pitzalis, Francesco Sinigaglia Milano and Pavia, Italy and London, United Kingdom

Objecitve: CD23 is expressed on numerous haematopoietic cells including B cells, T cells, monocytes and dendritic cells and mediates a variety of biological activities. It is involved in the regulation of inflammation, i.e blocking of CD23 can ameliorate experimental arthritis. High CD23 expression is a typical property of various chronic diseases, such as rheumatoid arthritis and lupus erythematodes. It is assumed that signalling through CD23 contributes to the pathogenesis of these diseases. CD23 expression is regulated by two different promoters, CD23a and CD23b. So far binding sites for STAT6, AP-1 and STAT1 and NF-kB have been described in one of these promoters. Since NF-AT-binding sites in many different cytokine promoters in T-cells have been described, we analysed the CD23 promoters for NF-AT binding sites. Results: Inspection of the promoter DNA sequences revealed three putative NF-AT binding sites, one within the CD23a promoter and two within the CD23b promoter. Whereas DNA/protein binding studies indicate the CD23a promoter site as a very weak (or no) NF-AT site, those from the CD23b promoter are bound by NF-AT factors with high affinity. In order to show whether these sites interact with NF-AT proteins we tested the binding of bacterially expressed chimeric GST-NF-ATp protein to the promoter in DNase I footprint protection assays. Several prominent footprints were detected. The most prominent one harboring the TGGAAAA core binding motif spans the nucleotides from position -160 to -141 and was designated as NF-AT-I. Moreover, we show that in transient transfection experiments using NF-ATp and STAT6 expression vectors the induction of the CD23b promoter is strongly enhanced after TPA and ionomycin treatment of cells. While NF-ATp or STAT6 expression alone resulted in a moderate 2-5 fold increase of CD23b promoter activity, coexpression of both factors led to a 15-30 fold increase. This effect could be reduced by addition of cyclosporin A, which inhibits activation of NF-AT proteins. Conclusion: These results show that (i) the CD23b promoter is a target for NF-ATp, and (ii) its induction is controlled by the concerted action of NF-ATp and STAT6 which both bind to the promoter with high affinity. We suggest that the cooperation between NF-AT and STAT factors might be an important molecular mechanism controlling gene activation. The impact of this finding for the pathogenesis of autoimmune disease is currently under investigation.

The mechanisms controlling the recruitment of Th1 cells to inflamed joints are not fully understood. Recent reports have documented the role of P- and E-selectin in the recruitment of Th1 cells. The functional activity of P- and E- selectin ligands is controlled by ␣(1-3) fucosyl transferases (FucT), in particular FucT-VII, that modify the carbohydrate moieties decorating PSGL-1. FucT-VII expression has recently been shown to be restricted to Th1. We have analyzed the role of FucT-VII in the recruitment of T cells to inflamed joints. By transfection of Jurkat cells with a vector coding for FucTVII, we show that expression of FucT-VII in T cells resulted in a 3-fold increase in the accumulation of these cells into RA synovial tissue grafted into SCID mice. Using kinetic RT-PCR, we demonstrate that the mRNA levels of FucT-VII are significantly (p⬍0.001) upregulated in T cells from synovial fluid (SF) (7844⫾6889 housekeeping normalized unit (HNU)) of patients with oligoarticular JIA (o-JIA) compared to paired peripheral blood (PB) T cells (2649⫾1568 HNU). The percentage of T cells binding P-selectin-Ig chimera, as well as the mean intensity of staining, was significantly (p⫽0.002) higher in SF (37.8⫾12.8 % T cells) than in paired PB samples (7.2⫾3.1), showing that higher expression of FucT-VII mRNA correlates with increased binding of SF T cells to P-selectin. Moreover, both FucT-VII expression and increased P-selectin binding capacity were found to be significantly increased in patients with extended o-JIA, showing their association with a more severe course of the disease. Our data indicate a critical role of FucT-VII in the enhanced homing of T cells to the inflamed synovium and suggest that inhibitors of FucT-VII enzyme activity may be of significant therapeutic value for the treatment of chronic arthritis. Disclosure:

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TRAFFICKING OF CD44-DEFICIENT LYMPHOCYTES IN NORMAL AND INFLAMMATORY CONDITIONS IN ARTHRITIS-SUSCEPTIBLE DBA/1 MICE. Reinout Stoop, Istvan Gal, Tibor T Glant, John D McNeish, Katalin Mikecz Chicago, IL and Groton, CT

INTRACELLULAR CYTOKINE PROFILES IN T-LYMPHOCYTES AND MONOCYTES OF RHEUMATOID ARTHRITIS PATIENTS, RECEIVING ANTI-TNF-␣ THERAPY. Annemie J Schuerwegh, Chris H Bridts, Wim J Stevens, Luc S De Clerck Belgium

CD44 is a transmembrane glycoprotein which can serve as a cell adhesion receptor for hyaluronan (HA). The functionally active (HA-binding) form of CD44 has been implicated in the recruitment of leukocytes at inflammatory sites in several models of autoimmune diseases including rheumatoid arthritis. Previous work from our laboratory showed a dramatic reduction of joint inflammation in mice with experimentally induced arthritis upon treatment with an anti-CD44 antibody. More recently, we have found significantly reduced incidence and severity of collageninduced arthritis in CD44 gene knockout DBA/1 mice. In the present study, we further substantiate the role of CD44 in lymphocyte trafficking under the condition of inflammatory arthritis. In naive (non-immunized) DBA/1 mice, CD44 deficiency resulted in a failure of leukocytes to adhere to immobilized HA, however, expression profiles of other major adhesion molecules did not differ significantly from those in wild type cells. Lymphocytes, harvested from naive CD44-knockout mice, homed to the lymphoid organs with normal kinetics following injection into naive recipient animals. Furthermore, immunization with type II collagen (to induce arthritis) elicited similar immune reactions in CD44-deficient and wild type DBA/1 mice. However, when lymphocytes from arthritic donors were transferred to arthritic recipients, the CD44-deficient and wild type cells showed different trafficking properties. As compared with wild type donor cells, lymphocytes from CD44-knockout donors preferentially homed to the lymph nodes, while their recruitment to the inflamed joints was delayed for at least 24 hours. The accelerated lymph node homing might be explained by a sustained expression of L-selectin on CD44-deficient leukocytes. L-selectin is thought to be the lymph node homing receptor for naive leukocytes, but is down-regulated on the cell surface (by shedding) upon activation. However, we found that L-selectin expression levels in lymphocytes from arthritic CD44-knockout donors were actually markedly lower than in those from arthritic wild type mice. We speculate that down-regulation of L-selectin expression in CD44-deficient cells could be a compensatory response in an effort to drive lymphocyte recruitment towards the inflammatory site instead of lymph nodes. We propose that CD44 directs leukocyte traffic to the site of chronic inflammation in part by acting as a negative regulator of lymph node homing.

Introduction It has been shown that T-lymphocytes, producing Th1 cyokines, and monocytes/ macrophages, producing pro-inflammatroy cyokines, play a pivotal role in the pathophysiology of rheumatoid arthritis (RA). In recent placebo controlled double-blind randomized studies, chimeric (human/mouse) TNF-␣ antibodies (cA2) proved to be very effective in improving parameters of clinical disease activity and inflammation. Objective To investigate whether anti-TNF-␣ therapy influenced the in vitro intracellular cytokine production in peripheral blood monocytes and T-lymphocytes of RA patients. Methods An intracellular flow cytometric technique was applied to measure IL-1␤, IL-6, TNF-␣, IL-10, IL-12 in monocytes and IL-2, IL-4 and IFN-␥ in T-lymphocytes of 15 patients, before, after 24 hours and after 6 months of therapy with monoclonal chimeric anti-TNF-␣ antibodies (3 mg/kg/bimonthly IV). All patients were on stable therapy with methotrexate (15-20 mg/week IM). The Friedman test and Wilcoxon rank test were applied to analyse differences of cytokine production in patients before and after therapy. Results After 6 months of therapy, a significant decrease of ESR and CRP was observed in all patients (p⫽0.01; p⫽0.02). Scores for Ritchie articular index and number of swollen joints were significantly decreased (p⫽0.001; p⫽0.03). Basal levels (at time zero), and levels (after 8 hours of culture without or with LPS) of IL-1␤, IL-6 and TNF-␣ were significantly decreased, 24 hours after the first administration of anti-TNF-␣ (for IL1␤ p⬍0.001; for IL6 p⬍0.04; for TNF p⬍0.001) and after 6 months of therapy (for IL1␤ p⬍0.003; for IL6 p⬍0.01; for TNF-␣ p⬍0.002). Basal IL-12 levels were significantly decreased after 24 hours and after 6 months, compared to base line levels (p⫽0.0001). In contrast, LPS stimulated levels of IL-10 increased significantly after 24 hours and after 6 months (p⫽0.02). The ratio of percentage IL-4/IL-2 CD4⫹ and IL4/IFN-␥ CD4⫹ T-lymphocytes was significantly increased after 24 hours and after 6 months of anti-TNF-␣ therapy (p⫽0.003; p⫽0.002). Conclusion Anti-TNF-␣ therapy downregulates the monocytic capacity to produce proinflammatory cytokines and induces a shift to a more pronounced anti-inflammatory Th2 cytokine balance.

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SELECTIVE INHIBITION OF IL-1␤ INDUCED PROINFLAMMATORY GENE EXPRESSION BY RECOMBINANT IL-1 RECEPTOR ANTAGONIST (IL-1RA; ANAKINRA) IN HUMAN WHOLE BLOOD CULTURES: CONTRAST TO OTHER ANTI-INFLAMMATORY AGENTS. Carl K Edwards, III, Robert F Baltera, Dennis M Miller, Michael J Gresser, Charles A Dinarello, Vivienne Marsh, Kathleen A Storm, Danute M Bankaitis-Davis, Michael P Bevilacqua Thousand Oaks, CA; Denver and Boulder, CO

HUMAN MHC CLASS II TRANSGENIC MICE RECOGNIZE PEPTIDE EPITOPES OF HUMAN CARTILAGE PROTEOGLYCAN (PG). Tamas Bardos, Anita Hanyecz, David S Chella, Tibor T Glant Chicago, IL and Rochester, MN

IL-1 is a key pro-inflammatory cytokine involved in multiple disease processes. A natural inhibitor, IL-1 receptor antagonist (IL-1Ra), blocks its effects. Studies in rheumatoid arthritis (RA) support the conclusion that recombinant IL-1ra (Anakinra) is clinically effective, slowing progression of bone damage measured radiographically. We compared the effects of IL-1ra and other anti-inflammatory agents on the expression of inflammatory genes (mRNA) in human whole blood. Blood from 4 healthy donors was cultured for 1 or 6 hr in the presence or absence of IL-1␤ (0.1, 1.0, or 10 ng/mL) or other stimuli (endotoxin and TNF) and clinically relevant concentrations (0.1-10mcg/ml) of IL-1ra. Selected pro-inflammatory genes, including IL-1␣, IL-1␤, TNF-␣, and COX-2, were measured using a quantitative PCR system optimized for precision (repeatability: %CV ⬍2). IL-1ra had little effect on gene expression in the absence of exogenous stimuli. Other therapeutic agents had reproducible effects on gene expression in unstimulated conditions. For example, methyl prednisolone inhibited expression of IL-1␣, IL-1␤, TNF-␣, and COX-2 by 50-80%. IL-1␤ stimulated blood demonstrated induction of gene expression ranging from ⬍10 to ⬎1,000 fold. In concentration studies, IL-1ra (0.1 to 100 mcg/ml) reduced gene expression in IL-1␤ stimulated blood. Inhibition of stimulated gene expression ranged up to 80% based on conditions and donors. IL-1ra had little effect on gene expression induced by other stimuli (e.g., endotoxin and TNF), whereas other anti-inflammatory agents, particularly steroids, substantially blocked the induced expression of pro-inflammatory genes by all of the stimuli. We conclude that IL-1ra blocks IL-1␤-induced gene expression in blood. Moreover, IL-1ra has little effect on gene expression in unstimulated blood or blood stimulated with endotoxin or TNF. Other anti-inflammatory agents reduced gene expression under all conditions tested. The selective pattern of IL-1ra activity supports its use as a long term therapy where maintainence of the patient’s immune response is critical.

Background. High-density cartilage proteoglycan (aggrecan), a major macromolecular component of cartilage, is highly immunogenic and induces progressive polyarthritis in genetically susceptible BALB/c and C3H/HeJCr mice. Cartilage PG can also be recognized by T cells in patients with rheumatoid arthritis (RA). Objective. 1) To study whether any of the predicted T-cell, epitopes of human cartilage PG induces immune response in mice expressing different MHC class II alleles, frequently associated with RA. 2) To investigate whether these HLA transgenic mice, as a response to PG-immunization, develop arthritis similarly to the susceptible mouse strains. Methods. Four HLA transgenic mouse lines lacking their own MHC class II molecules (HLA-DR2.Ab°, -DR3.Ab°, -DR4.Ab° and DQ8.Ab°) were immunized with human cartilage PG, and T-cell response (IL-2 production and proliferation) was determined in vitro in the presence of 15-mer synthetic peptides. A total of 143 T-cell epitopes (peptides) predicted along the entire PG molecule were tested. Some HLA-transgenic mice were further immunized and assessed daily for arthritis. Results and Discussion. We found different distributions of the T-cell epitopes among the different transgenic lines. We described all positive peptides, their locations and sequences, and we compared these HLA-presented T cell epitopes with those being presented by arthritis-susceptible murine strains [BALB/c mice (H-2d):27 T cell epitopes and C3H/HeJCr mice (H-2k):19 T cell epitopes]. Although 31 human MHC class II-restricted T-cell epitopes were identified by the four HLA transgenic lines, none of the transgenic mice developed arthritis in response to PG immunization. This observation indicates that the MHC alone (without a susceptible genetic background) is insufficient for the development of an autoimmune disease. To test this hypothesis, we recently backcrossed (i) HLA transgenic mice (ii) lacking the murine MHC class II (iii) into arthritis-susceptible BALB/c background.

Disclosure:

Disclosure: Supported by the NIH (AR-40310, AR-45652 and AI14764)

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INDUCTION OF ANTIGEN-SPECIFIC HUMAN CD4⫹ T CELL ANERGY BY PERIPHERAL BLOOD PLASMACYTOID DENDRITIC CELLS (PDCs). Masataka Kuwana, Junichi Kaburaki, Timothy M Wright, Yutaka Kawakami, Yasuo Ikeda Tokyo, Japan and Pittsburgh, PA

VARICELLA ZOSTER VIRUS SPECIFIC T CELL IMMUNE RESPONSES IN SLE AND THE EFFECT OF IMMUNOSUPPRESSION. Ki-Chan Kim, Hyung Bae Park, Tae-Hwan Kim, Jae-Bum Jun, Sang-Cheol Bae, Dae-Hyun Yoo, Seong Yoon Kim, Sungsoo Jung

Dendritic cells are antigen-presenting cells that are essential for initiation of T cell-dependent immunity and distinct dendritic cell subsets are known to direct different classes of immune responses. PDCs were recently identified as a Th2-skewing and IFN-␣-producing human dendritic cell subset. Since PDCs freshly isolated from peripheral blood express no or a minimal amount of CD80 and CD86, we hypothesize that failure of PDCs to deliver essential costimulatory signals during an interaction with T cells might result in a state of T cell anergy. To test this hypothesis, we prepared PDC-enriched fractions from peripheral blood mononuclear cells and evaluated their capacity to induce antigen-specific T cell anergy using human CD4⫹ T cell lines. Tetanus toxoid-specific CD4⫹ T cell lines incubated with tetanus toxoid-pulsed autologous PDCs failed to proliferate in secondary cultures with optimal antigenic stimulation. T cell anergy induction required T cell receptor engagement with antigen/major histocompatibility complex presented on PDCs. T cells rendered anergic lost IL-2 production but produced IFN-␥ and IL-10 upon stimulation. Upregulation of CD40 ligand was impaired in T cells after incubation with antigen-pulsed PDCs. The PDC-induced unresponsiveness was completely or partially reversible when a high concentration of exogenous IL-2 was added in the secondary cultures, but anti-CD28 antibody had less effect on reversal of an anergic state. Autoreactive CD4⫹ T cell clones specific for topoisomerase I derived from a patient with scleroderma were also rendered anergic after coculture with topoisomerase I-pulsed autologous PDCs, resulting in failure to proliferate or provide help to B cells. Incubation of topoisomerase I-specific Th0 clones with antigen-pulsed PDCs resulted in loss of IL-2 production and preferential expression of IL-10. Interestingly, this cytokine profile is consistent with that of type I regulatory T cells (Tr1 cells). These results suggest that PDCs are involved in maintenance of peripheral T cell tolerance and have potential for use in the suppression of pathogenic T cell responses in autoimmune diseases and organ transplantation.

Varicella Zoster virus (VZV) infections occur with increased frequency in patients with systemic lupus erythematosus (SLE) compared to the general population. In comparison with VZV infection, cytomegalovirus (CMV) infection in SLE is very rare. In spite of this, the precise mechanism of VZV infections in SLE is still unknown. To elucidate the mechanism of the abnormal immune responses to VZV infection in SLE and the effect of immunosuppression, we measured the virus specific T cell populations and the cytokines they secreted. The CD4⫹ and CD8⫹ T cells specific for VZV and CMV in SLE patients were compared with sexand age-matched healthy controls by intracellular cytokine staining. CD4⫹ T cells from normal individuals with a history of chicken pox or zoster infection predominantly produced IFN-␥ and TNF-␣ rather than IL-4 and IL-10. CD8⫹ T cells behaved similarly. In contrast, VZV specific CD4⫹ T cells and CD8⫹ T cells from patients with SLE showed inappropriate responses. In particular, the IFN-␥ production rate of SLE CD4⫹ T cells to VZV antigen was lower than that of normal CD4⫹ T cells (lupus 1.69 vs normal 5.16, p⬍0.005). The same result was produced with TNF-␣ (lupus 1.86 vs normal 4.97, p⬍0.01). However, CD4⫹ T cells and CD8⫹ T cells stimulated with CMV antigen showed more abundant production of IFN-␥ and TNF-␣ in both groups.

Disclosure:

CD4⫹ T cell cytokine production reponse rate

SLE (n⫽9)

IFN-␥ mean⫾SE TNF-␣ mean⫾SE

VZV 1.69⫾0.30 1.86⫾0.32

NORMAL (n⫽7) CMV 37.52⫾17.41 30.04⫾8.79

VZV 5.16⫾1.05 4.97⫾1.18

CMV 35.76⫾21.83 34.87⫾9.94

We found that there are differences in T cell immune responses between SLE and normal controls in terms of antigen specific T cell populations and their cytokine secretions to VZV and CMV antigens. CD4⫹ T cells which secrete less Th1 cytokines seem to play an important role in the susceptibility to VZV infection in SLE patients.Whether this is due to the effect of immunosuppression or if an intrinsic abnormality in lupus T cells which predisposes to VZV infection will be further investigated.


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IMMUNOMODULATORY PROPERTIES OF HLA-DRB*0401-BINDING PEPTIDES. Eric J Culbert, Corinne M Savill, Roger Ferguson, Jacqueline Caddy, Andrea Bamford, Gillian Slynn, Diane Neerunjun, Ron Cotton, Richard W Luke Macclesfield, Cheshire, United Kingdom

GAMMA GLUTAMYL TRANSPEPTIDASE ACTIVITY ALTERS THE T CELL RESPONSE TO OXIDATIVE STRESS AND FAS-INDUCED APOPTOSIS. Margaret L Carlisle, Miranda R Wilkins, David R Karp Dallas, TX

There is a strong genetic association of chronic, erosive rheumatoid arthritis (RA) with specific alleles of the HLA-DRB1 locus, notably DRB1*0401, *0404 and *0101 in Caucasian populations. These share a common sequence of amino acids in the antigen-binding groove (“shared epitope”). The only known function of HLA-DR molecules is to present antigen peptides to helper T cells, which has led to the hypothesis that disease chronicity in RA is associated with deregulated DRB1 allele self-peptide presentation. Transgenic expression of human DRB1*0401 or *0101 alleles renders mice susceptible to collagen-induced arthritis. Based on the conserved structural features, we designed peptide mimetics which were predicted to bind selectively with high affinity to HLA-DRB1 alleles carrying the shared epitope. These antagonists inhibited antigen binding to purified DRB1*0401 molecules (IC50⬍0.1 ␮M), and suppressed antigen presentation to DRB1*0401-restricted, antigen-specific T cell hybridomas derived from DRB1*0401-transgenic mice. We examined the ability of several peptide mimetics to inhibit RA antigen-specific delayed type hypersensitivity responses in DRB1*0401 transgenic mice. All peptide mimetics provided near to maximal inhibition of foot pad thickening when administered by sub-cutaneous infusion at ⬍1␮g/kg/day. These data support the possibility that selective antagonists of RA-associated HLA-DRB1 molecules may offer a novel therapeutic approach in the treatment of chronic RA.

The ectoenzyme gamma glutamyl transpeptidase (GGT) is differentially expressed on T cell populations. GGT is low/absent on naive cells, upregulated 10-20 fold upon stimulation, and intermediate on resting memory T cells. In other tissues, GGT is essential for the recapture of the antioxidant glutathione (GSH). Therefore, T cells with different levels of GGT activity were examined for their ability to withstand oxidative stress. To create a model system that reflected the level of GGT on naive and memory T cells, Jurkat T cells were cloned and their GGT expression analyzed. Thiol-specific monobromobimane staining was used to see if Jurkat clones with higher levels of GGT activity were better able to recapture released GSH. The mean level of intracellular thiol (GSH) in the Jurkat clone expressing GGT at a level comparable to memory T cells was approximately 30% greater than that of the GGT-low variant (similar to naive T cells). The level of cellular hydroperoxides was measured by quantifying the level of dichlorofluorescin (DCF) staining. Treatment of the cells with peroxide increases ROS in both cells, although the level seen in the GGT-high Jurkat is only 43% that in the GGT-low variant. Despite this protection from oxidative stress, the GGT-high Jurkat were found to be 2-3 fold more sensitive to Fas-induced apoptosis, as measured by staining with FITC-conjugated Annexin-V and propidium iodide. Gel shift assays, immunoblots and caspase activity assays indicated that the redox regulated Nf␬B pathway is activated in GGT-low cells, resulting in higher levels of cIAP-1/2 proteins that limit caspase activity. These findings suggest that upregulation of GGT may promote Fas-induced killing, hence, providing one way of eliminating memory/effector T cells at the end of an immune response.

Disclosure: All authors are employees of AstraZeneca Pharmaceuticals

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ANTIGEN-SPECIFIC SUPPRESSION OF HUMAN ALLOGENEIC T CELLS BY DENDRITIC CELLS. Sabine Hoves, Stefan Krause, Ulf Muller-Ladner, John D Mountz, Jurgen Scholmerich Regensburg, Germany and Birmingham, AL

THE T CELL RESPONSE TO TYPE II COLLAGEN IN HLA-DR1 TRANSGENIC MICE IS RESTRICTED TO DISTINCT CDR3 REGION THAT CORRELATE WITH FINE SPECIFICITY. Xiaowen He, Linda K Myers, Rosloniec F Edward, Wei Wang, Andrew H Kang, John M Stuart Memphis, TN

Objective: Activation of autoreactive T cells by Dendritic Cells (DC) has been implicated in several autoimmune diseases including RA and SLE. DC can be also used to suppress T cells, which has been demonstrated by expression of FasL (CD95L) in DC, or treatment of DC by IL-10 in animal models. However, the suppressive capacity of human DC has not been determined. Methods: Human monocytes were differentiated in vitro into DC in the presence of IL-4 and GM-CSF. At day 5 of culture IL-10 or TNF were added, and a first mixed-lymphocyte-reaction (MLR) with allogeneic T cells was established 2 days afterwards in the presence of anti-Fas (CH-11) or control antibody. Proliferation of T cells was determined by 3H-Thymidine-incorporation, and apoptosis was measured by FACS-analysis. Results: The proliferative response of allogeneic T cells was significantly reduced in cultures with IL-10 treated DC compared to TNF-treated DC. However, the strongest suppression of allogeneic T cells could be observed in cocultures with TNF-treated DC in the presence of anti-Fas antibody which resulted in substantial apoptosis of the activated T cells. This deletion of activated T cells was leading to an alloantigen-specific tolerance as demonstrated by an unaffected proliferative response of “rescued” T cells in cocultures with allogeneic “third party” DC in a second MLR. Conclusion: The present results demonstrate that the interaction of human T cells and DC can be modulated to suppress T cells in an alloantigen-specific manner. Therefore, induction of antigen-specific tolerance by modified DC might be a promising strategy for the treatment of T cell-dependent autoimmune disease in humans.

We have characterized TCRs driven by immunity to type II collagen (CII) using HLA-DR1 transgenic mice. The HLA-DR1 transgene confers susceptibility to arthritis elicited with human CII. T cell responses in these mice are focused on a dominant determinant with a core of human CII(263-270). TCRs on 22 clonal T hybridomas were analyzed, all of which recognizing the dominant determinant. The T cell hybridomas used V␤14 (68%)and V␤8 (32%). V␣ usage was also restricted, but to a lesser extent. The CDR3 regions of the T cells frequently used a group of neutral and polar amino acids, including Gly, Ser and Thr. The hydrophobic amino acids were fewer and had a tendency to locate at certain positions of the CDR3 region. Structurally related amino acid motifs, some of which extended to the whole CDR3 region, were identified. To study the fine specificity of the TCRs, analog peptides were generated using alanine substitutions at each of the residues and other amino acid substitutions at selected residues on the dominant determinant. IL-2 production induced by the peptides presented by DR1 molecules was determined. The responsive pattern was different among the T cells with different CDR3 regions, but was highly conserved among the T cells with structurally related amino acid motifs. Except at residue hCII265, Ala substitutions at residues within the determinant core greatly reduced the responses in most, but not all of the hybridomas. Other substitutions at the same residues usually had different effects. Ala substitution outside the determinant core has less or no effects on the T cell responses. These data indicate that T cells that recognize the dominant determinant on CII presented by DR1 use highly restricted V segments and distinct CDR3 motifs that correlate with fine specificity.

Disclosure: Supported by the German Research Foundation (DFG), FL297/3-1

Disclosure: Supported by Office of Research & Development, Medical Research Service, Dept. of Veterans Affairs

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REMITTING OLIGOARTICULAR JIA: AN ACTIVE ROLE OF CD4CD25 REGULATORY T CELLS? Isme de Kleer, Leonie Taams, Sylvia Kamphuis, Wilco de Jager, Nico Wullfraat, Ger Rijkers, Salvatore Albani, Berent Prakken Utrecht, Netherlands and San Diego, CA

SELECTIVE CLONAL EXPANSION OF CII-REACTIVE T CELLS EXPRESSING CONSERVED TCR-CDR3 REGIONS IN PATIENTS WITH RHEUMATOID ARTHRITIS. Seung-Hoon Kim, Mi-La Cho, Jeehee Youn, Sung-Hwan Park, Sue-Yun Hwang Hwang, Ho-Youn Kim Seoul, Korea

Juvenile Idiopathic Arthritis is a disease characterized by chronic inflammation of one or more joints. Striking differences in clinical course are seen between the three main subtypes, oligoarticular-, polyarticular and systemic JIA. While oligoarticular JIA is a remitting disease with a relative benign clinical course, polyarticular and systemic JIA usually are non-remitting and crippling diseases. It is still largely unknown which pathogenetic factors determine the different outcome of the three subtypes of JIA. Recently it became clear that, also in human, functionally specialized regulatory T cells, expressing a CD4⫹CD25⫹CD45RO⫹ phenotype exist as part of the normal immune repertoire. Their immune suppressive function is dependent on signaling through the negative regulator of T cell activation, CTLA4. As the regulatory T cells found in animal models, also the human regulatory T cells seem to have an important role in controlling autoimmunity. We hypothesized if differences in CD4CD25 regulatory cells between oligoarticular and polyarticular JIA patients add to the differences in clinical course and determined number, phenotype and intracellular cytokine production of CD4CD25 regulatory T-cells in both groups of patients. During remission of disease the number of CD4CD25 cells (expressed as %CD4CD25 of CD4 cells) in peripheral blood of oligoarticular JIA patients is comparable to the number in polyarticular patients (resp: 12,9 ⫾ 2,2% vs. 10,2 ⫾ 0,5%). More than 98% of the CD4CD25 cells in blood is CTLA4⫹, CD69- and CD40L-, indicating that CD25 acts not merely an activation marker. During disease activation in both oligoarticular and poly-articular JIA patients the number of CD4CD25CD69CD40L- cells increases (to 17,5 ⫾ 0,6 and 15,5 ⫾ 4,6). However, CTLA4 expression becomes up regulated on CD4CD25- and CD4CD25⫹ cells in oligoarticular, but not in poly-articular patients. In synovial fluid of 4 OA-JIA patients the number of CD4CD25 cells is much higher than in peripheral blood, half of them being activated cells, also expressing CD69. Still, CTLA4 expression on all CD4CD25 cells in SF was more than an 8-fold higher compared to CD4CD25 cells in peripheral blood. We have indications that in oligoarticular JIA patients these cells are capable of producing IL10, even without in vitro stimulation. Conclusion: In oligoarticular JIA patients CD4CD25 regulatory cells seem to play an important role in controlling disease by up regulation of CTLA4 and local production of IL10 during disease activation. Polyarticular JIA patients were lacking this up regulation of CTLA4.

PURPOSE: To analyze the structure of type II collagen (CII)-specific T-cell receptors (TCRs) involved in the pathogenesis of rheumatoid arthritis (RA). METHODS: CII-reactive T-cells were expanded by repeated in vitro stimulation with bovine CII from 7 patients and normal individuals. mRNAs extracted from these T-cells were analyzed by RT-PCR/SSCP method in which the antigen-binding CDR3 region, encompassing the V-n(D)n-J junction, was amplified with 22 V␤ primers and the common C␤ primer and examined by subsequent SSCP analysis. SUMMARY: The SSCP band pattern of normal T-cells appeared as smears for all 22 Vb subtypes. In contrast, distinct SSCP bands were observed in patients’ sample, indicating oligoclonal expansion of T-cells expressing a particular V␤ CDR3 region. Among the multiple discrete SSCP bands of patients’ T-cells, some TCR subtypes showed bands with identical migration in more than one individual, suggesting that these molecules are involved in the recognition of CII. We further analyzed the DNA sequence of one common SSCP band representing the V␤14 CDR3 region that was shared by 4 patients, and identified two distinct nucleotide sequences encoding either V␤ 14(CASS)-LWS- J␤ 2.1 or V␤ 14(CAS)-LWSPN- J␤ 2.3, respectively. Clonal expansion of T-cells expressing V␤ 14-LWS motifs disappeared in patient’s samples taken during a remission period, but reappeared when the symptom recurred. Selective expansion of T-cells expressing the LWScontaining CDR3 motif persisted after the stimulating antigen was switched from bCII to the immunodominant human peptide (256-270), up to 150 days of in vitro culture.CONCLUSIONS: Characterization of CDR3 motifs that are shared among 4 out of 7 independent RA patients tested marks the first identification of a common molecular structure of CII-reactive T-cells that may contribute to RA pathogenesis. Information obtained in this study can be applied to develop reagents for a targeted immune therapy that specifically blocks the T-cell response toward type II collagen in the RA synovium.

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A NOVEL SPLICE VARIANT OF NKG2D WITH A POSSIBLE REGULATORY FUNCTION. Greig P Lennon, Laszlo Pazmany, Robert J Moots Liverpool, Merseyside, United Kingdom

ESSENTIAL ROLE FOR RELB IN THE ONTOGENY OF MUCOSAL T CELLS AND CD1d RESTRICTED NATURAL KILLER T CELLS. Dirk Elewaut, Raziya Shaikh, Andrew Leishman, Hilde De Winter, Olga Naidenko, Olga Turovskaya, David Lo, Carl Ware, Hilde Cheroutre, Mitchell Kronenberg San Diego, CA Whereas the role of the Rel/NF-␬B family of transcription factors in mediating inflammatory responses has been well studied, its role on the development and homeostasis of lymphocytes is largely unknown. In this study we examined the role of RelB in the ontogeny of innate immune cells. Previous reports have shown that RelB is required for the development of the thymic medulla and myeloid-related CD8␣- dendritic cells. Positive selection of both ␣␤ and ␥␦ T cells proceeds normally in RelB deficient mice, whereas negative selection may be impaired as evidenced by the presence of autoreactive T cells. Here we report that RelB is essential for the development of all subpopulations of intra-epithelial lymphocytes (IEL) and mouse CD1 (mCD1) restricted Natural Killer (NK) T cells. By contrast, CD1 independent NK T cells are not affected in RelB-/- mice. Positive selection of NK T cells by mCD1 is distinct from that of conventional T lymphocytes as a hematopoietic cell type is responsible for selection of NK T cells, whereas cortical thymic epithelial cells are responsible for the selection of conventional T cells. Interestingly, transfer of RelB-/- bone marrow into lethally irradiated RAG-2-/- recipients fully restored both IEL and NK T cell development, which indicates that a RelB⫹ radio-resistant stromal cell is indispensable for the development of both T cell populations. In additional experiments however, we demonstrated that development of CD8␣⫹ IELs required the presence of ␤2-microglobulin on this radio-resistent stromal cell, whereas the development of NK T cells did not. These findings underscore the unique developmental requirements of these specialized lymphoid subsets. Disclosure:

Disclosure:

S299


Background. Lymphocytes, including natural killer (NK) cells and NKT cells, have been proposed to play an important role in rheumatoid arthritis (RA). NKG2D is a lectin-like receptor found on a variety of lymphocytes, including NK cells, CD8⫹ T cells and ␥␦ T cells. Ligands for NKG2D include the stress proteins MICA and MICB. Ligation of NKG2D on NK cells overrides the HLA-dependent inhibitory signals which usually prevent NK cell activation, allowing the killing of target cells regardless of whether they express inhibitory HLA Class I alleles. The transmembrane (TM) region is required for NKG2D co-expression with the adaptor protein DAP10 and signaling via DAP10. Experimental. We have identified a novel mRNA variant of NKG2D. Cloning and sequencing showed the variant lacks the 36 base pair exon 7, and encodes a protein of 80 aa, against the 216 aa of NKG2D. The variant encodes the intracytoplasmic domain and all but one aa of the TM domain. RNA extraction and cDNA synthesis was carried-out from the purified peripheral blood lymphocytes of a normal donor. The cDNA was used in an RT-PCR with a fluorescently-labelled primer. Products were analysed on an automated sequencer, with the relative amounts of the two mRNAs measured by peak area. This showed seven-fold more NKG2D mRNA than variant mRNA. Purified CD4⫹ T cell and NK cell populations from a healthy individual were examined by western blotting. This revealed small amounts of a protein that was the correct molecular weight to be the variant protein in the NK cell samples. However, this band was several log orders weaker in intensity than the band corresponding to NKG2D. Also, NKG2D was present in CD4⫹ T cells, although at weaker intensity than in the NK cell samples. Conclusions. Since the variant encodes almost the entire TM region, it is possible that the variant could interact with DAP10, and thus affect the function and/or expression of NKG2D. Potentially, this could be a mechanism for the control of NKG2D activation. However, initial experiments suggest this is unlikely in the peripheral blood NK cells of normal individuals, due to the low levels of the variant protein detected. Further investigations will determine the levels of variant mRNA expression in other cell types and under different conditions, and confirm whether the mRNA is expressed as a protein and if this can affect NKG2D function. This is of particular interest in RA, where large numbers of NK cells deficient for the CD158a/b inhibitory receptors are present.

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CD28 LACKING THE CYTOPLASMIC DOMAIN COSTIMULATES IL-2 PRODUCTION IN THE T CELL HYBRIDOMA DO11.10 UPON BINDING TO ITS NATURAL LIGANDS. Axel J Hueber, Bernhard Manger, Joachim R Kalden, Thomas Nagel Erlangen, Germany

INTERACTION OF HUMAN POLYCLONAL T CELLS WITH ARTIFICIAL ANTIGEN PRESENTING CELLS (aAPC) MIMICKS THE PHYSIOLOGIC EVENTS LEADING TO FORMATION OF IMMUNE SYNAPSE. A TOOL TO CHARACTERIZE AND MODULATE ANTIGEN SPECIFIC T CELL RESPONSES IN AUTOIMMUNITY. Rodrigo Samodal, Francesca Giannoni, Joellen Barnett, Kun Bi, Berent Prakken, Francesco Ria, Tho Lee, Amy Paolino, Aaron Altman, Salvatore Albani La Jolla, CA; Catania, Italy During T cells activation, initial contact between T cells receptors and MHC/peptide molecules leads to the migration and re-organization of antigen receptors, coreceptors, adhesion and signaling molecules to form membrane microdomains (immune synapse, IS). Increasing evidence identifies this event as an essential requirement for T cell mediated immune responses. We have previously described (Nature Medicine, 6, 12, 1407-1410. 2000) a method to identify class II restricted, low affinity antigen specific T cell. Here, we explored the potential, which aAPC have in modulating ex vivo T cell responses. aAPC employed in these experiments contained micromembrane domains, expressing high density of MHC/peptide and costimulatory molecules. We evaluated CD69 expression and307-319 intracellular production of IL-2 by CD4⫹ sorted cells stimulated with aAPC peptide /autologous HLA complexes, in presence or absence of anti-CD28 expressing HA antibodies incorporated in the aAPC membrane microdomains. We also evaluated CD69 and IL-2 production 307-319 by the same CD4⫹ sorted cells stimulated with aAPC containing anti CD3 antibodies in /HLA complexes. We compared the results with those obtained by stimulating the spite of HA cells with either PHA or anti-CD3/anti CD28 bound to the colture plate. The following table summarizes the results:

Complete T cell activation is dependent on the antigen-specific signal mediated by the T cell receptor (TCR) and costimulation by CD28. We previously demonstrated that in the T cell hybridoma A1.1, which lacks relevant amounts of endogenous CD28, CD28 can be reconstituted by stable transfection. Upon stimulation with PMA/Ionomycin and anti-CD28 antibodies, strong CD28-dependent costimulation can be induced by the wild type (WT) receptor, but not by a construct lacking the cytoplasmic tail (CD28TL). In an antigen- and ligand-specific model of T cell activation in the same hybridoma, in which both the TCR and CD28 are engaged by their natural ligands (MHC⫹peptide and B7-1/B7-2), surprisingly CD28TL induces a vigorous costimulation, but not the WT molecule. Here, we confirm this result in a similar model system using a different hybridoma, DO11.10. Like A1.1, it lacks endogenous CD28 or CTLA-4 expression, and CD28WT and CD28TL were stably expressed. For costimulation assays, antigen⫹MHC and B7 ligands were presented by the MHC-compatible B cell line TA3, and supernatant IL-2 was measured by ELISA. Stimulation of CD28WT did not alter IL-2 production compared to TCR-only stimulated cells or untransfected controls, unlike CD28TL, which reliably induced a strong IL-2 response. This was both dependent on the antigen concentration and the expression levels of CD28TL in different clones. This effect was specific for the interaction of CD28 with B7. It was completely blocked by anti-CD28 or anti-B7 antibodies, but not anti-CTLA-4 or control antibodies. Similar to A1.1, stimulation with mitogens could be enhanced several-fold by anti-CD28 antibodies. We thus are able to demonstrate that this finding is reproducible in different hybridomas with different antigen specificities. In addition, similar results were obtained when a tailless CTLA-4 construct was transfected into both hybridomas.Three separate conclusions can be drawn from these experiments: first, antibody- and ligand-mediated stimulation can lead to profoundly different results; generalizations should therefore be done only with due caution. Second, we propose that engagement of CD28TL by its ligands enhances TCR activation by promoting adhesion between T and B cells or, less likely, recruitment of activating signaling molecules through the extracellular or transmembrane domain. Third, in the full length receptor an inhibitory signal mediated by the cytoplasmic domain overrides this activation signal.

%CD4⫹ CD69⫹

%CD4⫹ IL2 producers

8.3⫹1.2 9.29⫹0.8 6.41⫹1 32.18⫹1.3

20.08⫹3.1 37.13⫹7.6 5.33⫹2.1 19.5⫹0.3

T cell responses were strictly dependent on the formation of the immune synapse and correlated with migration to the area of interaction of PKC-␪, a final product of the WASP-mediated pathway of cytoskeleton re-organization. We will discuss the application of this method to identification of rare class II restricted antigen specific T cells in autoimmunity.

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INDUCTION OF IL-10 AND IFN-␥ BY PAN-DR BINDING HSP60 EPITOPES IN JUVENILE IDIOPATHIC ARTHRITIS. Sylvia Kamphuis, Wilco de Jager, Isme de Kleer, Grace Gordon, Eva Koffeman, Margerita Massa, Alberto Martini, Ger Rijkers, Willem van Eden, Wietse Kuis, Berent Prakken, Salvatore Albani Utrecht, Netherlands; La Jolla, La Jolla; Italy

INDUCTION OF TH2 CELL DIFFERENTIATION BY COGNATE INTERACTION OF CD4 MEMORY T CELLS. Alla Skapenko, Peter E Lipsky, Joachim R Kalden, Hendrik Schulze-Koops Erlangen, Germany and Bethesda, MD

Objective: Previously we showed T cell reactivity to human and bacterial heat shock protein 60 (hsp60) in patients with Juvenile Idiopathic Arthritis (JIA). Interestingly, T cell reactivity to self-hsp60 correlated with a good prognosis, corresponding to the findings in the rat model of Adjuvant Arthritis (AA) where the induction of T cell reactivity to a self-hsp60 epitope induced protection. In order to determine potential target epitopes for immunotherapy, hsp60 epitopes recognized by a majority of JIA patients needed to be identified. The polymorphic HLA background in JIA has hampered this before. Methods: With the usage of a novel computer algorithm (Sette, Eppimmune, La Jolla, CA), predicting potential pan-DR binding sites on a given protein sequence, 8 different T cell epitopes on both human and mycobacterial hsp60 were identified and tested for T cell recognition in JIA patients by measuring proliferative activity and cytokine production. Results: All 8 peptides yielded clear T cell responses, each in a majority (60-100%) of JIA patients. Interestingly we found production of TNF-␣, IFN-␥ and IL-10; the production of both IL-10 and IFN-␥ strongly suggests the induction of regulatory T cells. One of the identified mycobacterial epitopes had only minor changes compared to the peptide that induced protection in AA, and there was a clear correlation between the T cell response to this mycobacterial peptide and its human analogue. This underlines that we have found a subset of T cells cross-reacting between self and non-self hsp60. Conclusion: We have identified multiple hsp60 epitopes recognized by a majority of JIA patients. The cytokine response induced by these peptides suggests the induction of regulatory T cells. We strongly believe these peptides will be good candidates for antigen-specific immunotherapy in JIA. Disclosure:


Cell Colture Conditions aCD3/aCD28 on plate aCD3/aCD28 on aAPC 307-319 /HLA on aAPC HA307-319 /HLA/aCD28 on aAPC HA

Th2 cell differentiation can be induced from human CD4pos memory T cells by priming with anti-CD28 without engagement of the TCR via activation of p38 MAP kinase and initiation of IL-4 gene transcription. To test whether CD28 mediated Th2 differentiation from memory T cells was an effect of a particular anti-CD28 monoclonal antibody (mAb), Th2 differentiation was assessed after priming of purified CD4pos memory T cells with transfected myeloma cells expressing human CD80 and/or CD86. Whereas the mock control did not induce Th2 cell differentiation, priming in the presence of myeloma cells expressing CD80, CD86 or CD80 and CD86 significantly increased Th2 cell frequencies. These data indicate that the initiation of CD28 mediated Th2 differentiation from memory T cells might be induced by engaging CD28 with its natural ligands, CD80 and/or CD86. As it has recently been suggested that T cells upregulate CD80 after activation, we evaluated the possibility that activated T cells might prime bystander T cells for Th2 differentiation. CD4pos memory T cells were activated with mAbs to CD3 and CD28 for five days. Flow cytometric analysis revealed that in contrast to resting T cells, activated T cells expressed CD80, however, at a low level. CD80 expressing T cells were fixed and cocultured with freshly isolated, syngeneic, CD4pos memory T cells for five days in the presence or absence of exogenous IL-4, but without additional stimulatory signals. For control, priming was performed with fixed syngeneic T cells that were freshly isolated and, thus, negative for CD80. Priming with fixed, non-activated syngeneic T cells did not induce Th2 differentiation, even in the presence of exogenous IL-4. In marked contrast, priming with fixed activated T cells induced significant Th2 differentiation from CD4pos memory T. This effect, however, was dependent on exogenous IL-4, suggesting, that the low levels of CD80 expressed on activated T cells were not sufficient to induce IL-4 transcription in CD4pos memory T cells but might have been sufficient to activate CD28 to induce signals that complement IL-4 mediated signals to induce Th2 differentiation. However, the data further indicate, that Th2 differentiation might be induced from bystander T cells by recently activated T cells through cognate T-T cell interaction involving CD28. TCR independent generation of Th2 effectors might provide a way to control Th1 dominated cellular inflammation. Disclosure:

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PATHOPHYSIOLOGICAL FUNCTION OF CD30-EXPRESSING CD4⫹ T CELLS IN PATIENTS WITH RHEUMATOID ARTHRITIS. Akira Okamoto, Masahiro Yamamura, Tetsushi Aita, Mitsuhiro Iwahashi, Akiko Ueno, Hirofumi Makino

DOMINANT AND SUBDOMINANT EPITOPES OF THE NUCLEAR ANTIGEN LA MEDIATE IMMUNE TOLERANCE IN THE T HELPER COMPARTMENT. Karen Davis, Catherine Keech, James McCluskey, A Darise Farris Oklahoma City, OK and Melbourne, Australia

Objective. CD30-expressing CD4⫹ T cells have been suggested to have a counter-regulatory role in the chronic inflammation of rheumatoid arthritis (RA). The pathophysiological function of CD30⫹/CD4⫹ T cells from RA patients was investigated. Methods. CD4⫹ T cells were isolated from peripheral blood and synovial tissue of RA with anti-CD4 magnetic beads. The surface expression of CD30 on CD4⫹ T cells at day 2, 4, and 6 after stimulation with anti-CD3 plus anti-CD28 was detected by flow cytometry. The cytokine profile of CD30⫹/CD4⫹ T cells was compared with that of CD30-/CD4⫹ T cells by intracellular cytokine staining and RT-PCR. The effects of anti-CD30 on activation-induced cell death and proliferation of anti-CD3 plus anti-CD28-stimulated CD4⫹ T cells were determined by staining with Annexin-V and 7-aminio actinomycin D and 3H-thymidine incorporation, respectively. Results. Both blood and tissue CD4⫹ T cells from RA patients expressed CD30 significantly at day 6 after anti-CD3 plus anti-CD28 stimulation, but CD30 expression on blood CD4⫹ T cells from healthy controls was transiently increased at day 4. Synovial tissues from RA contained mRNA for both CD30 and CD30 ligand, and immunohistochemical staining showed infiltration of CD30expressing lymphocytes in RA tissues. By RT-PCR and intracellular cytokine staining, CD30⫹/ CD4⫹ T cells expressed more IL-4, IL-10, and IL-13 but less IFN-␥ and IL-17, as compared with CD30-/CD4⫹ T cells. Anti-CD30 was found by Western blotting to induce the phosphorylation of TRAF2 in CD4⫹ T cells, like TNF-␣. The addition of anti-CD30 reduced the proliferative response of anti-CD3 plus anti-CD28-stimulated CD4⫹ T cells, but prevented their apoptotic cell death. Conclusion. These results indicate that CD30⫹/CD4⫹ T cells, which are capable of producing Th2 cytokines and resistant to activation-induced cell death, may infiltrate the RA synovium as a counter-regulator of Th1 cell-mediated immune inflammation.

Nuclear antigens are the primary targets of humoral autoimmunity in patients suffering from systemic autoimmune diseases; however, the nature of immune tolerance to nuclear antigens is not understood. Moreover, it is not known whether primary defects in tolerance play a role in the etiology of these disorders. To elucidate normal mechanisms of tolerance to the La nuclear antigen, we are examining immunity to xenoepitopes of the human La (hLa) protein in mice transgenic for normal nuclear expression of hLa compared to their non-transgenic littermates. With this model we previously demonstrated that tolerance to La resides primarily in the T cell compartment. Using adoptive transfer we now show that T cells recognizing the two immunodominant (hLa 61-84 and hLa 288-302) and one subdominant (hLa 196-219) H-2a -restricted determinants of hLa are functionally tolerant in hLa transgenic mice. Thus, adoptive transfer of T cells primed to hLa 61-84, hLa 288-302, or hLa 196-219 from non-transgenic (non-tolerant) donor mice into unimmunized mice bearing the hLa transgene results in the production of IgG anti-hLa autoantibodies. In contrast, similar transfer of T cells from transgenic (tolerant) donors primed to hLa 61-84, hLa 288-302, or hLa 196-219, does not result in the production of anti-La autoantibodies. Thus, under conditions of normal levels of in vivo antigen expression, immune tolerance to dominant and subdominant T cell epitopes is intact. These observations are compatible with hypotheses that patients with systemic autoimmune diseases either i) experience primary defects in T cell tolerance pathways or ii) experience peripheral perturbations of natural self antigen processing by APC to reveal cryptic (not constitutively presented) T cell epitopes. Disclosure:

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ANALYSIS OF ALTERNATIVELY SPLICED CD1D ISOFORMS AND ANTIGEN RESPONSE OF NKT CELLS IN PATIENTS WITH AUTOIMMUNE DISEASES. Satoshi Kojo, Akito Tsutsumi, Kazuko Shibuya, Masaru Taniguchi, Takayuki Sumida Tsukuba, Ibaraki and Chiba, Chiba, Japan

RECOGNITION OF NOVEL PAN DR BINDING T CELL EPITOPES OF HUMAN AND MYCOBACTERIAL HSP60 IN PATIENTS WITH RHEUMATOID ARTHRITIS. Huib de Jong, Francesca Giannoni, Margje H Haverkamp, Wilco de Jager, Leonie S Taams, Floris PJG Lafeber, Hans WJ Bijlsma, Ger T Rijkers, Wietse Kuis, Berent J Prakken, Salvatore Albani Utrecht, Netherlands and San Diego, CA

Objective: NKT cells comprise a unique T cell subset expressing invariant TCR and recognizing a glycolipid antigen (␣-GalCer) presented by the CD1d molecule. It is suggested that this subset regulates autoimmune responses. Recentry, we showed that the antigen response of NKT cells is reduced in patients with some autoimmune diseases, and that the low responsiveness of these cells may be due to the dysfunction of NKT cells (Arthritis Rheum. 44:1127-1138, 2001). Furthermore, the altered presentation of antigen on APC was also indicated. In this study, to clarify the mechanisms of low presentation of antigen, we examined the expression of CD1d and alternatively spliced CD1d isoforms in patients with autoimmune diseases. Method: (1) PBMC from nine ␣-GalCer responders (RA: 3, SLE: 4, SSc: 1, SS: 1), eight non-responders (RA: 4, SLE: 1, SSc: 1, SS: 2), and three healthy donors were stained with anti-human CD1d mAb, and the expression of molecule on monocytes were analysed by flowcytemetry. (2) cDNA was prepared from fresh PBMC, and DNAs encoding CD1d was analyzed by nested PCR, Southern blot analysis and sequencing. (3) Alternatively spliced CD1d variants was quantificated by TaqMan PCR using variant specific TaqMan probe and primers. Results: (1) There were no significant differences in the expression of the CD1d on peripheral blood monocytes between ␣-GalCer responder patients, non-responder patients and healthy donors. (2) New eight forms of alternatively spliced CD1d mRNA were detected. Two of these variants (V1 and V2) conserved the intact antigen binding site. One variant (V1) lacks the ␤2-microglobulin binding domain, however, contains the transmembrane and the cytoplasmic tail. Another form (V2) lacked both the transmembrane and the cytoplasmic tail, possibly a soluble form of the CD1d molecule. (3) V2 mRNA was reduced in patients with autoimmune diseases compared to healthy controls, whreas V1 mRNA showed no significant difference. Conclusion: Alternatively spliced variants of the CD1d molecule, especially soluble CD1d molecule (V2), may function as regulator of NKT cell response to antigen.

Heat shock proteins (HSP’s) are proposed as possible regulators in the immune response in chronic arthritis. Previously, we documented T cell recognition of hsp60 in patients with rheumatoid arthritis (RA) (J Clin Invest 1997;100:459). However until now we were unable to identify T cell epitopes of hsp60 in RA. In the Adjuvant Arthritis (AA) model, induction of T cells recognizing a self-cross-reactive hsp60 epitope induced protection (J Exp Med 1995;181:940). This could be translated to juvenile idiopathic arthritis (JIA) patients, where T cell reactivity to self-hsp60 was associated with disease remission (Arthritis Rheum 1996;39:1826). Recently we identified eight pan DR binding epitopes of both the human and mycobacterial hsp60, by using a computer algorithm (A Sette, La Jolla, CA) and we showed their recognition in JIA. Here we present the recognition of these eight novel hsp60 T cell epitopes in 17 RA patients. A stimulation index (SI) ⱖ 2 in a T cell proliferation assay was defined as T cell specific recognition. Four of the eight peptides tested, showed T cell recognition in the majority of the patients (2 of human and 2 of mycobacterial origin). In addition, the m256-270 peptide, which is the protective epitope in the AA model, was even recognized in 80% of the patients (mean SI 4,5). Remarkably, the T cell response to the m256-270 sequence is clearly correlated with the homologous human variant (r⫽0.81; p⬍0.001) strongly suggesting cross recognition between self and non-self. Our results show recognition of pan DR binding peptides from the human and mycobacterial hsp60 protein in RA patients. Recognition is also seen of peptides derived from the protective epitope in the AA model, with strong indications for the presence of cross-reactive T cells in RA patients. The results depicted here, suggest a possible role of the peptides as immune modulators in rheumatoid arthritis, and might be important candidates for immunotherapy. Disclosure:

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THYMIC ACTIVITY AND T-CELL DIFFERENTIATION IN RHEUMATOID ARTHRITIS. Frederique Ponchel, Ann Morgan, Mark Quinn, Sarah Bingham, Philip Conaghan, Paul Emery, Robert Verburgh, Jacob Van Laar, John Isaacs Leeds, United Kingdom and Leiden, Netherlands

CHRONIC EXPOSURE TO TNF ACTIVATES TNF-R SIGNALING PATHWAYS IN T LYMPHOCYTES WHICH MAY FAVOUR T CELL SURVIVAL. Panagiotis Vagenas, Joanna M Clark, Manvinder Panesar, Andrew P Cope Hammersmith, London, United Kingdom

Rheumatoid arthritis is a chronic inflammatory disease. Paradoxically, there is also evidence for suppressed immune function in RA. Abnormalities of the naı¨ve T-cell compartment have suggested a defect in generating new T-cells. Recent thymic emigrants (RTE) can now be identified as T-cell receptor excision circle (TREC)-positive cells and provide a surrogate measure of recent thymic activity. RA patients possess a lower number of TREC total CD4 and CD8 T-cells compared to controls irrespective of age and disease duration. It is still not clear however, if there is an intrinsic thymic defect in generating new T-cells or if an increased turn over of naive T-cells is responsible for this finding. We analysed T-cell differentiation in RA patients. Using a combination of 4 cell surface makers we observed a significant decrease in naı¨ve cells in newly diagnosed patients and resistant disease patients compared to age matched controls. We also observed the presence of an atypical subset of CD4 T-cells whose frequency correlated with CRP levels. We measured TREC-content in sorted naive CD4 and CD8 T-cells. We observed fewer TRECs in resistant compared to early disease and inflammation was inversely correlated with TREC-content. These data therefore suggest that the thymic defect observed in RA patients is secondary to inflammation rather than an intrinsic disease factor. We also examined T-cell dynamics in lymphopenic RA patients following autologous stem cell transplantation. Following treatment, total T-cell counts never regained base line levels. Thymic activity was evident after therapy in most patients. However total CD4 counts were still low 2 years after therapy. We did not observe memory CD4 cells expansion. Naive CD4 cells accumulated slowly but decreased at times of flare, associated with a decrease in TREC-content and an increase in CRP levels. Total CD8 counts regained base line level by one month via large expansion of memory cells. We also observed a correlation between inflammation, circulating levels of L-6 and the proliferation of naive CD4T-cells and their differentiation into the atypical subset observed previously. These results demonstrate the presence of thymic function in RA patients following lymphodepleting therapy. At present we cannot comment whether this is reduced compared to normal, but an inflammation-driven acceleration of T-cell differentiation may contribute to the poor reconstitution following ASCT for RA.

Tumour necrosis factor alpha (TNF) is a multipotent cytokine. Its role in the pathogenesis of rheumatoid arthritis (RA) is now well established. However, the signaling pathways activated by TNF in the context of chronic inflammation are less well understood. We set out to explore how TNF signals in T lymphocytes, and in particular to determine whether chronic exposure to TNF activates a cascade of signaling pathways qualitatively or quantitatively distinct from those activated following short term stimulation. Using a murine T cell hybridoma model we demonstrate that both p55 and p75 TNF receptors (TNF-R) are expressed on T cells at levels similar to RA synovial joint T cells. Over a period of 8-12 days, repeated stimulation of T cells with low doses of TNF (⬍2.5 ng/ml) leads to chronic, stable cytokine expression throughout the culture period. Even at this low dose, NF-␬ B is activated, while activation of other signaling pathways is less pronounced. In control T cells, high dose TNF (50 ng/ml) induces strong activation of NF-␬ B, Erk 1/2, Jnk, and p38 MAPK pathways, as well as caspase activation, within minutes of TNF-R ligation. In contrast, TNF induced activation of Jnk, p38 MAPK and NF-␬ B pathways is attenuated in T cells chronically exposed to low dose TNF when compared to control T cells, while activation of Erk 1/2 is spared. Moreover, cleavage of poly ADP-ribose polymerase (PARP, a substrate of activated caspases and a marker of apoptosis) is also markedly reduced in TNF treated T cells. Studies in progress using T cells from TNF-R deficient mice will determine which TNF-R is necessary and sufficient for desensitization of selected signaling pathways. Together, the data indicate that the profile of signaling pathways in T cells chronically exposed to inflammatory cytokines may be quite distinct from those pathways activated by cytokines over much shorter periods. Furthermore, in diseases such as RA, T cell effector responses may be driven by a programme of cytokine induced gene transcription which is dominated by chronic Erk 1/2 and NF-␬ B activation. According to this model, selective activation of these TNF-R signaling pathways by chronic TNF stimulation could promote T cell survival at sites of inflammation, and provide a mechanism through which the inflammatory process may be perpetuated.

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IMMUNOGENIC MHC CLASS II-BOUND SELF PEPTIDE FROM AUTOIMMUNE MRLlpr MICE. Chang-Hee Suh, Valsamma Abraham, John F Freed, Robert A Eisenberg, Philip L Cohen Philadelphia, PA and Denver, CO

CASPASES PROMOTE T CELL PROLIFERATION VIA THE REGULATION OF CELL CYCLE PROGRESSION. Jian Zhang, Tamas Bardos, Katalin Mikecz, Tibor T Glant, Jianye Xu, Reinout Stoop, Alison Finnegan Chicago, IL

The epitopes recognized by pathogenic T cells in systemic autoimmune disease remain poorly defined. We previously identified MHC class II-bound self peptides from autoimmune MRL/lpr mice which were absent in C3H mice. MRL/lpr and C3H mice were tested for the presence of T cells reactive to these peptides. Thymidine incorporation of T cells from draining lymph nodes (LN) and spleen (SC) of immunized mice was evaluated after 5 days of culture and it was considered positive if the stimulation index (SI) was more than 2. Eleven of 16 peptides produced T cell proliferation in both MRL/lpr (SI 2.03-5.01) and C3H mice (SI 2.03-3.75) and generally the SC response was stronger than the LN. IL-2 and interferon-␥ production was also detected, but not IL-4. Four peptides induced SC proliferation of naive MRL/lpr mice but not C3H mice. These peptides are derived from RNA splicing factor SRp20, histone H2A.2, ␤2-microglobulin and MHC class II I-A␬␤. Histone H2A.2 protein produced SC proliferation, yet ␤2-microglobulin did not. These proliferative reponses were detected as early as 1 month of age. After depletion of T cells of the SC, the proliferative response was abolished. The proliferative response to peptide was inhibited by pretreatment of anti-MHC class II antibodies. The results show that in both normal and diseased mice, tolerance to MHC-bound self peptides can easily be broken by immunization with peptide and adjuvant suggesting the availability of substantial numbers of peripheral self-reactive T cells. The presence of spontaneous T cells reactive to certain MHC-bound self peptides in MRL/lpr mice suggests that these T cells actively participate in their systemic autoimmunity.

Recent studies indicate that caspases, which are key mediators in apoptosis, are also involved in T cell proliferation. However, whether and how caspase cascades promote T cell proliferation induced by TCR ligation is unknown. Here, we reported that the caspase-9 inhibitor LEHD did potently inhibit T cell proliferation induced by anti-CD3 stimulation, suggesting that the triggering of cytochrome-c/caspase-9 signaling pathway is involved in T cell proliferation. Consistent with this observation, cytochrome-c was released from mitochondria, which subsequently induced caspase-9 activation during the T cell proliferation. Cell cycle analysis indicates that the presence of caspase inhibitors LEHD and zVAD blocked most T cells at the G0/G1 phase. Further biochemical analysis in LEHD/zVAD-treated T cells showed decreased expression of cyclin-D2, cyclin-E, Cdk-2 and Cdk-4 concomitantly with increased expression p27Kip1 and hypophosphorylation of Rb. These data suggest that inhibition of caspases results in a T cell blockade at the early G1 phase of the cell cycle. Taken together, our data suggest that activation of caspases, especially caspase-9 or caspase-9-like caspases, may be secondary to T cell activation, and that caspases are required for the cell cycle progression during TCR-induced T cell proliferation. Disclosure: Supported in part by grants from the NIH (AR40310, AR45652, and AR47412)

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FLIP TRANSGENIC MICE MANIFEST INCREASED T CELL PROLIFERATION AND A TH2 CYTOKINE PROFILE. Ralph C Budd, Wenfang Wu, Susanne Lens, Takao Kataoka, Jurg Tschopp Burlington, VT; Lausanne, Switzerland and Tokyo, Japan

CELL SURFACE GLUCOCORTICOID RECEPTORS: FIRST EVIDENCE FOR PRESENCE AND INDUCED UPREGULATION IN HUMAN PBMC. Burkhard Bartholome, Desiree Kunkel, Timea Berki, Gerd R Burmester, Andreas Radbruch, Alexander Scheffold, Frank Buttgereit Berlin, Germany and Pecs, Hungary

FLIP is the natural inhibitor of Fas and other death receptors, and overexpression of FLIP inhibits cell death from Fas ligation. An unexpected finding was that FLIP overexpression in transgenic murine T cells also augments proliferation to low-dose anti-CD3 stimulation. This was accompanied by a bias of cytokine production by the CD4⫹ subset toward increased IL-4 and decreased IFNg . These findings add to our previous observations that increased FLIP expression can divert Fas signaling from the caspase pathway toward NF-␬B and the MAP kinase, ERK, and that Fas can costimulate with TCR to promote T cell proliferation. Disclosure:

In the field of glucocorticoid research one of the most intriguing issues is currently the study of membrane bound glucocorticoid receptors (mGCR). These surface receptors are suggested to mediate rapid nongenomic effects. However, the existence of mGCR receptors has been shown so far only for amphibian neuronal membranes, rat and human lymphoma cells. Therefore, we aimed at answering the key question whether these receptors also exist in humans under physiological and/or pathophysiological, but non-malignant situations such as rheumatoid arthritis (RA). We have used high-sensitivity immunofluorescent staining (Nature Med,2000,6:10) to analyse the expression of mGCR on peripheral blood mononuclear cells (PBMC) obtained from healthy controls and from patients with RA. CCRF-CEM lymphoma cells (FASEB J,1993,7:1283) served as positive controls. This new method uses antibody-conjugated magnetofluorescent liposomes which can increase fluorescence signal intensity up to 1000-fold compared with conventional methods and allow the detection of 50-100 target molecules per cell. Using this technique we were able to demonstrate for the first time a significant expression of mGCR on human PBMC from healthy controls (n⫽11), which were undetectable by conventional technologies. Monocytes (4.7⫾1.2%), B lymphocytes (2.8⫾1.2%), but not T lymphocytes were positive for mGCR. Interestingly, vaccination of healthy controls significantly increased the percentage of mGCR positive monocytes up to 17-22%. Moreover, 16 patients with inactive and 12 patients with active RA were studied. All patients met the 1987 ACR criteria and disease activity was estimated according to clinical and laboratory criteria. The number of mGCR positive cells was significantly increased (up to 23%) in active patients, and statistical analysis of the data revealed a strong positive correlation between the number of mGCR positive monocytes and disease activity (C reactive protein, swollen/tender joints, VAS score for well-being, DAS). In conclusion, mGCR are physiologically present in healthy blood donors and upregulated during vaccination and in active RA. In RA patients the number of monocytes expressing mGCR is significantly correlated with various parameters of disease activity. Further experiments are underway to clarify the regulation of mGCR expression and to identify their (patho)physiological role. Disclosure: This study was supported by a grant from the Deutsche Forschungsgemeinschaft.

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T CELL FAS SIGNALING MEDIATES A COSTIMULATORY FUNCTION IN THE GENERATION OF AN IN VIVO CTL RESPONSE. Marc Roger Chevrier, Andrei Shustov, Phuong Nguyen, Charles Sanford Via Baltimore, MD

IL-10 AND TNF-␣ INCREASE Fc␥-MEDIATED PHAGOCYTOSIS IN HUMAN NEUTROPHILS VIA MODULATION OF Fc␥RIIA AND IIB. Kristina B Belostocki, Mee Soon Park, Jane E Salmon, Luminita Pricop New York, NY

Fas/Fas Ligand [FasL] mediated apoptosis plays an important role in lymphocyte homeostasis through activation induced cell death. Recent work in vivo suggests that T cell Fas/FasL signaling early in an immune response results in the delivery a costimulatory signal which is required for optimal T cell proliferation and maturation. To determine if Fas signaling in vivo has a costimulatory function, we investigated the ability of Fas defective T cells (B6.lpr) to induce acute GVHD in the parent-into-F1 model in both B6D2F1 and B6C3F1 hosts. Typically acute GVHD is characterized by profound elimination of host B cells by donor anti-host CTL which kills by both a perforin and Fas/FasL effector mechanisms. Importantly, FasL upregulation on donor CD8⫹ T CTL contributes to enhanced killing while Fas upregulation on host B cells promotes their elimination. To address the role of Fas signaling on antigen specific T cells, normal host mice were injected with either 50 x 106 wild type B6 (wt-⬎F1) or Fas defective B6.lpr donor cells (lpr-⬎F1). All donor mice were ⬍ 8 weeks old. Negative controls consisted of uninjected F1 mice. [At two weeks after cell transfer, mice were analyzed for splenic donor T cell engraftment and evidence of acute GVHD]. Characteristic features of acute GVHD were observed in wt-⬎F1 mice at two weeks after cell transfer e.g., significant engraftment of donor CD4⫹ and CD8⫹ T cells, host lymphocyte depletion, upregulation of FasL on donor T cells and Fas upregulation on host B cells. By contrast, lpr-⬎F1 mice exhibited reduced engraftment and expansion of donor CD4⫹ T cells and nearly absent donor CD8⫹ engraftment and expansion. Moreover, Fas upregulation on host B cells and FasL expression on donor T cells was reduced to near background levels. These results indicate that T cell Fas signaling early in an immune response is critical for optimal CD8⫹ T cell expansion, CTL maturation and subsequent Fas/FasL upregulation.

Introduction: In Rheumatoid Arthritis (RA), binding of immune complexes to the stimulatory Fc␥ receptor (Fc␥RIIA) results in activation of polymorphonuclear neutrophils (PMN). PMN activation can be attenuated by the inhibitory Fc␥R, Fc␥RIIB. Pro-inflammatory cytokines such as TNF-␣ are potent mediators of inflammation in RA. Cytokines with predominantly anti-inflammatory effects, such as IL-10, are also present in large amounts in RA synovium, but fail to limit joint inflammation and articular destruction. Given this paradox, we examined the effect of these cytokines on expression of stimulatory Fc␥RIIA and inhibitory Fc␥RIIB and their functional consequences with respect to Fc␥RII-mediated phagocytosis. Methods and Results: We extracted RNA from PMNs incubated with TNF-␣, IL-10, TNF-␣⫹ IL-10, and medium. cDNAs were obtained by reverse transcription and amplified with Fc␥RIIA, Fc␥RIIB, and actin primers. PMN cultured for 18 hrs with TNF-␣ expressed reduced levels of Fc␥RIIB transcripts compared to control as determined by densitometry (46.8⫾13.7%, n⫽5, p⫽0.029). This reduction remained significant even with the addition of IL-10 to TNF-␣ (44.4⫾10.0%, n⫽6, p⫽0.006). IL-10 alone had no significant effect on Fc␥RIIB, but increased Fc␥RIIA transcripts when compared to control (120.6⫾7.6%, n⫽4, p⫽0.030). To examine the functional consequences of Fc␥R isoform modulation, we measured phagocytosis of bovine red blood cells opsonized with Fc␥RII-specific probes (E-IV.3). IL-10 increased phagocytosis compared with PMN cultured in medium alone (117.1⫾4.2%, n⫽8, p⫽ 0.004). TNF-␣ also increased phagocytosis as compared with medium (207.8%⫾22.1%, n⫽9, p⬍0.001). Incubation of PMN with a combination of TNF-␣⫹IL-10 further increased phagocytosis compared with TNF-␣ alone (143.8⫾16.2%, n⫽9, p⫽0.012). Conclusions: The decrease in inhibitory Fc␥RIIB by TNF-␣ and increase in stimulatory Fc␥RIIA by IL-10 may augment PMN activation and amplify immune complex-triggered inflammation. IL-10 can increase Fc␥RII-mediated phagocytosis in the presence of TNF-␣ in an additive manner. Identifying factors that regulate opposing Fc␥R systems may lead to novel approaches to limit immune-complex triggered inflammation in RA.

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THE S128R POLYMORPHISM OF E-SELECTIN ALTERS THE SPECIFICITY OF ITS INTERACTIONS WITH LYMPHOCYTES. Ravi M Rao, Dorian O Haskard, R Clive Landis London, United Kingdom

IgG-MEDIATED ACTIVATION OF LEUKOCYTES IS INDEPENDENT OF Fc␥ RECEPTOR POLYMORPHISM. Agnieszka A Rarok, Hilde M Dijstelbloem, Minke G Huitema, Cees GM Kallenberg, Jan GJ van de Winkel, Pieter C Limburg Groningen and Utrecht, The Netherlands

Objectives: E-selectin is a cytokine-inducible endothelial cell adhesion molecule that contributes to leukocyte emigration from the blood into inflamed tissues. A serine to arginine (S128R) polymorphism has been reported to be present at increased frequency in patients with SLE and in atherosclerosis. The usual (wild type, WT) E-selectin binds a restricted population of lymphocytes, which are Th1 memory cells bearing the Cutaneous Lymphocyte Antigen (CLA). We tested the hypothesis that S128R E-selectin has different ligand binding characteristics and might contribute to a broader population of lymphocytes being recruited into inflamed tissues. Methods/Results: Lymphocytes were perfused over Chinese Hamster Ovary (CHO) cell monolayers expressing either WT or S128R E-selectin at equal density in a parallel plate flow chamber. More peripheral blood lymphocytes (PBL) interacted with S128R compared to WT (22.0 ⫾ 3.56 vs. 16.22 ⫾ 2.36 cells/filed, p ⬍ 0.05). Removal of CLA⫹ lymphocytes using anti-CLA-coated magnetic beads abolished rolling and arrest on WT E-selectin, but left a residual population that interacted with S128R (9.62 ⫾ 0.85 vs. 2.15 ⫾ 0.57 cells/field, p ⬍ 0.005). CLA⫹ and CLA- lymphocytes were also generated in culture using serum-free and serum-enriched medium, respectively. While there was no significant difference in the number of CLA⫹ cells interacting with either WT or S128R E-selectin, more CLA- lymphocytes interacted with S128R compared to WT (29.12 ⫾ 2.33 vs. 13.56 ⫾ 2.75 cells/field, p ⬍ 0.05). To assess whether this population of cells was of a Th1 or Th2 phenotype, lymphocytes were grown in medium containing IL-2 (Th0 ), supplemented with either IL-4 (Th2 ) or IL-12 (Th1). More Th0 and Th2 , but not Th1, cells interacted with S128R compared to WT (Th0: 26.9 ⫾ 1.07 vs. 14.00 ⫾ 0.64 cells/field, P ⬍ 0.01; Th2: 16.87 ⫾ 0.17 vs. 5.88 ⫾ 1.06 cells/field, p ⬍ 0.05). In addition, there was an increase in the number of CD45RO cells interacting with S128R compared to WT (21.6 ⫾ 2.51 vs. 14.57 ⫾ 2.33 cells/field, p ⬍ 0.01). No significant interactions of CD45RA cells were observed on either monolayer. Conclusion: These results demonstrate that the S128R polymorphism extends the range of lymphocytes interacting with E-selectin to include CLA- and Th2 memory cells. This property might result in the “wrong mix” of lymphocytes subsets interacting with endothelium and entering tissues in inflammation, and may contribute to the link between this polymorphism and vascular inflammatory diseases.

Introduction: Ligation of Fc␥ receptors for IgG (Fc␥ R) can trigger potent effector cell responses. Genetic polymorphisms of these receptors have been shown to modify IgG binding, and influence internalization of immune complexes. Indeed, in patients with infectious or autoimmune diseases, skewing towards low-binding Fc␥ R alleles has been demonstrated. The objective of this study was to investigate the influence of Fc␥ R polymorphism on leukocyte activation. Methods: We analyzed activation of neutrophils and monocytes stimulated by aggregated or solid phase-coated IgG1, IgG2, and total IgG. Neutrophil donors were selected based on their Fc␥ R genotype and homozygous for either Fc␥ RIIa-H131 / Fc␥ RIIIb-NA1 (HH-NA1/1) or Fc␥ RIIa-R131 / Fc␥ RIIIb-NA2 (RR-NA2/2). Monocyte donors were homozygous for either Fc␥ RIIa-H131 / Fc␥ RIIIa-V158 (HH-VV) or Fc␥ RIIa-R131 / Fc␥ RIIIa-F158 (RR-FF). Binding of immunoglobulins to lymphocytes was determined by flow cytometry. Activation of neutrophils was measured as the production of reactive oxygen intermediates (ferricytochrome c reduction), degranulation (lactoferrin release), and cytokine production (IL-8). TNF␣ secretion was used as a measure of monocyte activation. Results: As determined by flow cytometry, IgG1 aggregates firmly bound to neutrophils of both types of donors, albeit more avidly to donors expressing HH-NA1/1 alleles. In contrast, IgG2 aggregates firmly bound to HH-NA1/1 Fc␥ R neutrophils only. This binding could be blocked by pre-incubation of neutrophils with Fc␥ RIIa and Fc␥ RIIIb blocking antibodies. Despite the differences in binding of IgG subclasses to HH-NA1/1 and RR-NA2/2 neutrophils, we observed no differences in their activation as measured by oxygen radicals production, lactoferrin release and IL-8 production. Activation of both types of neutrophils with IgG1 or IgG2 aggregates could be at least partially blocked by the addition of Fc␥ R blocking antibodies. Similar to neutrophils, HH-VV and RR-FF monocytes were not distinguishable in their response to IgG, IgG1, and IgG2 as measured by TNF␣ release, although RR-FF monocytes do not bind IgG2 complexes. Conclusion: We conclude that although IgG-mediated activation of leukocytes is dependent on Fc␥ R, it does not appear to be influenced by Fc␥ R polymorphisms. These results are in favour of a new mechanism for IgG-mediated leukocyte activation, in which even a short interaction between IgG and Fc␥ R is sufficient to generate an appropriate inflammatory response. This may have important implications for inflammatory responses in infectious and autoimmune diseases. Disclosure:

Disclosure: This work has been funded by the Arthitis Research Campaign

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Fc␥ RECEPTOR EXPRESSION IN INFLAMMATORY NEUTROPHILS: ROLES OF CD32 AND CD16. Steven W Edwards, Gianluca Fossati, Roger C Bucknall, Robert J Moots Liverpool, United Kingdom

ROLE OF A NOVEL MYELOID ADAPTER PROTEIN IN DIFFERENTIATION AND SIGNAL TRANSDUCTION. Sally A Dmowski, Erik J Peterson, Christel Moog-Lutz, Peter G Lutz, Andrew Singer, Yvon E Cayre, Gary A Koretzky Philadelphia, PA and Paris, France

Blood neutrophils express cell surface Fc␥RII (CD32) and Fc␥RIIIb (CD16) which bind Ig-opsonised bacteria. In autoimmune disease such as RA, these receptors also contribute to host tissue damage via immune complex-(IC) mediated activation of neutrophils to release cytotoxic products. We determined the roles played by Fc␥RII and Fc␥RIIIb in activation of neutrophils by IC (of the type found in RA) and in bacterial killing. Insoluble IC activated reactive oxygen metabolite (ROM) generation in unprimed blood neutrophils. These IC were phagocytosed and the ROM were generated intracellularly. In contrast, soluble IC failed to activate unprimed blood neutrophils, but activated the release of ROM from cytokine-primed neutrophils. These IC also activated release of myeloperoxidase and lactoferrin from primed, but not unprimed neutrophils. Homotypic cross-linking Fc␥RII or Fc␥RIIIb activated ROM production and degranulation in primed (but not unprimed) neutrophils. Fc␥RIIIb ligation resulted in the production of significantly higher levels of ROM compared to ligation of Fc␥RII (p ⬍ 0.05, n⫽10) and most of the ROM were released extracellularly. Removal of surface Fc␥RIIIb by PI-PLC completely abrogated the ability of primed neutrophils to release ROM in response to soluble IC, but did not affect their ability to phagocytose and kill serum-opsonised S. aureus. In conclusion, homotypic ligation of Fc␥RII or Fc␥RIIIb activates cytokine-primed neutrophils to secrete ROM or granule enzymes. Fc␥RIIIb plays a major role in secretion of ROM and granule enzymes in response to soluble IC, but only a minor role in the phagocytosis and killing of serum-opsonised bacteria. Therapeutic blocking of Fc␥RIIIb function may limit tissue damage in disease but have minor impact on host defence. Disclosure:

During inflammatory responses, myeloid cells participate in the containment and elimination of the source of pathologic distress. Our laboratory has identified a novel, myeloid-restricted adapter molecule designated PML-RAR␣-Regulated Adapter Molecule-1 (PRAM-1). PRAM-1 was identified based on its sequence homology to SLP-76-Associated Protein of 130 kDa (SLAP-130 or Fyn-binding Protein, Fyb). SLAP-130, which is expressed in multiple lineages of hematopoietic cells, is tyrosine phosphorylated in response to T cell receptor stimulation in T cells and integrin stimulation in macrophages. PRAM-1, whose expression is restricted to neutrophils, is inducibly tyrosine phosyphorylated following pervanadate treatment demonstrating that it, also, is the substrate of tyrosine kinases. Additionally, SLAP-130 and PRAM-1 both constitutively interact with SKAP-hom and inducibly with SLP-76 following tyrosine phosphorylation. Our laboratory and others have exposed a role for SLAP-130 in TCR-induced inside-out signaling which upregulates T cell integrin receptor activity. Given the critical role of adhesion-based signaling in neutrophil activation, these data suggest a potential role for PRAM-1 in regulating neutrophil activity. Additional data suggests an alternative, but not mutually exclusive, role for PRAM-1. The expression of PRAM-1 is known to be transcriptionally repressed by the oncogenic fusion protein PML/RAR␣. PML-RAR␣ suppresses neutrophilic differentiation in acute promyelocytic leukemia. Pharmacologic levels of the differentiation agent all trans retinoic acid (ATRA) overcome the effects of the fusion protein which allows neutrophilic differentiation. Acute promyelocytic leukemia cells from patients or cell lines demonstrate PRAM-1 upregulation concordant with their ATRA-induced neutrophilic differentiation. Here we demonstrate that PRAM-1 expression is upregulated early during differentiation of murine embryonic stem cells under myeloid producing-conditions and immediately following G-CSF-induced differentiation of the murine myeloid cell line 32D Cl3. Additionally, preliminary findings indicate that overexpression of PRAM-1 in an embryonic stem cell differentiation assay may alter macrophage development. These observations strengthen the hypothesis that PRAM-1 modulates myeloid differentiation. Experiments are underway to define the roles of PRAM-1 in myeloid development and function. Disclosure:

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EXPRESSION OF MHC CLASS II MOLECULES BY SYNOVIAL FLUID NEUTROPHILS. Andrew Cross, Roger C Bucknall, Robert J Moots, Steven W Edwards Liverpool, United Kingdom

SKEWED BALANCE OF INHIBITING vs ACTIVATING Fc␥RECEPTORS BY MACROPHAGES OF CIA-SENSITIVE MICE AFTER IMMUNE COMPLEX STIMULATION. Arjen B Blom, Peter L van Lent, Astrid E Holthuysen, Cor Jacobs, Wim B van den Berg Nijmegen, Netherlands

In rheumatoid arthritis (RA) the joint is heavily infiltrated with neutrophils that can inflict tissue damage via their ability to secrete reactive oxygen metabolites and granule enzymes. Here, we report that infiltrating synovial fluid neutrophils (SFN) can express surface MHC class II molecules, and therefore may play a hitherto unrecognised role in RA by presenting antigen to CD4⫹ cells. Peripheral blood neutrophils of healthy volunteers or patients with RA, did not express MHC Class II molecules on their cell surface and no protein or mRNA for these molecules could be detected by western or northern blotting. After overnight incubation with cytokines, there was a small but significant (p ⬍ 0.05, n ⫽ 30) increase in surface expression of MHC Class II. Freshly-isolated SFN did not express MHC Class II molecules on their cell surface (as detected by flow cytometry), but did express cellular protein and mRNA. Strikingly, when these SFN were cultured ex vivo in the absence of exogenous cytokines, surface expression was markedly increased to levels as high as those detected on synovial fluid macrophages. SFN are thus triggered to express MHC Class II molecules on their cell surface. Curiously, although freshly isolated SFN express protein (presumably intracellular) and mRNA for MHC Class II, only low surface levels are detected. Ex vivo culture is required to detect surface expression. Thus, once SFN express surface MHC Class II molecules, they move from the synovial compartment to another joint location. The capacity of SFN to express class II MHC molecules suggests that they have the ability to present antigen to T cells. SFN may also process and present novel or cryptic antigens by virtue of their unique proteolytic activity. Disclosure:

Background: In a previous study we found that knee joints of mice, susceptible for collagen type II arthritis (DBA/1, B10.RIII) display enhanced sensitivity to immune complexes (ICs) compared to other strains (C57BL/6, BALB/c). Higher and prolonged inflammation and more severe cartilage damage was observed. Murine synovial macrophages as well as IL-1 determine IC mediated arthritis and these cells communicate with ICs using three classes of Fc␥ receptors mediating intracellular signalling: Fc␥RI and III are activating and Fc␥RII is an inhibitory receptor. Purpose: To investigate whether macrophages of mice that are prone to develop collagen type II arthritis are hypersensitive to ICs by differential Fc␥R expression and regulation. Methods: Basal expression of Fc␥R on macrophages of all strains was determined by immunohistochemistry on naive whole knee joint sections and FACS-analysis using 2.4G2 antibody. Peritoneal macrophages were stimulated with ICs and cells were processed for RT-PCR. Using primers specific for Fc␥RI, II or III semiquantitative RT-PCR was performed. In addition, peritoneal macrophages of all strains and of mice lacking functional Fc␥RI and III (FcR ␥-chain-/-) were stimulated and tested for the production of IL-1. Results: Basal expression of Fc␥R was significantly higher on synovial and peritoneal macrophages of DBA/1 and B10.RIII mice (mean fluorescence resp 440⫾50 and 360⫾30) if compared to C57BL/6 and BALB/c (mean fluorescence resp 240⫾30 and 280⫾30). After stimulation with ICs, mRNA levels of Fc␥RI and III were significantly higher in DBA/1 and B10.RIII mice. At day 2, Fc␥RI expression was 50x higher and Fc␥RIII was 10x higher. At day 3, Fc␥RI and III expression were both 10x higher. In contrast, Fc␥RII mRNA levels were downregulated 50x for DBA/1 and 10x for B10.RIII if compared to C57BL/6 and BALB/c. When DBA/1 and B10RIII peritoneal macrophages were stimulated with ICs (100 ␮g/ml), a twofold higher and prolonged production of IL-1 was measured if compared to other strains. Macrophages of FcR ␥-chain-/- mice showed no detectable amounts of IL-1 after IC stimulation, indicating the role of Fc␥R in stimulation of cells using ICs. Conclusion: Collagen type II arthritis sensitive mice are hyperreactive to IC through higher basal expression of Fc␥R on macrophages of these strains and/or stronger upregulation of stimulatory as well as downregulation of inhibitory Fc␥Receptors, resulting in higher and prolonged expression of IL-1. Disclosure:

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ENHANCED CD16 EXPRESSION ASSOCIATED WITH HIGH TGF-␤1 AND GM-CSF LEVELS SUGGESTS POLYMORPHONUCLEAR CELLS ACTIVATION IN CHRONIC RHEUMATIC FEVER. Martha C Rivero, Viviane MM Torres, Vilma ST Viana, Cleide Belleza, Ernesto Dallaverde, Cristiane Y Fukuda, Maria Helena B Kiss, Renato C Monteiro Sao Paulo, SP, Brazil and Paris, France

ACETYLCHOLINE SUPPRESSES MONOCYTE ACTIVATION VIA ␣3␤2 AND ␣5␤2 NICOTINERGIC RECEPTORS. Peer Hauck, Ute Ungethuem, Alex Scheffold, Christoph GO Baerwald, Matthias Wahle, Andreas Krause Berlin and Leipzig, Germany Transmitters of the autonomic nervous system are able to influence immune responses directly. While the effect of catecholamines is relatively well characterized, only little is known about the impact of acetylcholine (Ach) on the function of immmune cells. The study therefore aimed at investigating the effect of Ach on monocyte activation and the expression of Ach receptors (AchR) on monocytes. Mononuclear cells from 24 healthy donors were stimulated with LPS (1␮g/ml) and co-incubated with Ach in final concentrations between 10-5and 10-13 mol/l. After 24 hours, IL-6 as a marker of monocyte activation was determined in culture supernatants. The results showed that in a subgroup of 5 subjects Ach had a dose-dependent strong inhibitory effect on monocytes activation with decreased IL-6 concentrations by 70% (p⬍0.001). In another subgroup (n⫽5) Ach only had moderate inhibitory effects (maximum IL-6 decrease 35%) while monocytes from the remaining 14 subjects did not respond to Ach at all. Interestingly, monocytes from the latter group became moderately Ach responsive after 3 days of in vitro cultivation. Ach effects in the responder groups could be blocked by hexamethonium but not by atropin, indicating that Ach effects were mediated via nicotinergic AchR (nAchR). Using flow cytometry and monoclonal antibodies against ␣3 and ␣5 nAchR subunits it could be demonstrated that monocytes indeed express nAchR. Moreover, it could be shown that these nAchR did not bind a-bungarotoxin (bgt) and therefore belong to a special subgroup of non-bgt binding nAchR. Correspondingly, mRNA for the ␣3, ␣5, and ␤2 (but not ␤4) subunits of nAchR could be detected in mononuclear cells by molecularbiological methods. Taken together, this study demonstrates for the first time that Ach suppresses monocyte activation via non-bgt binding nAchR of the ␣3␤2 and ␣5␤2 subtypes. So far, these nAchR have only been found on ganglionic postsynaptic membranes and keratinocytes. Further studies on the regulation of AchR expression on immune cells and the possible role of Ach in regulating immune responses under physiologic and pathologic conditions are currently under way.

Disclosure: FAPESP grant #95/9348-0

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S303


Introduction: Rheumatic fever (RF) is an autoimmune sequelae triggered by a group A streptococcus infection. A persistent production of antibodies against bacterial and selfcomponents from heart, brain and joints has been implicated in tissue injury. Fc receptors (FcR) amplify the immune response against bacterial infection and their expression is modulated by different cytokines. In this aspect, TGF-␤ 1 and GM-CSF, both multifunctional cytokines, have been related to polymorphonuclear cells survival and functional status in inflammatory process. Objective: To determine whether cells of the innate immune system contribute to the chronic phase of inflammation in rheumatic fever. Methods: Heparinized blood samples from 33 patients in the chronic RF and 20 normal individuals with mean age of 15 ⫾ 2 and 14 ⫾ 3 yrs old, respectively, were analyzed. The cellular expression of three FcR for IgG: the high affinity Fc␥ RI (CD64), low affinity RII (CD32) and RIII (CD16), as well as, one FcR for IgA, the Fc␣ RI (CD89) on polymorphonuclear cells and monocytes were analyzed by FACS using mouse mAbs (PharMingen). Cytokines serum levels, TGF-␤1, GM-CSF, IL-6 and TNF-␣, were measured by ELISA (R&D Systems). Data from each group were compared statistically by Mann-Whitney non-parametric test. Results: There was no difference in the expression of Fc␥ and Fc␣ receptors on RF monocytes as compared to normals. In contrast, polymorphonuclear cells from RF patients presented a significant increased expression of CD16 as compared to control group (FI ⫽ 486 ⫾ 47 and 329 ⫾ 49, respectively, p⬍0.008). There was no correlation between IgG serum levels (1435 ⫾ 95 mg/dl) and ASLO with CD16 expression on RF polymorphonuclear cells. Cytokines profile analysis showed higher serum levels of GM-CSF and TGF-␤1 in RF patients as compared to controls (2.3 ⫾ 0.8 and 0.5 ⫾ 0.06 pg/ml for GM-CSF; 40.3 ⫾ 3.5 and 29.2 ⫾ 0.49 pg/ml for TGF-␤1, respectively). Additionally, TNF-␣, IL-6 and IFN-g serum levels, were consistently within normal range . Conclusions: Polymorphonuclear cells are activated in chronic RF as indicated by enhanced expression of CD16 receptor. Moreover, increased expression of TGF-b 1 and GM-CSF may explain continued activation of polymorphonuclear cells in the chronic stage of the disease.

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IMMUNE COMPLEXES (IC) DECREASE CHOLESTEROL 27-HYDROXYLASE AND ATP BINDING CASSETTE TRANSPORTER 1 (ABCA1) MESSAGE AND INCREASE ATHEROMATOUS FOAM CELL TRANSFORMATION IN MURINE MACROPHAGES FOLLOWING LIPID LOADING. Allison B Reiss, Nahel W Awadallah, Edwin SL Chan, M Carmen Montesinos, Charmela D Jagroop, David L Delano, Bruce N Cronstein

Sa ANTIGEN IS AN APOLIPOPROTEIN-A-1-BINDING PROTEIN. Milagrosa Escalona, Francisco J Lopez-Longo, Margarita Rodriguez-Mahou, Carlos M Gonzalez, Indalecio Monteagudo, Rosa del Castillo, Natividad Caro, Carmen Gonzalez-Montagut, Laura Cebrian, Luis Carreno Madrid, Spain

Background: Premature atherosclerosis is a significant cause of morbidity and mortality among patients with systemic lupus erythematosus (SLE). Immunological mediators, e.g. immune complexes (IC), are often found in the circulation of SLE patients and may influence critical metabolic events in the vessel wall, thus contributing to the development of atherosclerosis. We have found that IC which have fixed complement markedly diminish expression of the cholesterol-degrading enzyme cholesterol 27-hydroxylase and a protein that controls a cellular pathway for secretion of cholesterol and phospholipids for transport to the liver, ABCA1. Imbalances in the influx and efflux of cellular cholesterol, particularly within macrophages of the arterial wall, lead to the formation of lipid-laden foam cells, a critical step in the pathogenesis of atherosclerosis. We therefore determined whether cholesterol 27-hydroxylase expression and ABCA1 expression and foam cell transformation are modulated by IC in cultured mouse macrophages. Methods: Peritoneal macrophages were obtained from Balb/C mice by peritoneal lavage 4 days after intraperitoneal injection of 1 ml of 10% thioglycolate broth. Macrophages were incubated for 48h (37°C, 5% CO2) in medium containing 50% normal human serum (NHS) and 50␮g/ml acetylated LDL alone or in the presence of IC (0.48 mg/ml). Following incubation mRNA was isolated from the cells and reverse transcribed. The resulting cDNA was subjected to PCR using ABCA1- and GAPDH-specific primers. In other experiments foam cell transformation was quantified by counting cells containing oil red O-stained globules and expressing the results as the percentage of total cells present. Results: IC, in the presence of NHS, downregulated mRNA levels of ABCA1 and cholesterol 27-hydroxylase mRNA (28⫾3% and 33%⫾9% downregulation, respectively, p⬍0.002, n⫽3 and 6, respectively) in murine macrophages. After 48h in culture 1⫾1% of control cells formed foam cells but IC stimulated 94⫾6% of macrophages to undergo transformation to foam cells (p⬍0.001, n⫽3). Conclusion: These results demonstrate that immune mediators found in the circulation of patients with autoimmune disorders downregulate the enzymes involved in reverse cholesterol transport and promote the formation of lipid-laden foam cells, a critical step in the pathogenesis of atherosclerosis.

Anti-Sa antibodies are a novel serological marker highly specific for rheumatoid artritis. Objectives:To identified Sa antigen (ag). Methods: A saline extract from human placenta was used as antigenic source to purify the Sa ag by immunoaffinity chromatography.The purified antigen was electrophoresed and blotted onto PVDF membrane. Sequencing of the 68 kD-Sa, 50 kD-Sa and 29 kD band was performed in a Procise 494 sequencer. SwissProt and TrEMBL databases were searched for the resulting sequences to identify the proteins. Ligand blotting was performed to demonstrate the binding of apolipoprotein A1 to the Sa ag. Electrophoresis of the Sa ag was performed in reducing and non-reducing conditions, the proteins were transferred to a nitrocellulose membrane, blocked and incubated with apolipoprotein A1 for 8 hours at room temperature. After washing, was incubated overnight with an anti-apolipoprotein A1 monoclonal antibody, then with an anti IgG/peroxidase conjugate, and peroxidase staining was performed. Results: The purified Sa ag contained five bands, of 68, 50, 46, 29 and 10 kD. Only the 68, 50 and 46-kD bands were reactive by immunoblotting using anti-Sa sera. The 50 kD band could not be directly sequenced, probably due to amino terminal blockage.The sequence of the 68 kD band corresponds to DEPKXEVP, where X represent a residue which could not be determined. This sequence is not compiled in the databases searched, either as amino-terminal or internal fragment. Finally, the sequence of the 29 kD band was DEPPQSPWDXVKDLA, which was identified as the amino terminal sequence of human apolipoprotein A1. The 50 kD-Sa band binds apolipoprotein A1 by ligand blotting in non-reducing conditions. The electrophoretic pattern gave a high molecular weight band, around 120 kD. Anti-Sa sera reacted to this high molecular weight complex in immunoblotting. In reducing conditions, no binding could be detected and anti-Sa sera reacted to the 68 kD and 50/46 kD-Sa bands. The 68 kD-Sa band did not bind apolipoprotein A1. Conclusions: The Sa antigen contains two antigenic bands, of 68 kD and 50/46 kD. The amino terminal sequence of the 68 kD band is not compiled in SwissProt or TrEMBL databases, which made not possible its identification. It binds apolipoprotein A1 by ligand blotting in non-reducing conditions, and could be related to the placental apolipoprotein A1 binding protein described by Keso et al.

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1510

IMMUNE COMPLEXES (IC) IN THE PRESENCE OF NORMAL HUMAN SERUM (NHS) DOWNREGULATE ATP BINDING CASSETTE TRANSPORTER 1 (ABCA1) PROTEIN MRNA AND PROMOTE ATHEROMATOUS FOAM CELL TRANSFORMATION IN THP-1 HUMAN MONOCYTOID CELLS. Allison B Reiss, Nahel W Awadallah, Edwin SL Chan, M Carmen Montesinos, David L Delano, Bruce N Cronstein

DOES THE NUCLEAR AUTOANTIGEN LA/SS-B FUNCTION BY BINDING/CHELATING DIVALENT CATIONS? Michael P Bachmann, Biji T Kurien, Joanne D Gross, Judith A James, Robert H Scofield, John B Harley Oklahoma City, OK

Background: We previously reported that IC (in the presence of C1q and/or NHS) diminish expression of the anti-atherogenic enzyme cholesterol 27-hydroxylase in cells relevant to atherogenesis. The protein ABCA1 controls a pathway that secretes cholesterol and phospholipids to lipid-poor apolipoprotein and was recently identified as a critical regulator of macrophage cholesterol and phospholipid transport. ABCA1 is one of a group of sterol-sensitive proteins induced by lipid loading or specific oxysterols (e.g. 27-hydroxycholesterol). Diminished ABCA1 activity leads to premature atherosclerosis (Tangier Disease). Because circulating IC are commonly present in patients with SLE and RA, diseases associated with premature atherosclerosis, we studied the effect of IC, in the presence of complement, on expression of mRNA for ABCA1 in the human monocytoid cell line THP-1. Methods: THP-1 monocytoid cells and THP-1 differentiated macrophages (phorbol dibutyrate, 300 nM) in culture were incubated for 3h (37°C, 5% C02) with 50% NHS or 50% heat-inactivated serum (HIS, 56°C for 30 min) alone or in the presence of IC (0.48 ␮g/ml). Following the incubation, mRNA was isolated and reverse transcribed. The resulting cDNA was subjected to PCR using human ABCA1- and GAPDH-specific primers. THP-1 macrophages were incubated with 50% NHS and acetylated LDL (50␮g/ml) alone or in the presence of IC (48h). Formation of foam cells was quantitated by counting cells containing oil red O-stained globules and expressing the results as % of total cells present. Results: IC, in the presence of NHS, significantly downregulated ABCA1 mRNA expression in THP-1 monocytoid cells (31%⫾7% downregulation, p⬍0.002, n⫽6) but not in the presence of HIS (98%⫾3% of control, n⫽3). Similarly, IC, in the presence of NHS, downregulated ABCA1 mRNA in phorbol-differentiated THP-1 macrophages (33%⫾4% downregulation, p⬍0.001). Upon exposure to acetylated LDL, IC, in the presence of NHS, also increased (by 34⫾9%, p⬍0.02, n⫽3) foam cell transformation by phorbol-differentiated THP-1 macrophages. Conclusion: IC, in the presence of complement, decrease ABCA1 mRNA expression in THP-1 monocytes and THP-1 macrophages and promote foam cell formation. These findings are consistent with the hypothesis that circulating IC promote the development of premature atherosclerosis in patients with autoimmune disorders such as SLE by affecting expression of genes involved in cholesterol homeostasis.

The nuclear autoantigen La/SS-B is a major target of autoantibodies in sera of patients with systemic lupus erythematosus and Sjogren’s syndrome. Numerous functions have been assigned to the La protein, including a role in transcription/termination of RNA synthesized by polymerase III, and internal initiation of translation of viral and mitogen-stimulated mRNAs. Viral mRNAs usually contain a series of AUG codons upstream of an internal ribosomal entry site (IRES) and the initiation AUG codon. Divalent cations seem to stabilize the secondary structure of IRES elements. The La protein was proposed to bind to the initiation codon. It is, however, unclear how the La protein can differentiate between the AUGs upstream of the IRES and the initiation AUG codon. We describe a new feature of the nuclear autoantigen La/SS-B. This ability to bind divalent cations could explain the multiple functions of the La protein. Analyzing the expression of the human La gene we found that La mRNAs contain an IRES element. Recombinant human La protein allowed the translation of La mRNAs to switch from a cap-dependent to a cap-independent, internal initiation mechanism. Interestingly, the effects caused by the La protein could be partially mimicked by EDTA. Therefore, we addressed whether or not the La protein can bind to divalent cations. We identified a calcium binding site which is located between aa 278 and 374 of the La protein. Full length La protein only weakly binds calcium ions while (C-terminal) fragments of La protein strongly bind the ions. From our data we propose the following model. Virus infections or cell conditions requiring cap-independent translation allow proteolysis of La protein. Thereby, the C-terminal, calcium binding fragments are released. Their ability to bind Ca2⫹ and other divalent cations allows the binding of these La peptides to the (divalent cation containing) IRES elements and thereby helps the ribosome find the internal initiation AUG codon. Disclosure:


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1511

GAMMA GLUTAMYL TRANSPEPTIDASE INHIBITION ALTERS U-937 CELL LINE RESPONSE TO OXIDATIVE STRESS AND NF-␬B ACTIVATION. G Andres Quiceno, Margaret L Carlisle, David R Karp Dallas, TX

NOVEL COVALENT U1RNA-PROTEIN COMPLEXES ARE GENERATED IN CELLS BY ULTRAVIOLET B (UVB) IRRADIATION: IMPLICATIONS FOR AUTOIMMUNITY. Felipe Andrade, Livia CasciolaRosen, Antony Rosen Baltimore, MD

The ectoenzyme gamma glutamyl transpeptidase (GGT) is essential for the recapture of glutathione (GSH), the major intracellular non-protein thiol antioxidant. The function of GSH is to protect cells from oxidative stress. Reactive oxygen species (ROS) are generated at inflammatory sites and influence cellular events such as apoptosis and signal transduction. Monocytes both generate and respond to ROS. The purpose of this study was to determine the role of GGT in the response of monocytes to oxidative stress and its role in the regulation of NF-␬B activation. We evaluated the monocytic cell line U-937, which, like peripheral blood monocytes, expresses GGT. The inhibition of GGT with acivicin caused up to an 86% increase in intracellular ROS, as measured by dichlorofluorescin (DCF), in the absence or presence of exogenous stress. This finding suggests that a high expression of GGT helps U-937 cells maintain their redox state when subjected to oxidative stress or intracellular generated peroxides. In monocytes, TNF-␣ receptor activation results in the generation of second messenger molecules including ROS, and induces IL-8 through a transcriptional mechanism regulated by NF-␬B. I␬B-␣ proteolysis was evaluated by western blot in U-937 cells treated with acivicin, showing that GGT inhibition induces proteolysis of I␬B-␣. IL-8 production was evaluated by ELISA in cells treated with TNF-␣ and peroxide. The combination of acivicin and peroxide led to a 69% increase in IL-8 production in TNF-␣ stimulated cells over TNF-␣ plus peroxide alone. These data indicate that GGT activity serves to limit NF-␬B transcriptional activation in monocytes, by limiting the response to endogenous and exogenous oxidative stress.

Sunlight (particularly ultraviolet (UV) radiation) plays a pathogenic role in several autoimmune diseases (including lupus and dermatomyositis), and has been implicated in disease initiation as well as cutaneous and systemic disease flares. Modification of autoantigen structure induced by UVB irradiation is a mechanism potentially involved in this process. In these studies, we have used human autoantibodies to ribonucleoprotein autoantigens to search for novel UVB-induced autoantigen modifications. We have found that human sera which are monospecific for U1-70kDa by immunoblotting recognized multiple novel species (160, 150, 130, 110, 76 and 59kDa) when they were used to immunoblot lysates of keratinocytes or HeLa cells undergoing UVB-induced apoptosis, under conditions that minimize the activity of endogenous or exogenous ribonucleases. These species were not present in control cells, and were not observed when apoptosis was induced by Fas ligation or cytotoxic lymphocyte granule contents. Biochemical analysis using multiple assays revealed that these novel UVB-induced species correspond to RNA/protein complexes, which result from the covalent crosslinking between U1-RNA and U1-RNA-associated proteins. Further analysis showed that: 1) the 130 and 110kDa complexes are generated during apoptosis by cleavage of the 160 and 150kDa complexes; 2) identical products (130 and 110kDa) are generated in vitro by caspase 3-mediated cleavage of the 160 and 150kDa complexes, 3) U1-70kDa is contained in the 160 and 150kDa complexes, and is likely the target of caspase-3 cleavage; 4) U1-RNA cleavage does not occur during apoptosis; and 5) recognition of U1RNA-protein complexes by human sera appears to be mediated predominantly by antibodies recognizing U1RNA, as the majority of the immunoreactivity of the complex disappears when membranes are first incubated with RNAse A prior to immunoblotting. Interestingly, when apoptotic surface blebs were separated by centrifugation, the U1-RNA-protein complexes were highly enriched in these structures. Taken together, these data demonstrate that UVB irradiation can directly induce novel structural changes of autoantigens, which are recognized by human autoantibodies. As previously described for other autoantigens, these covalent complexes between RNA and proteins may have important consequences in terms of antigen capture and processing.

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ANTIBODIES TO APOPTOTICALLY MODIFIED U1-70kD BUT NOT INTACT U1-70kD CAN BE DETECTED IN A SUBSET OF HUMAN AUTOIMMUNE ANTISERA. Sriya Ranatunga, Mark F Foecking, Robert W Hoffman, Eric L Greidinger Columbia, MO

ANALYSIS OF THE STRUCTURE OF PROTEASOME-PCNA MULTIPROTEIN COMPLEX AND ITS AUTOIMMUNE RESPONSE IN LUPUS PATIENTS. Yoshinari Takasaki, Kazuhiko Kaneda, Ken Takeuchi, Ran Matsudaira, Keigo Ikeda, Hirofumi Yamada, Masakazu Matsushita, Hiroshi Hashimoto Tokyo, Japan

Apoptotically modified forms of autoantigens have been proposed to act as immunogens in the development of lupus. The U1-70kD (70k) molecule is a target of lupus autoimmunity that is modified in apoptosis, displays epitopes on its 40kD apoptotically cleaved form that are distinct from those on the intact protein, and is often one of the first targets of anti-RNP immune responses. To assess the pathologic significance of immunity to apoptotic 70k, this study tested for antibodies to intact and apoptotic 70k in a cohort of RNP-positive patients with clinical manifestations of lupus, in whom multiple serum samples were available. In a sample of 18 patients, 2.9 ⫾ 1.4 serum samples (mean ⫾ S.D.) collected over a span of 4.1 ⫾ 3.9 years were tested using immunoblot and ELISA techniques. No anti-70k responses were found in 4/18 subjects. Intact and apoptotic 70k antibodies were detected concurrently in 13/18 subjects. However, in one subject, assays of the initial available serum sample detected antibodies to the apoptotic but not the intact form of 70k. In subsequent sera from this patient, both intact and apoptotic 70k antibodies were observed. Assays also detected antibodies to the apoptotic but not the intact form of 70k in two of 21 additional RNP-positive patients in whom only a single serum sample was available. No sera tested had intact 70k antibodies in the absence of apoptotic 70k antibodies. To exclude the possibility that antibodies to the apoptotic form were detected in isolation because they persist longer than antibodies to the intact form, the presence of antibodies in subsequent sera after the detection of both intact and apoptotic 70k antibodies was assessed. Antibodies to the apoptotic form were lost in 6/24 subsequent sera (with concurrent loss of antibodies to intact 70k in each case), while loss of only antibodies to the intact form occurred in 0/24 cases-a significant difference at the 95% confidence interval. These results suggest that in subjects with lupus-like disease, immune responses to apoptotic 70k may precede responses to intact 70k but that immunity to intact 70k does not precede apoptotic 70k immunity. These results also identify a previously undescribed group of rheumatic disease patients with antibodies to the apoptotic form of 70k in whom assays for intact 70k antibodies can be negative.

We have recently found that PCNA multiprotein complexes (PCNA complex) associated with cell proliferation can be purified using monoclonal antibodies (mAbs) to PCNA (J. Immunol. 166, 2001). We attempted to analyze the structure of the complex and found that proteasome that has bee know as a ATP-dependent proteolytic enzyme involved in antigen presentation on class I major histocompatibility molecules, selective protein quality control, apoptosis, signal transduction, and cell cycle regulation is one of elements of the PCNA complex. And we also found that there was a linkage of autoimmune response between PCNA and proteasome. The PCNA complex was purified by affinity chromatography using mAbs to PCNA, TOB7. The PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with antibodies against p21, replication protein A, DNA helicase II, CDK4, CDK 5 and Topoisomerase I in immunoblotting (IB). These findings suggest that the PCNA complexes comprise the “protein machinery” for DNA replication and cell cycle regulation. At the same time, it was found that anti-Ki (a proteasome activator, PA28␥) sera from lupus patients reacted with the 32 kDa polypeptide in the PCNA complex. In addition to Ki, 20S proteasome ␣ and ␤ subunits, p52 subunit and ATPase subunit of another proteasome activator, PA700, were detected in IB. The immunoprecipitation study using mAbs to ␤ subunit and PA700 ATPase subunit, and the purified PCNA complex showed that “hybrid type proteasome” that consisted of PA700, 20S proteasome and PA28␥ is involved in cell cycle regulation or proteolysis of PCNA. Because it has been reported that there was a linkage of autoimmune response between PCNA and Ki, we tested the reactivity of 30 anti-Ki lupus sera to the proteasome, and found that 2 out of them could react with 20S proteasome a subunit and one could react with ␤ subunit, whereas 40 lupus sera negative for anti-Ki did not. These results suggested a novel cellular function of the hybrid type proteasome such as cell cycle and/or PCNA regulation, and the linkage of autoimmune response among them suggested that “antigen driven” system is playing an important role to induce the autoimmune response to the proteasome-PCNA complexes.

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IDENTIFICATION OF hnRNP I AND hnRNP K AS NOVEL COMPONENTS OF RO RIBONUCLEOPROTEINS. Guenter Steiner, Reinout Raijmakers, Silvia Hayer, Michael A Fouraux, Elisabeth Hoefler, Christof Zimmermann, Ger JM Pruijn, Gustav Fabini Vienna, Austria; Nijmegen, Netherlands

DO “ANTI-ENDOTHELIAL” ANTIBODIES BIND TO ENDOTHELIAL CELLS ? Ayhan Dinc, Sandor S Shapiro

Objective. The structure of Ro ribonucleoproteins (RNP) is still not fully resolved and their function has remained enigmatic. It was our aim to identify proteins that in addition to Ro60 and La are associated with Ro RNP complexes. Results. Reconstitution experiments performed with biotinylated hY RNAs and HeLa cellular extracts revealed the presence of several proteins in reconstituted Ro RNP complexes. By mass spectrometric analysis two of them were identified as the heterogeneous nuclear (hn) RNPs I and K. A monoclonal antibody (ab) to hnRNP I coprecipitated hY1 and hY3 RNA (though less efficiently than monoclonal anti-Ro60 or anti-La ab). Furthermore, both anti-Ro60 and anti-La ab coprecipitated hnRNP I from HeLa S100 extracts, while coprecipitation of hnRNP K was not observed. On the other hand, both proteins interacted to a similar degree with hY1 and hY3 RNA in a yeast 3-hybrid system. Immunoblot analyses performed with sera of patients with SLE, MCTD, scleroderma (SCL), poly/dermatomyositis (PM/DM), Sjoegren syndrome (SS), reactive arthritis (REA) and healthy controls revealed autoantibodies (aab) to hnRNP K and I in 44% and 35%, respectively, of SLE patients and less frequently in other diseases (Table). In SLE patients, anti-K and anti-I aab correlated with each other but not with anti-Ro or anti-La aab or with clinical features.

n hnRNP-K aab hnRNP-I aab

SLE

MCTD

SCL

PM/DM

SS

RA

HC

70 44% 35%

22 14% 5%

45 15% 18%

17 6% 18%

21 10% 15%

102 14% 8%

25 -

Conclusion. hnRNP I and K appear to be associated with a subpopulation of Ro RNPs and constitute autoimmune targets, particularly in SLE. Thus, these data may provide novel clues towards understanding structure, function and antigenicity of Ro RNPs.

Among the methods used to detect anti-endothelial cell antibodies (AECA), the enzyme-linked immunosorbent assay (ELISA) has been generally accepted as the preferred technique. In this study, we have attempted to localize the binding site of those antibodies detected with an ELISA technique. After coating 96-well microtiter plates with gelatin, half of each plate was seeded with human umbilical vein endothelial cells (HUVEC). The seeded and unseeded wells were further sub-divided into those exposed to the lifting buffer and those that were left intact. Thus, on each line (12 wells) four conditions were studied:gelatin-coated wells exposed or not exposed to lifting buffer, and HUVEC-covered wells exposed and not exposed to lifting buffer. All conditions were studied in triplicate. Wells were washed twice with washing buffer [phosphate buffered saline (PBS), 1% BSA] and either a study serum, a monoclonal antibody (mAb) or a purified IgG was added, followed by one hr incubation. For each condition, the blank consisted of diluent alone in other words, including every step except addition of study serum. The plates were then washed four times and incubated for 1hr with alkaline phosphatase-conjugated goat anti-human or anti-mouse IgG (diluted 1:2000). After two washes, a lifting buffer (including 10mM EDTA) was added to half the wells, while phosphate-buffered saline was added to the others. After two more washes with PBS alone, p-nitrophenyl phosphate was added and absorbance at 410 nm was read after 40 minutes. All stages of the ELISA were performed at room temperature. Results were calculated as absorbance of the serum sample minus that of the corresponding blank. Normal mouse IgG and mouse monoclonal antibodies against VLA2, ␤1 and ␣5 integrins did not bind to the gelatin-coated wells. Anti-integrin monoclonal antibodies, but not normal mouse IgG, bound significantly to HUVEC-coated wells; this binding was decreased significantly after exposure of these wells to lifting buffer. In contrast to the murine monoclonal antibodies and normal murine IgG, purified human IgG and five normal sera bound significantly to the gelatin-coated wells, but not to the HUVEC-coated wells. Of interest, a decrease in binding of these immunoglobulins was seen when the gelatin-coated wells were treated with lifting buffer. We considered that the main part of the AECA activity obtained in an ELISA technique may be related to the gelatin-bound antibodies instead of the HUVEC-bound ones. Disclosure:

Disclosure:

1514

1517

DETECTION OF ANTI-U1 RNP ANTIBODIES USING THE COMPLEX RECONSTITUTED BY U1 RNA AND RECOMBINANT PROTEINS OF U1 RNP. Yoshinari Takasaki, Akihiro Murakami, Kazuo Kojima, Kazuhiko Ohya, Koji I Mamura, Hiroshi Hashimoto Tokyo and Nagoya, Japan

IN VIVO SELECTIVE INHIBITION OF ERK 1/2 IN RABBIT EXPERIMENTAL OSTEOARTHRITIS IS ASSOCIATED WITH A REDUCTION IN THE PROGRESSION OF ARTICULAR TISSUE STRUCTURAL CHANGES AND METALLOPROTEASE INHIBITION. Jean-Pierre Pelletier, Florina Moldovan, Julio C Fernandes, Denis Schrier, Craig Flory, Johanne Martel-Pelletier Montreal, Quebec, Canada and Ann Arbor, MI

Disclosure:

In this study, we investigated using a rabbit experimental osteoarthritis (OA) model, the in vivo effect of a new and highly selective ERK 1/2 inhibitor, PD 198306, on the progression of articular tissues structural changes and on the level of the metalloprotease (MMP)-1 in cartilage. The OA model was created by sectioning with a stab wound the anterior cruciate ligament (ACL) of the right knee joint of the rabbit. Rabbits were separated into experimental groups: I) operated (OA) animals that received placebo; II) and III) operated animals that received oral treatment (gavage) with PD 198306 at therapeutic concentrations of 10 or 30 mg/kg/daily/po, respectively, starting immediately after surgery. The animals were killed 8 weeks after surgery. Macroscopical and histological studies were performed on the cartilage and synovial membrane. Tissue distribution of MMP-1 and phospho-ERK 1/2 were evaluated by immunohistochemistry using specific antibodies, followed by morphometric analysis. Osteoarthritic rabbits treated with the ERK 1/2 inhibitor showed a marked and dose dependent decrease in the size (p⬍0.001) and grade (p⬍0.02) of cartilage microscopic lesions. Histologically, the severity of the cartilage lesions and synovial inflammation were also reduced (p⬍0.004; p⬍0.02, respectively). In OA cartilage, the percentage of chondrocytes staining positive for phospho-ERK 1/2 and MMP-1 were high, and specimens from rabbits treated with PD 198306 demonstrated a reduction in the level of these two factors which was dose-dependent. This study demonstrates for the first time that in vivo, in an experimental OA model, the selective inhibition of ERK 1/2 and the subsequent reduced production of MMP-1 can result in a marked decrease in the progression of structural changes in OA. Disclosure: Supported by a grant from Pfizer Global Research and Development, Ann Harbour, MI, USA.

S305


Objective. Because some anti-U1 RNP positive sera in double immunodiffusion (DID) could not be detected by the enzyme-linked immunosorbent assay (ELISA) using recombinant 70 kDa, A and C proteins, we have attempted to develop a new type ELISA using in vitro transcribed U1 RNA in addition to recombinat proteins. We also demonstrate that some anti-U1 RNP sera specifically recognize the the conformation-structure that is altered by binding of 70 kDa and A polypeptide to the U1 RNA. Methods. Sera from 199 patients with mixed connective tissue disease, 119 with systemic lupus erythematosus, 20 with scleroderma, 25 with polymyositis/dermatomyositis and 30 with rheumatoid arthritis were used to test the reactivity in two ELISAs, one is using recombinant 70 kDa, A and C proteins as antigen source, and the other using in vitro transcribed U1 RNA in addition to those proteins. The specificity of those sera was also tested by DID and immunoprecipitation (IP). Results. When only the recombinant proteins were used as antigen source in ELISA (P-ELISA), 13 out of 246 sera that were positive for anti-U1 RNP in DID could not react, whereas all sera positive in DID reacted with the ELISA using the U1 RNA in addition to the proteins (R-ELISA). The reactivity of these 13 sera to the U1 RNA was tested by IP and ELISA, and only 3 out of them reacted with the U1 RNA. These results indicated that the increased reactivity in R-ELISA was not mainly due to the U1 RNA itself. Because we have found that the U1 RNA and, the 70 kDa and A proteins were directly binding to the U1 RNA by IP using antibodies to His-tag, we have tested the reactivity of 13 sera to the U1 RNA-70 kDa and the U1 RNA-A protein complexes by IP. Then, it was found that all 13 sera could reacted with the 70 kDa-U1 RNA complex and 2 of them were reactive with the U1 RNA -A protein complex. Conclusion. These results suggest that some anti-U1 RNP sera specifically recognize the conformation-structure that is altered by binding of U1 RNA to the proteins, and the ELISA using U1 RNA and recombinant proteins is useful to detect anti-U1 RNA the same as DID.

1518

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INCREASED EXPRESSION OF BONE MORPHOGENETIC PROTEIN-2 AND-4 IN OSTEOARTHRITIC LESIONS OF STR/1N MICE. Peter M van der Kraan, Alwin Scharstuhl, Henk M van Beuningen, Elly L Vitters, Wim B van den Berg Nijmegen, The Netherlands

RESTORATION OF LARGE FEMORAL TROCHLEAR SULCUS ARTICULAR CARTILAGE LESIONS USING A FLOWABLE POLYMER - AN EXPERIMENTAL STUDY IN SHEEP. Douglas W Jackson, Jeffrey C Felt, Yong Song, David C Van Sickle, Timothy M Simon Long Beach, CA; Minnetonka, MN and West Lafayette, IN


The aim of this study was the evaluate the expression of TGF beta 1, 2 and 3 and BMP2, 4 and 6 in spontaneous osteoarthritis of STR/1n mice. Knee joints were dissected from 28 STR/1n mice ranging in the age from 2 to 16 months. After dissection knee joints were decalcified in EDTA/PVP for 2 weeks. Following decalcification knee joints were extensively rinsed and subsequently cryosections were cut. The cryosections were shortly fixed with fomaldehyde and endogenous peroxidase activity was blocked by incubation of the sections with peroxide in ethanol. TGF beta1 was detected using a polyclonal chicken antibody (IgY) while polyclonal goat antibodies were used to detect TGF beta2 and 3 and the BMPs. In male STR/1n mice the development of osteoarthritis starts around the age of 50 days at the insertion site of the cruciate ligaments on the medial tibia. In this study only growth factor expression at the medial tibia is discussed. The incidence of osteoarthritis is much lower in female mice and starts at a later age. Therefore the medial tibial cartilage of two months old female mice was considered as control. TGF beta1 showed moderate expression in normal articular cartilage. Approximately 20% of the cells were positive. In chondrocytes bordering OA lesions little change in TGF beta1 expression was detected. Only a few cells showed increased expression compared to non-affected cartilage. TGF beta2 was very little expressed in both normal and OA cartilage. In contrast, TGF beta3 expression was clear in a majority of the cells in deep layers of non-calcified cartilage. A clear upregulation was detected in part of the cells at lesion sites. Expression of BMP2 and BMP4 was strongly co-regulated. Both factors were expressed in chondrocytes in control cartilage and showed a very pronounced upregulation in chondrocytes neighbouring OA lesions. Also histological normal cartilage in the vincity of the lesions showed very strong BMP2 and 4 expression. Chondrocytes further removed from the lesions showed background staining. BMP6 showed strong expression in hypertrophic chondrocytes. A portion of the chondrocytes at lesion sites showed increased BMP6 expression. Our data show that chondrocytes neighbouring OA lesions in STR/1n mice have an increased expression of anabolic growth factors, mainly BMP2 and 4, but also TGF beta3. The strongly increased expression of these factors around lesion sites is most likely related to an attempt of the chondrocytes to repair the damaged cartilage matrix.

The study was undertaken to evaluate a new technique for resurfacing the femoral trochlea for advanced patellofemoral osteoarthritis.In the sheep model the authors: 1. developed a standardized femoral trochlea articular cartilage lesion, 2. established the natural history of this articular cartilage lesion in the sheep model, 3. evaluated the biocompatibility and biodurability of an in situ curable polymer to resurface these large defects in the femoral trochlea under in vivo loading conditions. A total of 45 skeletally mature ewes were divided into 5 groups. Each group had 9 sheep (6 had polymer implant and 3 had control [empty] defects only). All the sheep had aseptic unilateral surgery performed to create a single large subchondral defect in the proximal trochlear sulcus (6 x 15 x 2.5mm in W, L, D). The flowable polymer was injected into the defect and after 35 seconds became firm. The patella was then reduced and the knee joint cycled resulting in anatomic restoration of the sulcus. The groups were evaluated at 3, 6, 12 and 24 mos after surgery. Gross analysis demonstrated the polymer remained in place and filled the defect with intact polymer at each evaluation period. There was minimal visible wear noted on the opposing articulating articular cartilage of the patella. Histologic analysis demonstrated the adjacent articular cartilage edges remained non-reactive. At 3 months there was a 2 component fibrous membrane that formed between the implant and the underlying trabecular bone. The most superficial component was a thin fibrous layer that contained no polymeric particles or inflammatory cells. The deeper layer contained polymeric particles encapsulated in thin giant cells. Macrophages with foamy cytoplasm were found in the connective tissue matrix adjacent to the polymeric particles. The macrophages did not appear to cause disturbance to adjacent tissue. Gait analysis of peak loading demonstrated a return to pre-operative loading levels after surgery with little observable difference between control and implanted animals at the 3 months test point. At 1 and 2 years the polymer surface retained a smooth surface. This work suggests using a polyurethane polymer as an articular cartilage substitute for local resurfacing and restoration of the gliding surface of the femoral trochlea may be a significant benefit. Although the long-term durability of this device has not been established, polymers may offer minimally invasive restoration of degenerative articular cartilage surfaces. By relieving the pain, it could postpone or delay the need for a total joint replacement in adult osteoarthritic patients with localized patellofemoral osteoarthritis.

Disclosure:

Disclosure: This study was funded in part by Advance BioSurfaces, Inc.

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IN VIVO INHIBITION OF TGF-␤ ACTIVITY INHIBITS OSTEOPHYTE FORMATION AND DECREASES CARTILAGE PROTEOGLYCAN CONTENT AND SYNTHESIS. Alwin Scharstuhl, Elly L Vitters, Henk M van Beuningen, Peter M van der Kraan, Wim B van den Berg Nijmegen, The Netherlands

DOES JOINT DISTRACTION RESULT IN ACTUAL REPAIR OF CARTILAGE IN EXPERIMENTALLY INDUCED OSTEOARTHRITIS? A CA Marijnissen, P M van Roermund, N Verzijl, J WJ Bijlsma, F PJG Lafeber Utrecht and Leiden, The Netherlands

The objective of this study was to determine the role of endogenous TGF-␤ on osteophyte formation and on cartilage proteoglycan (PG) content during an experimental model of osteoarthritis (OA). The soluble TGF-␤ type II receptor, lacking cytoplasmic and membrane domains, was used as a TGF-␤ antagonist. For the production of recombinant solRII, the methylotropic yeast Pichia pastoris was used. Intra-articular injection of papain was used as a model for experimental OA. The effect of systemic delivery of solRII on endogenous TGF-␤ was investigated in this model. Therefore, Alzet osmotic pumps containing either 10 mg of solRII or pumps containing solvent were implanted i.p. in C57Bl/6 mice. One day later, a 1.2% papain solution was injected in the right knee joint. PG synthesis was studied via incorporation of radioactive sulphate. Knees were dissected on day 7 or 14 and processed for routine histology. Injection of papain into murine knee joints resulted in the formation of osteophytes located on the tibia and femur. Papain injection also caused a decrease in PG content of non-calcified articular cartilage. Blocking of endogenous TGF-␤ via systemic administration of solRII resulted in a 75% reduction of osteophyte size (femur and tibia) compared to controls. Moreover, cartilage PG content was significantly decreased in solRII treated animals compared to control animals at day 7 and 14. PG synthesis was also decreased in the solRII treated animals compared to solvent treatment. This study shows for the first time that inhibition of endogenous TGF-␤ leads to a reduction in osteophyte formation during experimental OA. This finding implicates a direct role for TGF-␤ in the development of osteophytes, a major characteristic of OA. Furthermore, an important role for TGF-␤ in the maintenance of cartilage PG’s is indicated, since systemic solRII treatment resulted in a decrease in PG content and synthesis. In conclusion, endogenous TGF-␤ seems essential in osteophyte formation and protects against cartilage damage during experimental OA.

Joint distraction in the treatment of severe osteoarthritis (OA) results in prolonged (up to 7 yrs) clinical benefit, including an increase in joint space width (JSW). The latter suggests actual repair of cartilage. The absence of mechanical contact of the articular surfaces while intra-articular intermittent fluid pressure is maintained during walking is thought to be involved. Since actual cartilage repair is difficult to evaluate in humans, it was evaluated in a model of OA. In 14 Beagles, OA was induced in one knee by damaging the articular cartilage followed by intensified loading of the joint (groove model; A&R 2000;43:349). Ten weeks after induction, in 6 dogs articulating distraction of the knee was carried out for 8 weeks. Twenty weeks after the end of treatment, cartilage and synovium were analyzed biochemically and histologicatly. OA induction resulted in changes in cartilage proteoglycan (PG) turnover, collagen damage and mild synovial inflammation, which were all characteristic of OA. The treated dogs tolerated the method of joint distraction differently. Dogs with considerable joint use of the treated joint showed significant better outcome than animals with poor joint use. Dogs with significant joint use showed an increase in PG-synthesis from 31% (average control OA) to 40%, a decrease in loss of newly formed and the total amount of PGs (31% to 8% and 60% to 37%) and even an increase of PG content (from -15% to ⫹23%). In contrast, dogs with poor joint use showed deterioration of all parameters (PG-synthesis increase was ⫹88%, release of the newly formed PGs and total PGs was ⫹104% and ⫹167%, and PG-content was further decreased (-29%). Synovial inflammation and macroscopic cartilage damage (scored blinded by two observers) showed on average a better outcome after joint distraction (pⱕ0.02 and 0.01, respectively). Based on the increase in JSW in the clinical study and the observation from this animal study, joint distraction might indeed change cartilage beneficially. Joint use during distraction (possibly resulting in better nutrition of the cartilage as a result of intermittent fluid pressure) would beneficially influence outcome.

Disclosure:

Disclosure: The Dutch Arthritis Association supported this study

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ROLES OF AUTOIMMUNITY TO CARTILAGE INTERMEDIATE LAYER PROTEIN (CILP) IN RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS. Zhenyu Yao, Tomohiro Kato, Manae Kurokawa, Kayo Masuko-Hongo, Junichiro Tshuruha, Masakiro Sakata, Hiroshi Nakamura, Kusuki Nishioka Kawasaki, Kanagawa, Japan

PERFORMANCE OF A COLLAGEN/HYALURONATE IMPLANT FOR CARTILAGE REPAIR. Harold M Aberman, Timothy M Simon, Simon Smith, Peggy A Lalor, Robert C Spiro, Victor Shen, Douglas W Jackson Los Alamitos, Long Beach and Mountain View, CA; Bothell, WA;

Objective: To investigate whether the autoimmunity to cartilage intermediate layer protein (CILP) exist and is involved in the pathogenesis of arthropathy Materials and Methods: The cDNA fragments encoding the first and second half of the NTPPHase non-homologous region of CILP were subcloned and prepared as a ␤-gal or MBP fusion protein in E.coli (C1 and C2, respectively) respectively. Further, three different parts of the C2 region were prepared similarly (C2F1, C2F2 and C2F3 respectively). Autoantibodies against these proteins in serum samples from patients with OA or RA were investigated by ELISA and western blotting. To assess arthritogenicity of CILP, four strains of mice were immunized with mixture of C1 and C2 proteins. Further more, C57BL/6 mice were immunized separately with C1, C2, C2F1, C2F2 and C2F3 fragments. Cell proliferation experiment was performed in CILP immunized C57 BL/6 mice. Results: Autoantibodies against the C2 region were detected in 10% of the tested OA patients and in 7.9% of the RA patients. Within the C2 region, all fragments of C2F1, C2F2, and C2F3 were found to carry autoepitopes. All mice immunized with the CILP proteins developed long-lasting chronic arthritis, in particular, the ICR mice developed polyarthritis characterized by infiltration of mononuclear cells in the synovium and exfoliation of the surface of cartilage. The mice immunized with C2 fragment developed severe arthritis than those immunized with C1 or the mixture of C1 and C2, There was lymphocyte proliferation to human C2 in the human C1 immunization mice, but human C2 could not cause this effect. C2F1, C2F2, C2F3 caused lymphocyte proliferative response similarly as C2 immunized mice, but the immunization of C2F1, C2F2, and C2F3 caused only mild arthritis. Conclusion: The immune response to CILP may play a role in the pathogenensis of chronic arthrpathy such as OA and RA. Suppression of the immune responses to various cartilage-derived components, including CILP, may have a therapeutic benefit in these arthropathies. Disclosure:

It was the purpose of this study to test a unique implant device to determine if it is effective as an aid to healing of a clinically relevant full thickness cartilage defect. In this study, the use of a novel biological osteochondral implant made of collagen and hyaluronate was examined1. The implant matrix was intended to serve as a scaffold which promotes cellular ingrowth and differentiation. Efficacy to repair the bone and cartilage was assessed using histological and biomechanical examination of the repair tissue. A subchondral defect was created in the weight bearing region of the medial femoral condyle of skeletally mature goats. Repair tissue was assessed at 6 and 26 weeks. Non-destructive articular cartilage load and stiffness measurements were obtained of the repair site and the intact contralateral control. This was followed by decalcified histological processing.

6 week 26 week

Maximum Load (% of intact)

Stiffness (% of intact)

Energy (% of intact)

11.61 ⫾ 2.51 69.45 ⫾15.25*

10.24 ⫾ 0.69 77.61 ⫾ 16.65*

12.34 ⫾ 3.48 65.00 ⫾ 15.68*

* ⫽ pⱕ0.01, compared to same treatment group at the 6 week time period. Histological evaluation showed at 6 weeks that there was no marked cellular response, nor was there any regeneration of the chondral tissue. A small amount of GAG production associated with endochondral bone formation was observed. By 26 weeks, a marked amount of regeneration was seen with the region of the defect composed of bone and chondral tissue. A calcification tide mark was observed histologically in the regenerated tissues. Historically in this goat model2, empty defect controls show that after 26 weeks, this 6 mm x 6 mm cylindrical osteochondral defect created in the medial femoral condyle is a critical size defect that does not heal. The data from this study provides evidence to indicate that the treatment of a 6 mm x 6 mm osteochondral defect with collagen/hyaluronate matrix promotes regeneration of both the bone and chondral tissue. There is a significant improvement in the biomechanical properties of the repair tissue replacing the matrix from 6 to 26 weeks, and histologically there is tide mark formation and integration of the regenerated chondral tissue with original cartilage following implantation of the collagen/ hyaluronate matrix in this model. References: 1.Liu L-S, Thompson AY, Heidaran MA, Poser JW and Spiro RC. An osteoconductive collagen/hyaluronate matrix for bone regeneration. Biomaterials, 1999, 20:1097-1108. 2. Jackson DW, Lalor PA, Aberman HM, and Simon TM. Spontaneous repair of full-thickness defects in articular cartilage in a goat model. J Bone Joint Surg, 2001, 1:53-64. Disclosure: The authors were either an employee of or paid for contracted services by the funding corporation.

S306

1524

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IN VIVO INTERLEUKIN-1 CONVERTING ENZYME MODULATION BY NITRIC OXIDE IN THE DOG EXPERIMENTAL OSTEOARTHRITIS MODEL. Jean-Pierre Pelletier, Christelle Boileau, Florina Moldovan, Jean-Yves Jouzeau, Patrick Netter, Pamela T Manning, Johanne Martel-Pelletier Montreal, Quebec, Canada; Vandoeuvre-Les-Nancy, France and St. Louis, MO

BISPHOSPHONATES STRUCTURALLY SIMILAR TO RISEDRONATE (ACTONEL) SLOW DISEASE PROGRESSION IN THE GUINEA PIG MODEL OF PRIMARY OSTEOARTHRITIS. Joan M Meyer, Susan M Dansereau, Ralph W Farmer, Gwen L Jeans, Marla C Prenger Mason, Ohio

The aim of this study was to investigate the relationship between two key mediators implicated in cartilage osteoarthritis (OA): nitric oxide (NO) and interleukin (IL)-1 converting enzyme (ICE). IL-18 was also studied and served as reference to the effect of ICE. For this study, an OA model was created by sectioning the anterior cruciate ligament (ACL) of the right stifle joint of dogs by a stab wound. Dogs were separated into experimental groups: I) unoperated dogs that received no treatment; II) operated dogs (OA) that received no treatment, and III) operated dogs (OA) that received oral N-iminoethyl-L-lysine (L-NIL, a specific iNOS inhibitor; 10 mg/kg/day) starting immediately after surgery. The OA dogs were sacrificed 12 weeks after surgery. In a second set of experiments, ACL injury was induced in dogs for 12 weeks, and femoral condyles dissected and incubated with specific inhibitors of important signaling pathways involved in the OA process, namely SB 202190 (10 ␮M, p38 MAP kinase inhibitor), PD 98059 (100 ␮M, MEK 1/2 inhibitor), NS-398 (10 ␮M, specific cyclooxygenase-2 inhibitor), SN-50 (50 ␮M, NF-␬B inhibitor) and L-NIL (50 ␮M). Specimens were processed for in situ hybridization or immunohistochemistry for ICE and IL-18, followed by a morphometric analysis. Data showed that ICE and IL-18 were both present in vivo in dog cartilage. Compared to normal, their levels were significantly increased in the OA dog cartilage. L-NIL treatment induced a decrease (p⬍0.001) of ICE and IL-18 levels throughout the OA tissue for both the femoral condyles and tibial plateaus; values were similar to normal dogs. This effect, however, appeared to occur posttranscriptionally as L-NIL did not affect ICE mRNA levels as demonstrated by in situ hybridation. Interestingly, in vitro experiments demonstrated a significant inhibition of the ICE levels by the above inhibitors except L-NIL, indicating that in vivo the NO effect is not direct but requires intermediaries. In this study, we demonstrated the in vivo effectiveness of a specific NO inhibitor in completely reversing the OA enhanced ICE and IL-18 synthesis levels. Data further suggests that the in vivo NO inhibition of ICE levels requires intermediary factors. Disclosure: This study was funded in part by Pharmacia Corporation, St. Louis, MO.

Most animal models of osteoarthritis involve chemical or surgical initiation of the disease, although most human OA is considered primary. The Duncan-Hartley guinea pig is a model of primary OA and mimics human disease in many aspects. Cartilage lesions are bilateral & primarily on the medial tibial plateau. Chondrocyte cloning, osteophytes & tidemark duplication can also be seen. This may be a useful model for testing potential structure modifying OA drugs. We have shown previously the bisphosphonate risedronate but not alendronate statistically significantly reduced cartilage lesion size & severity in this model. In this study, we investigated a series of BPs at 1 or more doses. Compounds were selected that were both similar and different in structure to risedronate & in antiresorptive potency. Animals were randomized into the treatment groups shown in Table 1 (N⫽ 15). Test compounds were dissolved in sterile isotonic saline (vehicle control) and administered via s.c. injection every day for 14 days and every other day thereafter for a total of 24 weeks. At sacrifice, the stifle joint was disarticulated and stained with Evans blue dye for cartilage damage assessment, including absolute lesion area, % lesion area of the medial tibial plateau and mean lesion severity score (0-4 scale). Mean values for each parameter were compared across treatment groups via least squares analysis.

Treatment

Visual Lesion Score (mean ⫾ SEM)

Lesion Area (mm2)

% Lesion Area (mean⫾SEM)

vehicle NE-97221 (0.14mg/kg) NE-58051 (0.14mg/kg) NE-11429 (0.14mg/kg) NE-10575 (0.015mg/kg)

3.62⫾0.27 2.87⫾0.44* 2.89⫾0.29* 3.60⫾0.27 2.89⫾0.34^

6.46⫾0.46 4.82⫾0.92* 5.89⫾0.98 6.87⫾0.69 6.07⫾0.54

32.4⫾1.61 23.0⫾4.11* 26.9⫾3.48 31.6⫾2.72 31.3⫾2.21

* p ⬍ 0.05 vs. vehicle control ^ p ⬍ 0.06 The compounds which demonstrated a statistically significant effect, NE-97221, NE-58051, and NE-10575 are structurally similar to risedronate. NE-11429, and other compounds structurally dissimilar to risedronate have not shown efficacy in this model. These data suggest not all bisphosphonates are efficacious in this model of primary OA, & this effect appears to be unrelated to antiresorptive potency. Further studies underway may provide insight into the mechanism of action of these compounds as structure modifying OA drugs. Disclosure:

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1528

IS THE NOTOCHORD CELL THE KEY TO INTERVERTEBRAL DISC HOMEOSTASIS? W Mark Erwin, Paul Doherty, Robert D Inman Toronto, Ontario, Canada

THE IN VIVO DUAL INHIBITION OF CYCLOOXYGENASE AND LIPOXYGENASE BY ML-3000 REDUCES THE PROGRESSION OF EXPERIMENTAL OSTEOARTHRITIS: SUPPRESSION OF COLLAGENASE-1 AND INTERLEUKIN-1␤ SYNTHESIS. Jean-Pierre Pelletier, Julio C Fernandes, Francois-Cyril Jolicoeur, Pascal Reboul, Stefan Laufer, Susanne Tries, Johanne Martel-Pelletier Montreal, Que´bec, Canada; Tubingen and Blaubeuren, Germany

Purpose: Rabbits, cats, and mongrel dogs maintain notochord cells within the intervertebral discs throughout life and do not develop degenerative disc disease. Purebred dogs and humans do not maintain these cells and do develop degenerative disc disease. Our hypothesis is that intervertebral disc integrity is dependent upon factors produced locally be notochord cells that interact with the disc nucleus matrix. Methods: Canine notochord cells were obtained from mongrel dogs (ages 1-3 years) and were cultured in alginate beads in serum-deficient conditions to produce notochord-enriched media (NCEM). NCEM was added to cultures of bovine disc chondrocytes in which proteoglycan production was determined by 35S labeling, column chromatography, and scintillation counting. Using 35S incorporation, proteoglycan production in the further-removed matrix (FRM) was also measured. FRM was isolated by treatment with hyaluronidase and the cells were pelleted by centrifugation; the supernatant was subjected to SDS-PAGE the autoradiography of the gel. The proteins in NCEM were analyzed by 1-D and 2-D gel electrophoresis. Results: There was a dose-dependent relationship between the amount of NCEM added and the proteoglycan production by the chondrocytes. Specifically, sulfate incorporation was 900 cpm for DMEM alone, and 1600, 3200, and 3700 cpm for NCEM from notochord-alginate beads at 20, 30, and 40 beads per well respectively. Autoradiography of the FRM chondrocyte matrix after culture wiht NCEM revealed high molecular bands consistent with the large aggregating proteoglycan aggrecan. 1-D SDS-PAGE of NCEM revealed several proteins in the 40-73 kD range, with the predominant bands in the 66-80 kD range and the 40-45 kD range. In 2-D gel electrophoresis the 40-45 kD proteins were represented in the pH range of 4.3-7.0. The proteins in the 67 kD range were largely in the neutral pH range. Conclusions: Intervertebral disc chondrocytes when cultured with notochord cell supernatant demonstrated a dose-dependent upregulation of proteoglycan production, including proteoglycans of the further-removed matrix. NCEM contains several proteins in the 40-80 kD range with acidic to neutral pI. These studies may offer important insights for novel therapeutic approaches to degenerative disc disease. Disclosure:

We investigated the therapeutic effectiveness of ML-3000, a new anti-inflammatory drug which has balanced dual inhibitory activity against 5-lipoxygenase and cyclooxygenase, on the development of lesions in an experimental osteoarthritis (OA) dog model. The anterior cruciate ligament of the right joint of mongrel dogs was sectioned with a stab wound. Dogs were divided into 3 groups: group 1 received placebo, groups 2 and 3 were treated with therapeutic dosages of oral ML-3000; 2.5 and 5 ␮g/kg/day, respectively. The dogs received medication the day after surgery and were killed 8 weeks later. Evaluations were made of the size and grade of cartilage erosions on both condyles and plateaus. Histologic examinations of cartilage lesions and synovial inflammation were performed. Levels of collagenase-1 in cartilage and IL-1b in synovial membrane were measured by immunohistochemistry. Levels of PGE2 in the synovial fluid and LTB4 in conditioned medium of cultured synovial membrane were also determined using specific EIA assays. Serum levels of ML-3000-treated dogs were within therapeutic range. Macroscopically, ML-3000 significantly decreased the size (condyles, 2.5 mg/kg, p⬍0.05; 5.0 mg/kg, p⬍0.04; plateaus, both concentrations, p⬍0.01) and grade (plateaus, 2.5 mg/kg, p⬍0.004, 5 mg/kg, p⬍0.02) of the cartilage lesions compared with the placebo-treated OA dogs. Histologically, the severity of cartilage lesions was also decreased in the ML-3000-treated dogs versus the OA dogs both on the condyles (5 mg/kg, p⬍0.03) and the plateaus (2.5 mg/kg, p⬍0.002; 5 mg/kg, p⬍0.02). All 3 OA groups showed a notable and similar level of synovial inflammation. ML-3000 significantly decreased the level of PGE2 in synovial fluid and LTB4 production by synovial membrane. It also markedly decreased the levels of collagenase-1 in the cartilage and IL-1␤ in synovial membrane. ML-3000 significantly reduced the development of lesions in experimental dog OA. The drug acts by reducing the synthesis of inflammatory mediators, PGE2 and LTB4, and catabolic factors such as collagenase-1 and IL-1␤, playing an important role in the OA lesions. The effect of the drug on catabolic factors can possibly be related to its inhibitory action on LTB4 synthesis. Disclosure: Supported by grants from Merckle GmbH, Ulm, Germany.

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A NEW METHOD TO MONITOR OSTEOARTHRITIC CARTILAGE IN ANIMAL MODELS. Scott Martin, Deborah Stamper, Nirlep Patel, James Fujimoto, Mark Brezinski Boston and Cambridge, MA; WIlkes-Barre, PA

SELECTIVE COX-2 INHIBITION IS BENEFICAL FOR MATRIX TURNOVER IN OSTEOARTHRITIC CARTILAGE; A HUMAN IN VITRO STUDY. S C Mastbergen, J WJ Bijlsma, F PJG Lafeber Utrecht, GA, The Netherlands

Currently, most therapeutic interventions in animal models of osteoarthritis are monitored by sacrificing the animal at time points. Problems with this include requiring a significant number of animals, wasting scarce therapeutics, and the inability to sequentially follow changes. The ability to progressively monitor changes in cartilage response would be a powerful tool for the evaluation of therapeutic agents for osteoarthritis. Recently, optical coherence tomography (OCT) has been introduced for the high resolution assessment of osteoarthritis (Journal of Rheumatology 1999; 26(3): 627-635). In these studies, OCT was able to identify cartilage thinning, collagen disorganization, fibrosis, and disruption of the bone/cartilage interface. OCT is analogous to ultrasound, measuring the intensity of backreflected infrared light rather than sound (Science 1997;276:20372039). OCT has several advantages over current imaging technologies. It has a resolution between 4-20 mm, up to 25X anything available in clinical medicine, and is high speed, with current systems near video rate. OCT can also be combined with a range of spectroscopic techniques, including polarization spectroscopy. By using polarization spectroscopy, changes in articular cartilage collagen disorganization can be identified. Preliminary studies using OCT to assess normal and osteoarthritic rat and rabbit cartilage show that normal rat cartilage has substantially more polarization sensitivity than rabbit or human cartilage. Cartilage thinning as well as loss of polarization sensitivity could be identified in osteoarthritic samples. OCT represents a promising new technology for monitoring osteoarthritic therapeutics in animals.

In treatment of osteoarthritis (OA), nonsteroidal anti-inflammatory drugs (NSAIDs) are very useful in reducing inflammation and pain. But assuming inflammation to be secondary to the degenerative process of cartilage damage, their direct effect on cartilage may be at least as important in the final outcome of the treatment. Although the selective COX-2 inhibitors suppress inflammation with minimal gastrointestinal side effects, their direct effects on cartilage have been less well explored. In the present work we investigated the effect of a selective COX-2 inhibitor, celecoxib, on matrix turnover of osteoarthritic cartilage. Normal and clinically defined osteoarthritc human articular cartilage was exposed for 7 days in culture to celecoxib (0.01, 0.1, 1, and 10 ␮M). Changes in cartilage matrix turnover (proteoglycansynthesis [35SO4 incorporation], -retention [release of 35SO4 labeled GAGs], -release [Alcian Blue precipitated and stained GAG released] and -content [Alcian Blue stained and precipitated GAGs] were determined. On normal cartilage, no significant, and thus no adverse, effects of celecoxib were found. Osteoarthritic cartilage showed the characteristic changes in proteoglycan turnover (inhibition of synthesis, diminished retention, enhanced release, and a diminished content; all p⬍0.05). Celecoxib was able to reverse these adverse changes by increasing synthesis (39%) and retention (27%), diminishing release (29%) and enhancing content (17%) in a dose dependent manner with an optimum at 1 ␮M (all pⱕ0.05). These results show that in human osteoarthritic cartilage COX-2 plays an important role in disturbed cartilage matrix turnover. This corroborates our previous findings demonstrating an important role of COX-2 in IL-1␤/TNF␣ treated normal cartilage (Mastbergen et al. A&R. 2000;43:349; ms submitted). Celecoxib normalizes proteoglycan turnover of osteoarthritic cartilage, as was demonstrated for the IL-1␤/TNF␣ treated normal cartilage. This suggests celecoxib to be disease modifying and makes celecoxib suitable for long-term treatment of joint disorders including osteoarthritis.

Disclosure: Mark Brezinski and James Fujimoto are both founders of LightLab, Inc. Mark Brezinski and James Fujimoto both have equity and are salaried by LightLab, Inc.

Disclosure: This study was supported by Pharmacia

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NITRIC OXIDE-INDUCED CELL DEATH IN HUMAN OSTEOARTHRITIC SYNOVIAL FIBROBLASTS: A TYROSINE KINASE, COX-2 MEDIATED PROCESS. Jean-Pierre Pelletier, Dragan Jovanovic, Francois Mineau, Kohei Notoya, Johanne Martel-Pelletier Montreal, Que´bec, Canada

EFFECTS OF ROFECOXIB ON METALLOPROTEINASES AND NITRIC OXIDE PRODUCTION BY HUMAN OSTEOARTHRITIC SYNOVIAL TISSUE AND CARTILAGE CULTURES. Ilana Yaron, Idit Shirazi, Rahel Judovich, Michael Yaron Tel-Aviv, Israel

This study explored the major pathways involved in the regulation of the osteoarthritic (OA) synovial fibroblasts nitric oxide (NO)-induced cell death. Human OA synovial fibroblasts incubated with the NO donor, sodium nitropruside (SNP, 0.5 mM), decreased the cell viability by more than 50%. This was associated with increased levels of both COX-2 synthesis and PGE2 production. Selective inhibition of COX-2 by NS-398 (50 ␮M) significantly inhibited SNP-induced cell death, even in the presence of exogenously added PGE2 (100 ng/ml). Further experiments revealed that SNP-treated cells expressed increased levels of active caspase-9 and -3, while Bcl-2 was down regulated. Incubation of these treated cells with inhibitors of caspase-9 (Z-LEHD-FMK; 100 ␮M) or caspase-3 (Z-DEVD-FMK, 100 ␮M) protected SNP-treated OA synovial fibroblasts viability, indicating that NO-mediated cell death is mainly related to apoptosis rather than necrosis. This is also confirmed by the DNA fragmentation found in these cells (TUNEL method). Data also showed that SNP induces the activation of the kinases ERK 1/2, p38, and tyrosine kinases. Specific inhibition of tyrosine kinases completely abrogated the SNP-induced cell death. In turn, this cell death protection was associated with a complete inhibition of caspase -9 and -3 as well as COX-2/PGE2 production. Together these findings indicate that these kinases are likely mediators of NO-mediated apoptosis. Additional experiments were performed: i) inhibition of the scavengers of NO (carboxy-PTIO), peroxinitrite (uric acid) and superoxide peroxide (taxifolin), ii) suppression of inducible NO synthase (L-NIL), and iii) chelation of iron (deferoxamine mesylate). Data demonstrated that NO-induced cell death was not related to NO or peroxynitrite, but rather to the generation of hydrogen peroxide and iron-sulphur enzymes. Our results provide strong evidence of the role of tyrosine kinases and MAPK activation via the upregulation of COX-2 expression in NO-induced OA synovial fibroblast death. Moreover, the generation of hydrogen peroxide and iron-sulphur enzymes appears to be a major factor in the death of these cells.

Objective: To evaluate rofecoxib effects on collagenase (MMP-1), stromelysin (MMP-3) and nitric oxide (NO) production by human synovial and cartilage cultures in the presence of IL-1␤. Methods: Synovial tissue and cartilage were obtained in the operation theater from patients undergoing total knee replacement for osteoarthritis, cut into small pieces and cultured in the presence or absence of rofecoxib and IL-1␤. MMP-1 and MMP-3 in culture media were measured by ELISA and NO with the Griess reagent. Cartilage synthesis was evaluated by 35S incorporation. Results: Rofecoxib (0.45␮g/ml, 1.4␮g/ml) 4.2␮g/ml) inhibited IL-1␤ (1ng/ml) stimulated MMP-1 production by synovial tissue and cartilage cultures in a dose dependent manner, (p ⬍ 0.05 at 0.45 ␮g/ml). IL-1␤ (1ng/ml) stimulated MMP-3 production by synovial tissue was inhibited by rofecoxib in a dose dependent manner (p⬍0.05 at 0.1␮g/ml). IL-1␤ stimulated NO production was inhibited by rofecoxib in a dose dependent manner (p⬍0.05 at 0.1␮g/ml in synovial tissue cultures and at 4.2␮g/ml in cartilage cultures). Rofecoxib reversed IL-1␤ (1ng/ml) inhibition of cartilage synthesis in a dose dependent manner (p⬍0.05 at 0.45␮g/ml). Conclusions: Rofecoxib may have a chondroprotective effect by inhibiting MMP-1, MMP-3 and NO production by IL-1 stimulated synovial tissue and cartilage and by reversing IL-1 inhibition of articular cartilage synthesis. Disclosure:

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ROLE OF LEUKOTRIENE B4 IN ABNORMAL FUNCTION OF OSTEOARTHRITIC OSTEOBLASTS. Daniel Lajeunesse, Yosabeth Paredes, Frederic Massicotte, Johanne Martel-Pelletier, Stefan Laufer, Jean-Pierre Pelletier Montreal, Que´bec, Canada and Tubingen, Germany

THE FATE OF ORAL GLUCOSAMINE TRACED BY 13C-LABELING IN THE DOG. George R Dodge, Ravinder R Regatte, Jeffery O Hall, Arijit Borthakur, Akella V Sarma, Dale A Callaway, Ravinder Reddy Wilmington, DE; Philadelphia, PA and Logan, UT

Recent data indicate a direct intervention of subchondral bone remodeling in the initiation and/or progression of osteoarthritis (OA). We showed abnormal cell metabolism in human OA subchondral osteoblasts (Ob) including increased alkaline phosphatase activity (ALPase), osteocalcin release (OC) and prostaglandin E2 (PGE2) levels compared to normal Ob. Non-steroidal antiinflammatory drug (NSAIDs) inhibit the production of PGs by acting on cyclooxygenase (COX), but could also lead to a shunt to the 5-lipoxygenase (LO) pathway, producing leukotrienes (LTs). Here, we compared the effect of therapeutic levels of ML-3000, a dual inhibitor of COX and LO, NS-398 (COX-2 inhibitor) and BayX-1005 (inhibitor of LO activating protein) on the production of leukotriene B4 (LTB4), PGE2, ALPase and OC by human OA Ob. Data showed that OA Ob produced variable levels of PGE2 and of LTB4 compared to normal Ob. ML-3000 (0.3 to 3 mg/ml) inhibited the production of PGE2 and LTB4 by OA Ob reaching a maximum of 61.2 ⫾ 6.4% and 67 ⫾ 7.6% respectively. NS-398 reduced PGE2 production by 75.8 ⫾ 5.3% while BayX-1005 inhibited LTB4 production in OA Ob by 38.7 ⫾ 14.5%. ML-3000 dose-dependently stimulated 1,25(OH)2D3-induced ALPase while it inhibited OC release. BayX-1005 partly reproduced this effect on OC and ALPase, while NS-398 failed to affect these activities. LTB4 dose-dependently (10-13 to 10-9 M) inhibited ALPase in OA Ob while its effect on OC release depended on endogenous LTB4 levels in these cells. In normal Ob, LTB4 dose-dependently stimulated OC release, whereas it failed to influence ALPase. These results show that ML-3000 inhibits both LTB4 and PGE2 production by OA Ob, and has selective effects on ALPase and OC at least via its role on LTB4 production. As abnormal subchondral bone remodeling may be linked to OA initiation/progression, these results suggest ML-3000 could potentially be very attractive for the treatment of OA.

Interest in new therapies for arthritis is timely and the search for chondroprotective agents is the subject of many studies. Amongst the most notable is glucosamine (GlcN)(i.e. glucosamine-SO4, glucosamine-HCl) with the variety of studies demonstrating efficacy at modulating chondrocyte function and joint pathology. It has remained ambiguous as to whether ingested GlcN makes its way to the joint. We built on our recent in vitro experiments (A&R 44:1089-1095) showing that cartilage explants cultured in medium containing 13C-GlcN result in the detection of 13C in galactosamine moieties of chondroitin sulfate. We designed a study to trace the fate of orally dosed GlcN in the dog using 13C-GlcN and the sensitive techniques of inductively coupled plasma mass spectroscopy (ICP-MS) and NMR spectroscopy. Two dogs were treated with 13C-glucosamine-HCl by oral dosing (combined with food). Dog 1 was given 500 mg/dog/day (⬃50 mg/kg/day) for 2 weeks and dog 2, 250 mg/dog/day for 3 weeks. Cartilage was harvested from the femoral condyles, along with liver, spleen, heart, kidney, skin, skeletal muscle, lung, and costal cartilage Treated and control dogs). Tissues were prepared for ICP-MS (Perkin-Elmer Elan6000) by digestion with nitric acid and analyzed for 12C and 13C in triplicate (identical to 0.01%). The articular cartilage from dog 1 (high dose) showed an increase of 2.6% of 13C as compared to control and dog 2 (low dose) an increase of 1.6%. These data were confirmed by NMR spectroscopy where all five peaks in the anomeric region of 13C NMR spectra (GlcUA and GalNAc peaks from chondroitin sulfate(CS)-4 and CS-6 and keratan sulfate, respectively) were all elevated in both treated dogs compared to controls. The 12 13 C/ C ratios of other tissues were analyzed to determine the distribution of 13C-GlcN. As expected, the liver had the highest 13C percentage of the other tissues but all were less than articular cartilage. Considering the natural stability of 12C/13C ratios, rate of biosynthesis, and the single 13C per GlcN, the increases are well within the expected range if 13C-GlcN was incorporated into proteoglycans. These results for the first time provide convincing evidence that oral GlcN is bioavailable to chondrocytes in joint cartilage thereby potentially having an affect on the available GlcN for proteoglycan biosynthesis.

Disclosure: This study was supported in part by a grant from Merckle GmbH, Ulm, Germany.


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CYCLIC TENSILE STRETCH DECREASES PROTEOGLYCAN PRODUCTION BY ANULUS FIBROSUS CELLS IN VITRO THROUGH NO PRODUCTION. Francois Rannou, Pascal Richette, Valerie Genries, Michel Revel, Maite Corvol, Serge Poiraudeau Paris, France

GLUCOSAMINE SULFATE ON CARTILAGE: LAPINE STUDY. Roy D Altman, Herman Cheung Miami, FL

Context : Degeneration of the intervertebral disc is one of the factors associated with low back pain and is a prerequisite to disc herniation. Although mechanical forces are important modulators of remodeling of intervertebral disc tissue, the molecular mechanisms implicated are not known. Objective : To study the effects of cyclic tensile stretch (CTS) on proteoglycan synthesis and degradation by anulus fibrosus (AF) cells. Study design : In vitro culture of AF cells under CTS. Methods : After enzymatic digestion of rabbit intervertebral disc, AF cells were cultured at high density on a flexible substrate. CTS was applied using a pressure-operated instrument inducing the deformation of flexible-bottomed culture plates. The proteoglycans newly synthesized and secreted by AF cells were measured by labelling with 35S-sulphate in the absence or presence of cycloheximide or NOS inhibitors NG-methyl-L-arginine (L-NMA). Proteoglycan aggregability was studied by the elution profile on Sepharose 2B columns after 35S-sulfate incorporation. Cell content of aggrecan and biglycan mRNA was determined by Northern blotting. NO production was evaluated by Griess reaction. Results : Cells were confluent after 6 days. CTS was delivered at 1% and 5% at a frequency of 1 Hz during 30mn to 24h. No effect was observed at 1% elongation. At 5% elongation, a significant decrease in the neo-synthesized proteoglycans in the cellular pool and in the culture medium was observed after 8 hours stimulation (30% and 21% respectively). This decrease was time dependent until 24 hours of stimulation (43% and 41% respectively) and persisted in the presence of cycloheximide. No modification in physical properties of proteoglycans or aggrecan and biglycan expression was detected. A significant increase of NO production (350%) was observed after 8 hours elongation. L-NMA abolished the effects of CTS on NO and proteoglycan productions. Conclusion : CTS can participate in the regulation of intervertebral disc matrix by decreasing proteoglycan production through NO production. This result could be of interest to develop local therapeutic strategies aimed at inhibiting intervertebral disc degradation.

The objective was to determine structure modifying effects of orally administered glucosamine sulfate in an animal model. At baseline, 6 month old New Zealand white rabbits received a right medial hemimeniscectomy (Moskowitz model) and received no treatment (OA; n⫽13), glucosamine sulfate 100mg/kg/d po (GS100; n⫽11) or 200mg/kg/d po (GS200; n⫽11). A control group had no surgery and no glucosamine (C; n⫽10). Tissues were examined for anatomic changes and enzyme activity at 12 weeks. On gross examination, both GS100 and GS200 were both more similar to C than OA. Mankin histologic scores: OA 9.8⫾1.7; GS100 3.7⫾0.8; GS200 4.9⫾2.0; C 1.0⫾0.0. Immunohistochemistry revealed a dose response reduction in MMP-1 and MMP-3 in the GS treated animals. Substrate gel analysis revealed increased MMP-2 and MMP-9 in all operated groups. MMP-3 was less elevated in a dose response in the GS groups than the OA group. RT-PCR revealed no difference in MMP-1, MMP-3 and MMP-13 in all the operated groups. Orally administered GS was related to a more normal gross and microscopic appearance of cartilage in an animal model of osteoarthritis. Although there was no significant change with GS in mRNA level of MMP-3 in the cartilage, there was a immunohistochemical reduction in MMP-3. Despite the lack of effect on mRNA, an effect of GS on MMP-3 can not be excluded. Disclosure: Rotta Pharmaceuticals McNeil Consumer Products

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CLINICAL EFFECTS AND POSSIBLE MECHANISMS OF GLUCOSAMINE IN THE TREATMENT OF OSTEOARTHRITIS. Hiroshi Nakamura, Michiaki Tanaka, Kayo Masuko-Hongo, Tomohiro Kato, Kusuki Nishioka Kawasaki, Kanagawa, Japan

SERUM LEVELS OF TYPE IIB PROCOLLAGEN AMINO TERMINAL PROPEPTIDE (PIIBNP) ARE DECREASED IN PATIENTS WITH KNEE OSTEOARTHRITIS. Jean-Charles Rousseau, Pierre Miossec, Eric Vignon, Patrick Garnero, Pierre D Delmas Lyon and Pierre-Benite, France

Background: Glucosamine has been used for the treatment of arthritis as a supplement. A lot of clinical studies showed that glucosamine was effective for treatment of OA. A number of in vitro experiments using chondrocytes were reported, however the precise mechanism is still unclear. Purpose: To ascertain the clinical efficacy of glucosamine on OA and elucidate its mechanism by in vitro experiments. Method: (1) Clinical study; 40 OA patients with 62 knees were administered with glucosamine preparation (1500mg/day) after informed consent. Knee score, VAS for pain, Face scale and clinical symptoms were assessed for 3 months. Age-matched 15 OA patients with 25 knees were used for control. Serum sample was collected before and after the treatment. The concentration of YKL-40(Chondrex), PGE 2 , NO and titer of anti type II collagen antibody (COLIIab) were measured in serum. (2) in vitro study: PGE2, NO, MMP-1, 3, 13 and TIMP-1 concentrations were detected in the supernatant of IL-1 stimulated human chondrocytes isolated from OA and fractured patients.COX-2 and iNOS mRNA were analysed by RT-PCR. Results: Knee score, VAS, Face scale as well as clinical symptoms improved after the administration of glucosamine, while no improvement was found in the control group. Serum PGE2 concentrations and titer of anti COLIIab decreased to the level of healthy control after the treatment. Serum YKL-40 concentrations showed no difference during the treatment, however, strong relationship was found between YKL-40 concentration and the stage of OA. In in vitro studies, glucosamine suppressed the production of PGE2, NO and MMP-1 in a dose dependent manner. Conclusion: Glucosamine preparation was effective for treatment of OA as previous duble-blind control studies suggested. Moreover, our clinical and in vitro data suggested that the possible mechanism was anti-inflammatory, immune modifying and chondro-protective effects .

Purpose: Type II collagen, the major component of cartilage, is synthetized as a procollagen containing N and C-extension propeptides which are released in the circulation and can thus be used as serum molecular markers of type II collagen synthesis. N-propeptide exists as two forms: a short form, PIIBNP, synthesized by mature chondrocytes and a long form, PIIANP, containing an additional 69 amino-acid cystein rich domain encoded by exon 2, synthesized mainly by chondroprogenitor cells in developing skeletal tissues. To date no assay for serum PIIBNP has been developed. The aim of this study was to develop a specific assay for PIIBNP and to measure its serum concentration in healthy controls and in patients with knee osteoarthritis (OA) and rheumatoid arthritis (RA). Methods: We selected a synthetic amino-acid peptide located at the junction of the protein part encoded by exon 1 and exon 3 which is found only in PIIBNP and not PIIANP. This peptide coupled to bovine serum albumin was used as immunogen to raise polyclonal rabbit antibodies. Using this antibody (titer: 20000) and the six amino-acid sequence coupled to ovalbumin as standard, we developed a competitive ELISA and measured serum PIIBNP in 24 patients with knee OA (mean age, 65 yr, median disease duration 10 yr), 37 patients with RA (mean age 57; median disease duration 6 yr) and 42 healthy age-matched controls. Results: The ELISA for PIIBNP demonstrated adequate dilution recovery (mean 120%) and intra and inter-assay precision (CV⬍15%), allows accurate measurements of serum PIIBNP from 67 to 5500 pmol/L, and does not cross react with PIIANP.

Disclosure: This work is supported in part by Rohto Pharmaceutical Co., Ltd.

groups

Controls (n⫽42)

Knee OA (n⫽24)

RA (n⫽37)

Serum PIIBNP (pmol/L)

1280⫾833

672*⫾317

1139⫾422

*p⬍0.01 vs controls and vs RA As shown on the table, compared to controls serum PIIBNP was significantly decreased by an average 48% in patients with knee OA but not in RA patients. Conclusion: We have developed the first specific immunoassay for serum PIIBNP which exhibits adequate technical performances. Decreased serum PIIBNP in patients with knee OA suggests impaired reparative process of cartilage that may responsible in part for joint destruction. Disclosure:

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AUTOLOGOUS T CELL RESPONSE AGAINST CHONDTROCYTE WITH CD80/86 IN OSTEOARTHRITIS. Michiaki Tanaka, Masahiro Sakata, Kayo Masuko-Hongo, Hiroshi Nakamura, Atsuyuki Shibakawa, Jun-ichiro Tsuruha, Tomohiro Kato, Kusuki Nishioka Kawasaki, Kanagawa and Tokyo, Japan

AGGRECAN DEGRADATION PRODUCTS GENERATED BY AGGRECANASE AND METALLOPROTEINASE (MMP) ENZYMES IN THE SYNOVIAL FLUID OF PATIENTS WITH RHEUMATIC DISEASES. Sherine Chan, Rob Will, Neil Kent, Rainer Klocke Perth, WA, Australia; and Bath, United Kingdom

Background: We have proposed T cell mediated cartilage destruction in the pathogenesis of osteoarthritis (OA), based on the presence of immune response against the cartilage tissue derived peptide such as CILP and YKL-39. Objective: To investigate direct interactions of chondrocytes with autologous T cells in OA. Materials and methods: Chondrocytes and peripheral blood mononuclear cells (PBMCs) were obtained from 13 patients with OA (age 53-80, OA chondrocytes) and 7 patients with femoral neck fracture (age 55-88, normal chondrocytes). Chondrocytes were cultured in DMEM containing 10%FCS in 96-well plates. Autologous PBMCs or T cells, which were isolated from PBMCs, were co-cultured with irradiated chondrocytes and measured for 3H-thymidine incorporation in order to assay the response against chondrocytes. In addition, Immunocytochemical analysis was performed to demonstrate the expression of HLA-DP, -DQ, -DR, CD80, and CD86 on chondrocytes. Results: Both PBMCs and T cells responded against autologous chondrocytes significantly higher in OA (stimulation index ⫽ 7.22 ⫾ 2.43) compared to normal (1.38 ⫾ 0.23, p⬍0.01). This response was partially blocked by each anti-HLA class I, class II, CD4 and CD8 antibodies (Abs), and was completely inhibited by combination of anti-HLA class I and class II Abs, or anti-CD4 and CD8 Abs. Immunocytochemical analysis revealed that HLA-DP, -DQ, -DR, CD80, and CD86 molecules were expressed on the surface of OA chondrocytes, but not on normal chondrocytes. Conclusion: The present study demonstrated that OA chondrocytes expressed surface molecules implicated in antigen presentation and that T cells responded against autologous chondrocytes. These results suggested that T cells reacted with antigens derived from chondrocytes, and might support the presence of T cell mediated pathogenic process in OA. Disclosure:

We investigated the pattern of aggrecan products released into synovial fluid of 34 patients with a variety of rheumatic diseases to determine the relative importance of matrix metalloproteinases (MMP’s) and aggrecanase in the degradation of aggrecan and link protein. About 2-4 mls of synovial fluid was usually aspirated from an involved knee and in 5 patients with OA a contralateral knee. This was CsCl fractionated and 10␮g was treated with chondroitinase ABC and keratanase I and II overnight at 37°C. The proteoglycan was then dialysed and lyophilized and loaded onto 4-12% Tris-glycine gradient gels and transferred onto nitrocellulose. Bands were detected using the monoclonal antibodies (MAbs) BC-3 which detects a N-terminal ARGSV.. epitope generated at the Glu373-Ala374 by the action of aggrecanase, BC-14 which detects the N-terminal FFGVG.. epitope generated by MMP’s which cleave at the Asn341-Phe342 site and 8A4 which detects link proteins (LP1-3). Mutiple bands could be identified in most patients using the 3 MAb’s, with BC-3 the prominent bands were at 140, 65 and 55 kda. With BC-14 the prominent bands were at 68 kda and a doublet at 33kda. Multiple bands were often detected with 8A4 with the prominent bands occuring at 55,52 and 50 kda.In 2 patients with early RA (disease duration:⬍12 months) no bands with BC-14 were detected. Bands detected occured in the following descending order, BC-3⬎8A4⬎BC-14. This pattern occurred in all of our patients’ studies. There were often divergent patterns in patients with bilateral OA of the knee. Bands with BC-14 did not occur in 3 pts with gout, 1 with AS and 1 with subacromial bursitis. Where bands with BC-14 were detected, densitometric standardisation suggested much less epitope was present than with the BC-3 MAb. Conclusions: 1. The prominent degradative enzyme in the arthritic joint is aggrecanase and this acts in most patients. 2. The pattern of the catabolites generated in RA and OA using BC-3 appear to be the same. 3. More aggrecanase generated epitope seems to be present than MMP generated epitope and MMP’s may not be acting in early RA. 4. MMP’s are active in most patients with advanced RA and in OA but may not be active in other diseases. 5. There are differences in the catabolite patterns in contralateral OA knees.6. Link protein catabolites appear to be generated by aggrecanase. Disclosure:

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ASSOCIATION OF HLA-DRB1*02 WITH OSTEOARTHRITIS IN A COHORT OF 106 PATIENTS. Brigitte Mueller, Jill Menard, Verena Moos, Jochen Sieper Berlin, Germany

SERUM LEVELS OF COLLAGENASE-CLEAVED TYPE II COLLAGEN ARE RELATED TO AGEING AND DISEASE ACTIVITY IN PATIENTS WITH RHEUMATOID ARTHRITIS. T Kojima, N Ishiguro, T Oguchi, R C Billinghurst, M Ionescu, A R Poole Nagoya, Aicni, Japan and Montreal, Quebec, Canada

Disclosure: This work was funded by the DFG grant MU 844/4

INTRODUCTION: Degradation of type II collagen in cartilage, which causes cartilage damage in osteoarthritis (OA) and rheumatoid arthritis (RA), is initiated by cleavage of type II collagen by collagenases. Following this cleavage, enzymes such as gelatinases and collagenases digest the denatured type II collagen releasing the cleavage site into body fluids. Recently we developed a new monoclonal antibody to detect and quantify the cleavage neoepitope of type II collagen by collagenases(C2C). We demonstrated that its release into culture media, synovial fluid and serum was increased on induction of degradation in explant cultures and in experimental arthritis. We have now measured serum levels of the cleavage neoepitope in RA patients and healthy volunteers to determine whether the serum level of C2C is related to ageing and arthritis. MATERIALS AND METHODS: Serum samples were collected from healthy volunteers with no joint pain (81 Females, 76 Males, age 18-68) and female RA patients (n ⫽ 77, age 35-61). The level of C2C was determined by ELISA. For the RA patients, serum levels of CRP were also measured. RESULTS: Serum levels of C2C in a healthy younger group (age ⬍ 45) were significantly higher than that of an older group (age⬎45) in both females and males. In the younger group, C2C of females was higher than that of males while, in the older group there was no significant difference between sexes. According to CRP levels over one year, we identified two (low and high CRP) RA disease activity groups. C2C of the high CRP group was higher than that of the low CRP group and the age-matched healthy group. There was no significant difference in C2C between low CRP and healthy groups. DISCUSSION: In the high CRP/RA disease group, elevated levels of C2C suggest more cartilage damage based on our observations in experimental models. Serum levels of C2C in the low CRP group suggest less evidence for cartilage damage. Turnover is clearly not similar in disease-free persons and sex and age differences must be carefully controlled. CONCLUSION: The results suggest increased cleavage of type II collagen by collagenase with inflammation in RA. The C2C assay may be useful in monitoring cartilage damage in arthritis. Disclosure:

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statement of purpose: We have previously shown that the inflammatory cytokines TNFalpha and IL-6 or IL-1beta are upregulated in chondrocytes of osteoarthritis (OA) patients. However, the inflammatory responses associated with OA are low grade and restricted. To investigate an involvement of the immune system in the pathogenesis of OA we here analyzed patients for their HLA-DRB1 haplotypes. Methods: Combining SSO or SSP typing procedures, 139 randomly selected controls and two cohorts of 59 and 47 OA patients each were typed for their HLA-DRB1 alleles. Results: The combined analysis of both cohorts shows statistically significant differences in the frequencies of the DR2 and the DR5 alleles compared to the controls. While the DR2 allele is elevated among the OA patients, the DR5 allele is negatively associated with the disease. The P-values describing the differences to the controls are 0.0431 for the DR2 and 0.0386 for the DR5 allele and the odds ratios conferred by the DR2 and DR5 alleles are 1.58 and 0.54 with confidence bounds of 1.05 - 2.96 and 0.30 - 0.97, respectively. Conclusion: The association of DR2 and DR5 with OA might hint at a linkage disequilibrium between HLA-DRB1 genes and genes involved in the pathogenesis of OA. Alternatively, DR2 may directly play a role in restricting immunological responses to the low grade inflammation characteristic of OA.

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OSTEOGENIC PROTEIN-1 (OP-1)IN SYNOVIAL FLUID IS A POTENTIAL MARKER IN ARTHRITIS. Benjamin S Frank, Bhavna Kumar, Charis A Merrihew, Brian Fisher, David C Rueger, Joel A Block, Susan Chubinskaya Chicago and MA

VALIDITY OF ARTHROSCOPIC GRADING SYSTEM OF CHONDROPATHY IN OSTEOARTHRITIS OF THE KNEE: CORRELATION WITH HISTOPATHOLOGICAL FINDINGS. Carlos Acebes, Itziar Palacios, Gabriel Herrero-Beaumont, Emilio Delgado Madrid, Spain

The development of body fluid biomarkers for arthritis is, potentially, of immense utility in the treatment of these diseases. The molecular analysis of synovial fluid may yield substantially more information than methods routinely used today. The purpose of the current study was to investigate whether osteogenic protein-1 (OP-1) could be detected in synovial fluid, whether quantitative approaches could be adapted for the assessment of OP-1 in synovial fluid, and whether there are diffences in the levels of OP-1 comparing normal donors to patients with rheumatoid arthritis (RA) and osteoarthritis (OA). OP-1 is a member of the bone morphogenetic protein family, and has multiple anabolic effects in many tissues. We have previously demonstrated the expression both of OP-1 protein and message in different connective tissues including cartilage, synovium, tendon, ligament and meniscus. For the current study, synovial fluid was aspirated from subjects with OA and RA as well as from normal joints of human organ donors. Specimens from 40 joints were analyzed by Western blot with anti-pro and anti-mature OP-1 antibodies, and the concentration of OP-1 was measured by a sandwich ELISA method newly developed in our laboratory (1) which was tested and optimized for the analysis of synovial fluid. We found pro and mature forms of OP-1 simultaneously present in all tested samples. The distribution of immunoreactive bands was similar to that described for human articular cartilage. By ELISA, clear differences in OP-1 levels were identified between OA and RA groups. OP-1 concentration was higher in synovial fluid obtained from RA patients than in that from OA patients (p⬍0.04). Notably, the content of OP-1 in synovial fluid from organ donors was comparable to that detected in cartilage extracts from the same donors. The results of this study suggest potential for development of synovial fluid OP-1 as a diagnostic and prognostic marker. Reference: 1)Merrihew et al.(2001)Trans ORS 47,26:175.

Clinical research in osteoarthritis (OA) requires accurate and objective methods able to evaluate cartilage status. Arthroscopic evaluation of the articular cartilage (chondroscopy) would be a reliable instrument for the measurement of the quantity, integrity and quality of articular cartilage in knee OA. Objective:To determine the validity of the arthroscopic grading (AG) system for scoring chondropathy in osteoarthritis of the knee. Methods: Magnified articular cartilage image of medial femoral condyle and medial tibial plateau of three OA knee joints was directly evaluated and videorecorded using a 2,7 mm scope. After individualized and graded (0 to IV) according to the classification proposed by Beguin and Locker: 0, normal; I, softening and/or swelling; II, superficial fibrillations; III, deep fibrillations; IV, exposure of subchondral bone, chondropathy areas were registered in a grading scheme. Complete articular cartilage of femoral condyles and tibial plateaus were cut in 4 x 10 mm sections and processed for histopathological study. A total of 73 sections were graded twice by two observers according to the Mankin histological-histochemical grading (MHG) system (0-14). MHG system was mathematically categorized into five groups to correlate with AG system: normal (0-2), mild (3-5), moderate (6-8), severe (9-11) and very severe (12-14). Results: The intra and interobserver reliability of the MHG system was high for both observers: (r⫽0,895 and r⫽0,835) (r⫽0,906 and r⫽0,822). Average MHG system respect to AG system was: grade 0 (1,7 ⫾ 0,59 n⫽20), grade I (3,62 ⫾ 2,33 n⫽28), grade III (8,13 ⫾ 2,13 n⫽34) and grade IV (11,5 ⫾ 3,18 n⫽14). Correlation between AG and MHG system was calculated computing the kappa index for categorical variables for both observers: kp⫽0,50 and kp⫽0,58. Conclusion: Arthroscopic grading system is significantly correlated with histopathological grading system, and seems to be a feasible instrument for scoring chondropathy in OA.

Disclosure: This project is supported in part by a grant from Stryker Biotech (Hopkinton MA).

Disclosure: Financial support by a Gant of Spanish Health Institute (FIS 98/0626)

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MMP-3 IN SYNOVIAL FLUID OF PATIENTS WITH RHEUMATOID ARTHRITIS HAS POTENTIAL TO CLEAVE MEMBRANE BOUND FAS LIGAND. Hiroaki Matsuno, Kazuo Yudoh, Yasuhiro Watanabe, Fujio Nakazawa, Hiroyuki Aono, Tomoatsu Kimura Toyama, Toyama, Otaru, Hokkaido and Osaka, Osaka, Japan

THE COST-EFFECTIVENESS OF CELECOXIB AND ROFECOXIB IN PATIENTS WITH OSTEOARTHRITIS OR RHEUMATOID ARTHRITIS. Andreas Maetzel, Murray Krahn, Gary Naglie Toronto, Ontario, Canada; Toronto

Objective; To investigate the relationship between matrix metalloproteinases (MMP) and the soluble form Fas ligand (sFasL) in the synovial fluid (SF) of patients with rheumatoid arthritis (RA), and to determine which MMP have a major role in cleaving FasL. Materials and Methods; The concentrations of sFas and sFasL in SF from 48 patients with RA and 43 patients with osteoarthritis (OA) were measured using specific ELISA. The levels of different MMP (MMP -1,-2,-3,-7,-9) in SF were also measured by ELISA. The acactive forms of gelatinases were detected by gelatin zymogram. Human FasL-expressing transfected cells (hFasL/L5178Y) were used to investigate whether FasL is cleaved from membrane bound FasL. Results; Significantly higher levels of MMP -1,-3, and -9 were found in SF from RA patients compared to OA patients, but MMP-7 was not detectable in either group. The concentrations of sFas and sFasL in SF were also higher in RA than in OA patients. However, there was no relationship between the concentration of sFas and sFasL. Among MMP, MMP-3 concentrations in SF were closely correlated with the level of sFasL and with disease activity of RA. Enzymatic cleavage assay indicated that MMP-3 has potential to cleave the FasL expressed on hFasL/L5178Y cells and to produce sFasL. Conclusion; There was significant correlation between the concentration of sFasL and MMP-3 in SF of patients with RA. In addition, our data indicate that the sheddind of FasL may be regulated by MMP-3 in the joint of patients with RA. Disclosure:

Objective. To evaluate the cost-effectiveness of the cyclooxygenase-2 selective (COX2) nonsteroidal anti-inflammatory drugs (NSAIDs) rofecoxib in comparison to naproxen, and celecoxib in comparison to ibuprofen and diclofenac. Design. Cost-effectiveness analysis based on a 5-year Markov model. Probability estimates were derived from detailed data from two randomised trials, and a systematic search of the medical literature. Utility estimates were obtained from 60 randomly selected members of the general public. Cost estimates were obtained from Canadian provincial databases. Incremental costeffectiveness ratios ($CAD) were calculated for patients at average risk of upper gastrointestinal (UGI) events and for high-risk patients with a prior history of a UGI event. Setting and Participants. Subjects were patients with osteoarthritis (OA) or rheumatoid arthritis (RA) who do not require treatment with low-dose aspirin. The analysis was performed from the perspective of the Ontario Ministry of Health. Main Outcome Measures. Proportion of patients with clinical or complicated UGI events, quality-adjusted life-expectancy, life-expectancy. Results. Evaluation of rofecoxib vs naproxen in patients with RA at average risk resulted in costs per quality-adjusted life-year (QALY) gained of $271,188. Celecoxib was dominated by diclofenac in average-risk patients, and the cost-effectiveness of diclofenac compared with ibuprofen was $125,276 per QALY gained. Both celecoxib and rofecoxib are cost-effective in high-risk patients. Analyses by age groups and assuming a threshold of $50,000 per QALY gained, suggest that rofecoxib or celecoxib would be cost-effective in patients aged over 76 and 81, respectively, without additional risk factors. Conclusion. Both celecoxib and rofecoxib are economically attractive in high risk and elderly patients. They are not economically attractive in patients at average risk. Co-prescription of proton-pump inhibitors with COX2 NSAIDs is not economically attractive for patients at high risk. Insufficient evidence exists to evaluate the role of COX2 inhibitors in patients who also require ASA therapy.


Disclosure: The study was funded by the Canadian Coordinating Office for Health Technology. Dr. Naglie chairs the Drug Quality and Therapeutics Committee of the Ontario Ministry of Health and Long-Term Care. Dr. Maetzel has a longstanding working relationship with Dr. Claire Bombardier who consulted for Merck & Co. as the chair of the steering committee of the VIGOR study.

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MRI EVALUATION OF CHONDROPATHY IN OSTEOARTHRITIS OF THE KNEE: ARTHROSCOPIC CORRELATION. Carlos Acebes, Angel Robledo, Juan J Granizo, Gabriel Herrero-Beaumont Madrid, Spain

A PROSPECTIVE COMPARISON OF THE COSTS OF ILLNESS AMONG PATIENTS WITH RA, OA OR HYPERTENSION. Andreas Maetzel, Linda C Li, James Pencharz, Lyn Maguire, Claire Bombardier, The CHAP Team Toronto, ON, Canada

Arthroscopic evaluation (chondroscopy) of the articular cartilage is considered a relevant instrument of osteoarthritis (OA) assessment for research purposes. Nevertheless, availability of other accurate, but non-invasive methods for chondral evaluation in OA, such as MRI, would be necessary. Objective: To evaluate the validity and the reliability of fat-suppressed spoiled gradient-echo sequences (SPGR) of magnetic resonance imaging for quantification of articular cartilage in OA of the knee comparing with arthroscopic evaluation. Patients and methods: Twenty-four consecutive patients diagnosed of knee OA were referred for knee articular MRI of medial condyle and tibial plateau with fat-suppressed SPGR sequences before arthroscopic examination. MRI and arthroscopically evaluated lesions were registered in a grading scheme and total score of chondropathy was calculated using a planimetric software (autoCAD) by the two methods. Spearman test was used for calculating correlation for both evaluations. Results: Average arthroscopic and MRI scores were respectively 603,44 ⫾ 210,04 and 254,87 ⫾ 105,89. Significant correlation between arthroscopic score and MRI score for medial condyle (r⫽0,781) and tibial plateau (r⫽0,531) was observed. Intraobserver reliability of the arthroscopic score for condyle and tibia (r⫽0,806 and r⫽0,721 respectively) was similar to MRI score (r⫽0,781 and r⫽0,531 respectively). Conclusion: Quantification of chondropathy with fat-suppressed SPGR magnetic resonance imaging is feasible and well correlated with arthroscopic evaluation and seems to be a relevant non-invasive instrument to follow up cartilage lesions in OA of the knee. Disclosure: Financial support by a Grant of the Spanish Health Institute FIS 98/0626).

Purpose: To prospectively collect and compare direct and indirect costs of illness among patients with RA, OA or high blood pressure (HBP) recruited consecutively from family physicians’ and rheumatologists’ practices in Ontario. Methods: Consecutive patients were recruited by 53 rheumatologists (RA) and 76 family physicians (OA & HBP). All patients were interviewed at baseline and 3 months. Information on demographics, generic and disease-specific health status, and presence of comorbidities was collected. Patients completed a detailed, open-ended resource utilization questionnaire which inquired, at baseline and 3 months, about the type and intensity of visits to health professionals, type and number of tests, reasons for day surgeries and hospitalizations, use of community services, use of medications, patients’ time lost from work and performing chores, and expenses incurred covering lost time. Results: 292 patients with RA and 541 patients with OA or HBP were recruited, of which 253 (86.7%) and 470 (86.9%, OA:328 HBP:142), respectively, completed the baseline and 3-month interviews. Patients with RA incurred total semi-annual disease costs of $4,403CAD; higher than previously reported and exceeding HBP costs by more than 60%. Indirect costs estimated for RA exceeded those of OA and HBP by 70% and 80% respectively. Female patients incurred up to 6 times higer indirect costs, while direct costs were slightly higher for male patients. Semi-annual costs $1999 CAD: Mean (SD)

RA (nⴝ270)

OA (nⴝ328)

HBP (nⴝ142)

Direct Costs Hospital stays Health Professionals Test and Procedures Medications Indirect costs Total semi-annual costs

1, 899 (1,496) 214 (827) 457 (520) 230 (139) 998 (744) 2,504 (3,360) 4,403 (3,977)

1, 389 (1,624) 271 (1159) 291 (323) 92 (119) 735 (961) 742 (1,824) 2,131 (2,811)

1, 252 (1,813) 256 (1436) 253 (288) 81 (186) 662 (873) 508 (1,887) 1,760 (2,765)

Conclusion:This detailed prospective evaluation of semi-annual costs among patients with RA, OR or HBP showed that disease costs attributable to RA exceed those incurred by HBP by approximately 60%. Female patients incurred considerably higher indirect costs, in particular female patients with RA. Disclosure: Financial support was received from Merck & Co., Inc. and Merck Frosst Canada

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KOREAN COST-EFFECTIVENESS ANALYSIS OF NSAIDS, NSAIDS WITH CONCOMITANT THERAPY TO PREVENT GASTROINTESTINAL TOXICITY, AND COX-2 SPECIFIC INHIBITORS IN THE TREATMENT OF RHEUMATOID ARTHRITIS. Hyung Ran Yun, Kwang Taek Oh, Tae-Hwan Kim, Sung-Soo Jung, Dae-Hyun Yoo, Seong Yoon Kim, Sang-Cheol Bae Seoul, Republic of Korea

COST-EFFECTIVENESS OF 102-WEEKS OF INFLIXIMAB FOR RHEUMATOID ARTHRITIS. John B Wong, Ferdinand C Breedveld, Josef S Smolen, Arthur F Kavanaugh, Piet L van Riel, Johanna MW Hazes, Cindy J Wong, Brian G Feagan, Desiree M van der Heijde Boston, MA; Leiden, Nijmegen and Rotterdam and Maastricht, Netherlands; Vienna, Austria; San Diego, CA; London, ON, Canada

Objective: To assess cost-effectiveness of nonsteroidal anti-inflammatory agents (NSAIDs), NSAIDs with concomitant therapy to prevent gastrointestinal (GI) toxicity, and COX-2 specific inhibitor (COX-2) in the treatment of RA in Korea. Methods: Markov (state-transition) models were used to simulate a cohort of rheuamtoid arthritis (RA) patients with 50 years of age, taking disease modifying antirheumatic drugs, low dose steroid (prednisone ⱕ 10mg/day) and one of the following strategies: 1) NSAIDs without prophylaxis, 2) NSAIDs with misoprostol, 3) NSAIDs with proton pump inhibitor (PPI), or 4) COX-2. The outcomes (Markov states) included dyspepsia, GI complications, acute hepatic failure, acute renal failure, and death. Data on incidence and consequences of adverse events from treatments were taken from the literature. Direct medical costs were measured in 2000 Korean Won and health effects were expressed as quality-adjusted life years (QALYs). Costs and health outcomes were discounted at a rate of 3% per year. Sensitivity analyses were undertaken. Results: Among the strategies to prevent GI toxicity, PPI was the best strategy in Korea. The incremental C/E (cost/effectiveness) ratio between PPI and no prophylaxis was 28,987x103Won/QALY (24,565$/QALY). It differed from the result of our US cost-effectiveness analysis which showed COX-2 was the best option in the US (Arthritis Rheum 43:S474, 2000). The sensitivity analyses using probabilities of GI complications, age, medical costs of adverse events, discount rate, and utility were robust, except cost of COX-2 and NSAIDs. Strategies

Cost (x 103 Won)

QALY

⌬C/E ratio (x 103 Won /QALY)

⌬C/E ratio ($ /QALY)

NSAIDs Misoprostol PPI COX-2

17,780 22,116 27,256 32,464

11.46 11.55 11.79 11.70

Extended dominated 28,987 Dominated

Extended dominated 24,565 Dominated

Conclusion: Although PPI is the best option among the strategies to prevent GI toxicity in Korea, the incremental C/E ratio between PPI and no prophylaxis is above 28,000x103Won/QALY (24,000$/QALY). Disclosure: This work was supported in part by the research fund of the Korean Ministry of Health and Welfare (2001).

Introduction: In rheumatoid arthritis patients who respond partially to methotrexate, 102 weeks of infliximab improves ACR responses and stabilizes radiographs. A prior study suggests that the absence of radiographic progression should improve the mean HAQ by 0.27 after 3 years. Purpose: To examine the cost-effectiveness of 102 weeks of infliximab including benefit from radiologic stabilization. Methods: We projected the 102-week results from the ATTRACT randomized trial of infliximab into lifetime economic and clinical outcomes using a Markov computer simulation model. Direct and indirect costs, quality of life and HAQ estimates were based on ATTRACT trial results, ARAMIS outcomes and published data. We modeled radiologic stabilization as a higher likelihood of maintaining the same HAQ group. Results were discounted using the standard 3% rate. Cost-effectiveness ratios below $50,000-100,000 were considered to be “costeffective.” Results : Intention to treat with infliximab for 102 weeks had a marginal costeffectiveness ratio of $38,200 per discounted quality-adjusted life year (DQALY) gained versus methotrexate alone. When considering prevention of HAQ progression because of radiographic stabilization, the marginal cost-effectiveness ratio of infliximab improved to $6,200. For decreased annual likelihood of HAQ progression by 1-10% (because of stabilized radiographs), the marginal cost-effectiveness of infliximab ranged from $22,500 to $2000 (direct costs only) per DQALY gained. For indirect costs equivalent to direct costs, infliximab was cost-saving compared to methotrexate alone when the rate of progression decreased by more than 4%. In the basecase, the absolute HAQ benefit from infliximab was 0.37 after 1 year and 0.10 after 4 years. If radiologic stabilization improved this HAQ benefit to 0.27 after 4 years, infliximab had a cost-effectiveness ratio of $5100 per DQALY gained (direct costs only) and was cost-saving (extending life and reducing costs) when including indirect costs that are equal to direct costs. Conclusions: Our results suggest that 102 weeks of infliximab for rheumatoid arthritis who respond partially to methotrexate should be cost-effective especially when considering additional benefit from radiographic stabilization. Disclosure: Grant support from Schering-Plough

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COSTS AND EFFECTS OF LEFLUNOMIDE IN THE UK. Gisela Kobelt, Peter Lindgren, Adam Young Speracedes, France; Uppsala, Sweden and St Albans, United Kingdom

IN RHEUMATOID ARTHRITIS (RA) UTILITY CALCULATIONS SHOULD BE DERIVED FROM EQ5D RATHER THAN SF-36 DUE TO FLOOR EFFECTS. Nick Bansback, Alan Brennan, Alan Reynolds, Pete Conway, Tony Hammond Sheffield, Yorkshire, Maidenhead, Berkshire, Maidenhead, Berks and Maidstone, Kent, United Kingdom

STUDY OBJECTIVES: This study investigated the cost-effectiveness of leflunomide (LEF) compared to methotrexate (MTX) and sulphasalazine (SSZ) in the UK. MATERIAL AND METHODS: A Markov model was constructed using health states defined by HAQ score. The model is based on a cohort of patients with recently diagnosed definite RA who were followed for up to 15 years at 9 rheumatology clinics in the UK. Annual HAQ scores for 916 patients were used for the modelling with a mean follow-up period of 7.8 years. Costs and quality of life in each HAQ state were calculated from a cross-sectional survey in a sub-sample of the ERAS cohort. The treatment effect was calculated based on clinical trials comparing LEF to MTX (one international trial, N⫽999, and one US trial, N⫽482) and to SSZ (one international trial, N⫽354). Transitions between health states for the first two years of treatment were calculated from the clinical trials, while the extrapolation beyond the trial was based on the ERAS cohort, using an ordered probit model. This makes it possible to predict transitions for patients with similar characteristics (age, time since disease onset) as in the trials. Separate analyses were performed for each trial, and all analyses were done over a ten-year timeframe. As is standard practice in the UK, costs and effects were discounted at 6% and 1.5% respectively. RESULTS: When compared to MTX, the two trials gave differing results. Using the US trial, LEF cost less and led to greater benefits (GBP 44,017 and 4.824 QALYs, compared to GBP 44,988 and 4.733 QALYs for MTX). In the case of the international trial this case was reversed (GBP 34,070 and 5.238 QALYs for MTX compared to GBP 36,351 and 5.139 QALYs for LEF). When compared to SSZ, the cost of using LEF was slightly lower (GBP 35,855 compared to GBP 36,731 for SSZ). Quality of life was higher, and the number of quality-adjusted life-years was 4.646 for LEF, compared to 4.492 for SSZ. This made LEF the dominant strategy. CONCLUSION: When comparing the use of LEF to MTX, the two trials give differing results. One possible reason for this is that MTX patients in the US trial were given folic acid, whereas in the international trial folate supplementation was not mandated. This may have reduced the effectiveness of MTX. In the UK, it is standard practice to use folic acid with MTX, and therefore the results from the US trial may be more relevant for the UK. Compared to SSZ, the use of LEF appears to be cost-saving in the UK.

Background: Calculation of QALYs requires availability of utility scores. In RA the SF-36 generic quality of life (QOL) scale has been widely used, particularly in recent clinical trials. This questionnaire was developed for use in the general population but there is evidence that it may exhibit floor effects in RA patients (Ruta et al. Brit J Rheum. 37: 425-436, 1998). An algorithm to calculate utility scores has been developed by Brazier. An alternative instrument is the EuroQoL (EQ-5D) which was specifically developed to derive utilities in subjects with illness. Objectives: This study was designed to compare the utility scores obtained using the SF-36 and EQ-5D in a sample of 156 RA patients attending an hospital outpatient clinic. Additionally the relationship between the disease specific Health Assessment Questionnaire (HAQ) and the generic instruments was explored. Methods: All RA patients currently attending a hospital DMARD monitoring clinic were audited to determine their eligibility for anti-TNF␣ threatment, according to the British Society for Rheumatology guidelines, could be assessed. As part of their assessment patients were asked to complete the HAQ, SF-36 and EQ-5D. Results: The intercept and slope of the regression analysis of SF-36 and HAQ data were 0.7583 and -0.1136 respectively with an adjusted R squared value of 0.4810. This contrasts with intercept, slope and R squared values of 0.8285, -0.2344 and 0.3567 for the relationship between EQ-5D and HAQ. When utilities derived from the two generic instruments were compared it was evident that there was a significant floor effect with the SF-36. In the majority of patients with EQ5-D utility scores of 0.3 or below, the SF-36 derived values were 0.4 and above.

Conclusions: In RA, the use of the SF-36 appears to be insensitive to differences between disability states, particularly for the more severely affected patients. The EQ-5D is the preferred instrument for economic analyses in RA.

Disclosure: This work was funded by a grant from Aventis Pharma.

Disclosure: AVR and PC are employees of Wyeth Laboratories. TH was in receipt of an unrestricted grant from Wyeth Laboratories and the analyses undertaken by ScHARR were funded by Wyeth.

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COST EFFECTIVENESS OF CYCLOSPORINE TREATMENT IN SUBJECTS WITH EARLY ACTIVE RHEUMATOID ARTHRITIS. Lorenzo G Mantovani, Andrea Belisari, Roberto Ferrara, Ornella Della Casa Alberighi, For the GRISAR Group Milan and Origgio (VA), Italy

PATIENT TREATMENT PREFERENCES IN RHEUMATOID ARTHRITIS (RA). Liana Fraenkel, Sidney T Bogardus, David T Felson, Dick R Wittink New Haven and West Haven, CT and Boston, MA

Disclosure: Dr. Mantovani and Dr. Belisari have been consultants for Novartis Farma in several occasions and different fields including Rheumatic Diseases, Hypertension and Diabetes. The Center of Pharmacoeconomics has received research and educational grants from Novartis to perform research and education on Alzheimer disease and Oncology. Dr. Mantovani fellowship with the University of Milan has been financed with an unrestricted research grant of Novartis in the field of oncology. Dr. Mantovani and Dr.Belisari attendance to various Congresses has been financed with educational grants from Novartis. Dr. Della Casa and Dr. Ferrara are Novartis employees.

Multiple treatments are currently available for patients with RA. In this study, we used adaptive conjoint analysis (ACA), a validated interactive computer program, to rank patient preferences for methotrexate, gold, leflunomide, and etanercept, and to examine how varying benefit and co-pays influence treatment preference. We ascertained patient values (i.e. utilities) for 17 DMARD characteristics including amount of physician experience, route of administration, benefit (chance and onset of clinical and functional benefit, prevention of bone erosions), 11 adverse effects, and co-pays. Characteristics were decribed in lay terminology adapted from patient information sheets published by the Arthritis Foundation. ACA estimates utilities for specific medication characteristics through a series of ranking and paired-comparison tasks. Preferences for defined products are subsequently estimated using simulation models, analogous to sensitivity analyses in decision models, based on the utilities derived from the ACA questionnaire. One hundred RA patients were interviewed; mean (⫾SD) age ⫽ 68⫾10 years; 89% female, 75% had some college education; 87% had insurance coverage for medications, 59% were currently using a DMARD; 40% rated health status as poor or very poor; Using the first choice model, which assumes that respondents prefer the option with the highest utility, 93% of the patients surveyed preferred etanercept over the other DMARDs when all options were assumed to have equal monthly co-pays. Simulations modeling equal efficacy for all options decreased the proportion of respondents preferring etanercept to 81%. Simulations increasing the co-pay of etanercept by $10 and $30 relative to the other medications decreased the percent of patients preferring etanercept to 81% and 77% respectively. Monthly costs for uninsured patients were not modeled. Using the share of preference model, which reflects strength of preference, etanercept received 62% of the preference shares, leflunomide 16%, gold 13% and methotrexate 9% when all options were considered to have equal co-pays. Using a well-validated tool, we found that most of the RA patients surveyed preferred etanercept over methotrexate, leflunomide and gold injections. The finding that the majority of patients preferred etanercept when all options were considered to be equally effective, suggests that patients are more willing to accept the risk of unknown long-term toxicity in order to avoid the known risk of adverse effects associated with established medications. Disclosure:

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Objective To assess the economic profile of Cyclosporine A (CsA) in patients with early active Rheumatoid Arthritis (RA). Methods Technique: incremental cost effectiveness analysis (CEA). Source: the GRISAR study, a long-term RCT of CsA vs conventional DMARDs (CD). Perspective: society. Costs: direct medical costs using Italian National Health Service tariffs and indirect costs using the human capital approach. Costs are in US$ of year 2000. Effectiveness: Progression in the Damage Score (PDS), according to the Larsen-Dale method. Results 248 early active RA patients (F/M 196/52; mean age 48.7⫾11.9 yrs) were considered. The mean follow-up duration was 3.85 years. The main differences detected were: drug cost (10,490 CsA vs 1,105 CD, p⬍0.05), indirect costs (1,540 CsA vs 4,320 CD), average total cost (14,185 CsA vs 7,490 CD, p⬍0.05), average PDS (6.1 CsA vs 12.1 CD, p⬍0.05). The incremental cost effectiveness ratio (ICER) showed that the progression of one point in the damage score can be prevented at the cost of 1,115 US$ in a period of 3.85 years with the use of CsA vs. CD. 11/140 (7.8%) subjects in the CD group and 3/108 (2.7%) in the CsA group stopped their working activity because of RA (OR 3; 95% CI: 0.8-10.9; P⫽0.1 by Fisher exact test). DiscussionThis is one of the first prospective CEAs based on a long-term, randomised clinical trial with a pragmatic design in early RA. CsA treatment appears to reduce indirect costs by reducing the incidence of work abandon in relatively young subjects. When used in early active RA patients, CsA has favourable pharmacoeconomic profile as incremental effectiveness can be achieved at a reasonable incremental cost.

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UNDERSTANDING PATIENT PREFERENCES FOR THE TREATMENT OF LUPUS NEPHRITIS (LN) WITH ADAPTIVE CONJOINT ANALYSIS (ACA). Liana Fraenkel, Sidney T Bogardus, Dick R Wittink New Haven and West Haven, CT

HOW MUCH IMPROVEMENT IN RENAL SURVIVAL DO LUPUS PATIENTS REQUIRE BEFORE CHOOSING CYCLOPHOSPHAMIDE (CTX) OVER AZATHIOPRINE (AZA)? Liana Fraenkel, Sidney Bogardus, John Concato New Haven and West Haven, CT

Treatment for LN consists of corticosteroids in combination with cytotoxic therapy, most commonly cyclophosphamide (CTX) or azathioprine (AZA). CTX, although more effective is also more toxic than AZA. The purpose of this study was to evaluate patient treatment preferences for LN using ACA, a well-validated tool used extensively in market research. We used ACA to derive patient values (i.e. utilities) for nine medication characteristics using probability estimates reported in randomized controlled trials. We assumed that IV pulse CTX inferred the same risk of cancer as AZA. We calculated the relative impact of each characteristic on treatment preference, and ran simulations using ACA to estimate the percentage of patients preferring CTX under different risk-benefit scenarios. We interviewed 103 women with lupus. Of the medication characteristics studied, efficacy and risk of infection had the greatest impact on choice, each accounting for 20% of the variation in preferences. A 50% increase in the risk of reversible hair loss had the same impact on preference as the much rarer risk of cancer. Risk of zoster and nausea had a moderate impact on choice (each accounting for almost 10% of the variation in preferences), whereas the risk of hemorrhagic cystitis and oral ulcers had little influence on respondents’ decisions. For the base-case scenario, which modeled preferences for the maximum renal survival advantage of CTX over AZA and a low probability of adverse effects, premenopausal women wanting more children were less likely to choose CTX compared to their counterparts (56% versus 80%, p⫽0.04). Only in the hypothetical situation where CTX carried no additional risk of infertility were women wanting more children as likely to choose this drug as women not wanting more children. Modest changes in the probability of renal survival or major toxicity lowered the percentage of women preferring CTX by more than 20%, regardless of their desire for more children. The percentage of postmenopausal women preferring CTX was similar to that of premenopausal women not wanting more children across all simulations, thus supporting the validity of the questionnaire. The women surveyed in this study considered differences in the risk of infection equally as important as differences in the probability of renal survival. Our results suggest that without a substantial renal survival advantage, many women, especially those wanting more children, might refuse CTX in favor of a less effective, but less toxic therapy for LN.

The current standard of care for lupus nephritis includes high dose steroids in combination with cytotoxic therapy, most commonly CTX or AZA. CTX is more effective, but also more toxic than AZA. The objective of this study was to examine the amount of improvement in renal survival that lupus patients require before choosing CTX over AZA using a validated probability trade-off task. Consecutive lupus patients were presented with two medication descriptions, labeled A and B, representing CTX and AZA respectively. Each medication was described in terms of its route of administration, amount of monitoring required, and adverse effects. The content of the treatment descriptions was derived from randomized controlled trials (RCTs) and written using lay terminology. We described outcomes in terms of the number of people with “kidneys that work well” as well as the corresponding number of patients who “need to be on dialysis” to decrease framing bias. Renal survival probabilities were displayed using a 2⬘7⬘⬘ VAS scale ranging from 0 to 100. After reviewing the treatment descriptions, subjects were asked to indicate their preferred choice if both conferred an equal probability of renal survival. Strength of preference was assessed by systematically increasing the renal survival of the treatment not initially chosen until choice of medication switched. Treatment preferences were elicited for both 5 and 15-year renal survival. Ninety-three women were interviewed; mean⫾SD age ⫽ 40⫾7 years; 83% Caucasian; 62% college graduates. All but two participants initially chose AZA over CTX when both options were presented as equally effective. Of the 91 preferring AZA at baseline, 31% refused to switch to CTX for improved short-term renal survival, and 15% refused to switch from AZA to CTX for improved long-term renal survival, even when CTX conferred 100% renal survival. Of those initially preferring AZA, 44% demanded better short-term renal survival and 33% demanded better long-term renal survival than that actually conferred by CTX in RCTs before switching preferences. Higher education was positively associated with preference for CTX [adjusted OR (95% CI) ⫽ 3.7 (1.4-9.4)]. Age, duration of lupus, and self-reported health status were not associated with treatment preference. Although the majority of patients switched preferences to CTX for better renal survival, a substantial minority is unwilling to accept the toxicity associated with CTX even if it is much better than AZA at preventing renal failure.

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PATIENT PREFERENCES FOR TREATMENT OF KNEE OSTEOARTHRITIS (OA). Liana Fraenkel, Sidney T Bogardus, David T Felson, Dick R Wittink New Haven and West Haven, CT and Boston, MA

IMPACT OF KNOWLEDGE OF ARTHRITIS DIAGNOSIS ON THE UTILITY RATINGS OF FUNCTIONAL STATES. Maria E Suarez-Almazor, Barbara Conner-Spady, Mark Kuebeler Houston, TX and Houston, AB, Canada

There is an ongoing debate in the literature regarding which medications are best for knee OA. The objective of this study was to use adaptive conjoint analysis (ACA) to examine patient preferences for certain treatment options in knee OA (NSAIDs, COX-2 inhibitors, opioids, glucosamine, and capsaicin). Using ACA, we ascertained patient values (i.e. utilities) for seven medication characteristics including response rate, label (natural supplement, over the counter, prescription), common adverse effects, risk of ulcer, time to benefit, and monthly out-of-pocket costs. We then ran simulations using ACA, analogous to sensitivity analyses in decision models, to examine how varying efficacy and cost influenced treatment preference. Sixty patients were interviewed; mean (⫾SD) age ⫽ 71⫾7; mean (⫾SD) duration of knee OA ⫽ 12⫾11 years; health status ⫽ very well or well in 55%; 90% had a prescription drug plan; 80% were classified as prefering an active or collaborative role in medical decision-making. Assuming no prescription drug plan, 78% preferred capsaicin when all options were modeled to be equally effective, 13% preferred COX-2 inhibitors, 3% glucosamine, 3% opioids, and 2% NSAIDs. When capsaicin was modeled to be half as effective as the other options, 27% preferred capsaicin, 3% COX-2 inhibitors, 48% glucosamine, 17% opioids, and 5% NSAIDs. Increasing the response rate of NSAIDs and COX-2 inhibitors by 50% over the other options increased the percentage of respondents choosing NSAIDs and COX-2 inhibitors to 10% and 22% respectively. Assuming a $10.00 monthly co-pay for prescription drugs; 58% of respondents preferred capsaicin when all options were modeled to be equally effective, 28% preferred COX-2 inhibitors, 3% glucosamine, 10% opioids, and 0% NSAIDs. When capsaicin was modeled to be half as effective as the other options, 38% of respondents chose COX-2 inhibitors and 33% chose capsaicin. Increasing the response rate of NSAIDs and COX-2 inhibitors by 50% over the other options increased the percentage of respondents choosing NSAIDs and COX-2 inhibitors to 25% and 53% respectively. In summary, patient preferences for knee OA treatments are sensitive to variations in costs and probability of benefit. Without a prescription drug plan, a minority of patients choose COX-2 inhibitors, regardless of whether they were modeled to be more effective than other options. With a prescription drug plan, COX-2 inhibitors become the leading choice among patients with knee OA, assuming they are more effective than capsaicin.

OBJECTIVE: One of the proposed advantages of utility measures is that they can provide ratings on similar functional states across diseases without the need to use other natural measures that are often disease-specific. The purpose of this study was to evaluate the impact that knowledge about an underlying disease status may have on the ratings of functional health states using different preference-based elicitation techniques. METHODS: 43 health professionals were asked to rate 2 different scenarios of patients with arthritis: one mild and the other one severe. Both health states were based on the domains of the EQ5-D, an instrument commonly used to obtain utilities. Both scenarios described different levels of pain, functional abilities and psychological status. For each of the states, the health professionals were asked to give ratings assuming two different diagnoses: a)that the described patient had rheumatoid arthritis (RA), and b) that the patient had osteoarthritis (OA). Utility elicitation methods included visual analogue scales (VAS), time trade-off (TTO) and standard gamble (SG). RESULTS: Statistically significant differences were observed in the ratings of the scenarios depending on whether they described a patient with RA or OA. Mean utilities were higher for patients with OA compared to RA for both mild and severe scenarios, and for VAS and TTO methods (no differences were observed for SG). Individual differences in utilities comparing the same scenarios for RA and OA ranged between -0.15 and 0.35, and were larger for the scenario depicting severe disease CONCLUSIONS: These findings suggest that utility ratings of descriptions of generic functional states may not be appropriate to compare patients with different types of arthritis since knowledge of the diagnosis influences the ratings. Disclosure:


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UNDERSTANDING HOW RHEUMATOID ARTHRITIS (RA) PATIENTS VALUE THE RISKS AND BENEFITS ASSOCIATED WITH DMARDS USING ADAPTIVE CONJOINT ANALYSIS (ACA). Liana Fraenkel, Sidney T Bogardus, Dick R Wittink New Haven and West Haven, CT

ECONOMIC CONSEQUENCES OF THE PROGRESSION OF RHEUMATOID ARTHRITIS IN SWEDEN AND THE UNITED KINGDOM. Gisela Kobelt, Linus Jonsson, Peter Lindgren, Kerstin Eberhardt, Adam Young Speracedes, France; Uppsala and Lund, Sweden and St Albans, United Kingdom

Therapeutic decisions for RA involve complex trade-offs between the risks and benefits of multiple treatment options. The objective of this study was to use ACA, a well-validated tool, to evaluate how RA patients value the competing risks and benefits associated with DMARDs. We ascertained patient values (i.e. utilities) for specific medication characteristics including amount of physician experience, route of administration, benefit (chance and onset of clinical and functional benefit, prevention of bone erosions), and adverse effects. Changes in utilities reflect the value respondents associate with changes in specific medication characteristics. One hundred RA patients were interviewed; mean(⫾SD) age ⫽ 68⫾10 years; 89% were female, 75% had some college education; 40% felt that their arthritis related health status was poor or very poor. Average gains in utility units for improved benefit or reduced risk of toxicity are displayed in the figure below. On average, patients valued a reduction in the risk of toxicity more than an increase chance of benefit. For example, based on the utilities generated by ACA, respondents placed a much greater increase in value for a reduced risk pneumonitis, liver damage, and diarrhea (gain of more than 80 utility units for each) than they did for a 25% increased chance of responding to treatment (gain of 50 utility units), not developing erosions (45 units) or faster onset of response (gain of 50 utility units). Patients placed relatively little value on amount of physician experience with the medication. These results are in keeping with treatment preference studies in other patient populations suggesting that patient treatment preferences are influenced more by their perceived risk of toxicity than their perceived benefit.

STUDY OBJECTIVES: In view of the current debate regarding the efficient use of health care resources, it is important to estimate the long-term consequences of treatments in chronic diseases. We developed two simulation models (Markov models) for analysis of the cost-effectiveness of new treatments that affect the progression of rheumatoid arthritis. MATERIAL AND METHODS: We used data from two cohorts of patients followed since onset of the disease for up to 15 years. The Swedish study followed 183 patients at the Rheumatology unit at the Lund University Hospital for a mean of 11.3 years. The UK study (ERAS) followed 916 patients at 9 rheumatology clinics across the country for a mean of 7.8 years. Mean age at disease onset was 51.4 in Sweden and 54.1 years in the UK. Mean HAQ score at inclusion was 0.93 and 1.11 in Sweden and the UK respectively. Patients were grouped into six states according to HAQ at baseline and disease progression is modelled as annual transitions between states over 10 years. An ordered probit model was used to estimate transition probabilities conditional upon HAQ states, age, gender and time since onset of disease, to allow simulating different patient cohorts. Costs were estimated in Sweden from the cohort study, in the UK from a cross-sectional survey of a sub-sample of patients. Quality of life was estimated in both countries from cross-sectional surveys. RESULTS: Costs increase and quality of life decreases as the disease progresses. In the less severe states, costs are similar in the two countries, but costs in Sweden are higher in the more severe states, ranging from 780 $ to 24700 $ per patient and year, compared to 700 $ to 14200 $ in the UK. Cumulative costs over 10 years for patients starting in state 1 are 64 632 $ in Sweden and 39 529 $ in the UK. The cumulative number of quality-adjusted life-years (QALYs) is 5.145 and 5.501 respectively. Both costs and QALYs are discounted with 3%. CONCLUSION: The two models based on two different patient cohorts come to a similar conclusion in terms of the effect on quality of life (QALYs) over 10 years. Thus, they appear to accurately capture the effect of disease progression and can therefore be useful to estimate the effectiveness of new treatments in RA. Costs depend on country specific treatment patterns and cannot be directly compared.

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MEDICAL COSTS OF CARE IN PATIENTS WITH OSTEOARTHRITIS (OA) OF THE KNEE ARE STRONGLY PREDICTED BY PATIENT DISABILITY: A PROSPECTIVE STUDY OF 1,468 PATIENTS OVER 18 YEARS. Gurkirpal Singh, Bonnie Bruce, Tyson Hillard Holmes Stanford, CA Background: Osteoarthritis of the knee is one of the most common causes of disability and functional impairment in the US. There are few large longitudinal cohort studies that have examined long-term outcomes and costs in patients with OA of the knee. Objectives: To study long-term outcomes and costs of care in patients with OA of the knees. Methods: We studied 1,478 consecutively-enrolled patients with OA of the knee from four ARAMIS centers, followed for a total of 3,656 patients years of observation (mean follow-up 2.6 years). Data are based on semi-annual validated self-reports on functional status (HAQ disability index, VAS pain and patient global health assessment scales), health care resource utilization, and a review of hospital discharge summaries, procedures notes, and bills. Medical costs were computed as units of service multiplied by uniform costs (rather than charge). Medicare reimbursement rates were used to approximate costs for physician services; wholesale prices were used for medications. Results: At study entry, the patients were on an average 65 years old, 92% white, and had a mean disease duration of 15.1 years. The mean HAQ disability index (DI) was 0.87 (0-3 scale, where 0 is no disability and 3 is completely disabled). The average pain scale was 1.31 (0-3 scale, where zero is no pain and 3 is severe pain). Over the course of the study, within patients the DI increased by an average of 0.04 units per year, while the pain scale increased by an average of 0.01 units per year. The average annual direct costs (? standard error of mean) of medical care for patients with OA were $2,175 ⫾98, expressed in inflation-adjusted year 2000 dollars. Of these, the largest proportion (49%) was spent on surgical hospitalizations ($1,067 ⫾ 82]. There was a strong correlation between costs of care and patient’s DI, after controlling for covariates such as age, gender, and disease duration. For every unit increase in disability index, the annual costs increased by $976⫾169. Conclusion: OA of the knee is a progressive illness with substantial health care resource utilization. The direct medical costs of care are strongly correlated with disability levels. Since the prevalence of this condition is high and continues to increase, there are significant cost implications.

PREDICTING LONG-TERM HEALTH CARE COSTS AND HEALTH OUTCOME IN SLE. Ann Clarke, Michelle Petri, Susan Manzi, David Isenberg, Caroline Gordon, Jean-Luc Senecal, Yvan St. Pierre, John Penrod, Paul Fortin, Nurhan Sutcliffe, Jean Richard Goulet, Denis Choquette, Tamara Grodzicky Montreal, QC and Toronto, ON, Canada; Baltimore, MD; Pittsburgh, PA; London and Birmingham, England United Kingdom Aim: Health resource consumption and health status in SLE in 3 countries with different health funding structures were compared. Based on these data, we developed models to predict cumulative costs and health outcome for patients from each coutnry. Methods: 713 patients (Canada ⫽ 229, US ⫽ 269, UK ⫽ 215) were enrolled in a 4-yr study on health resource utilization and health status. We performed between country comparisons of 1) cumulative health care expenditure (expressed in 1997 Canadian $) and 2) SLE disease damage (SLICC/ACR DI) after 4 years of patient observation. Seemingly unrelated regressions were used for these 2 outcomes with an indicator variable for the country where the patient was receiving care with the US as the reference country. Study entry values of age, sex, race, education, marital status, disease duration, disease activity (SLAM-R and physician VAS), disease damage, health status (SF-36 subscales and patient VAS), social support, and patient satisfaction with health care were used as potential covariates. To develop predictions of either costs or damage, specific values of the model predictor variables were multiplied by their respective coefficients and the respective country coefficient was added. Results: At present, complete 4-yr data are available on 483 patients (Canada ⫽ 152, US ⫽ 167, UK ⫽ 164). 43 patients died (Canada ⫽ 15, US ⫽ 18, UK ⫽ 10), 63 withdrew (Canada ⫽ 37, US ⫽ 13, UK ⫽ 13), 51 were lost to F/U (Canada ⫽ 5, US ⫽ 34, UK ⫽ 12), and 73 have not yet completed the study (Canada ⫽ 20, US ⫽ 37, UK ⫽ 16). The following table shows a set of predictions based on a sample of actual patients’ characteristics. 4yr cum costs (103 $)

Baseline Values SF-36 GH* / PT VAS / SLICC 92 / 0.9 / 0 77 / 2.8 / 0 87 / 8.4 / 1 30 / 3.1 / 2 30 / 6.4 / 3 5 / 0.9 / 7 *GH ⫽ general health subscale of SF-36

CAN 6.4 6.9 14.7 22.4 27.9 44.9

US 7.0 7.6 16.3 24.8 30.8 49.6

SLICC at 4yr F/U UK 6.7 7.2 15.5 23.6 29.4 47.3

CAN 0.1 0.4 1.6 2.5 3.9 7.6

US 0.3 0.6 1.8 2.7 4.1 7.8

UK 0.1 0.3 1.6 2.4 3.9 7.6

Conclusion: There is substantial variability in costs and damage accumulation across SLE cohorts. We have developed a model incorporating health status, activity, and damage that can be used to predict long-term outcomes dependent on the country of care. Disclosure: This work has been supported by the Arthritis Society of Canada and the Fonds de la recherche en sante´ du Que´bec.

Disclosure: Research Support from Wyeth-Ayerst

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INDIRECT COST (IC) OF MUSCULO-SKELETAL DISEASE (MSD): A SENSITIVITY ANALYSIS. Yvan St-Pierre, Ann E Clarke, Viviane Adam, Bruno Fautrel, John R Penrod Montreal, QC, Canada

INDIRECT AND TOTAL COSTS OF EARLY RHEUMATOID ARTHRITIS: A RANDOMIZED COMPARISON OF COMBINED STEP-DOWN PREDNISOLONE, METHOTREXATE AND SULFASALAZINE WITH SULFASALAZINE ALONE. Ingeborg BC Korthals-de Bos, Maurits W Van Tulder, Maarten Boers, Arco C Verhoeven, Herman J Ader, Jack Bibo, Annelies Boonen, Sjef MJP van der Linden

OBJECTIVES: Assessment of productivity losses due to illness remains plagued with theoretical, ethical and methodological difficulties. We examine the potential variation of estimates for these ICs with respect to MSD and other chronic disease (CD). Relevant issues included self-report of disease and limitation, accounting for unpaid work and alternative treatment of wage differentials. DATA AND METHODS: Two Statistics Canada population-based samples were used for this study: the 1998 General Social Survey (GSS Cycle 12, N⫽ 10,749), inquiring about minutes for each activity on a random day, and the National Population Health Survey 1995-96 (NPHS, N⫽210,377), asking about disability days in the past two weeks. Both distinguish 7 disease categories if causing activity limitation, but only the NPHS identifies diagnoses independently. Various methods were used, from costing only paid labour loss based on self-reported limitation and using age-sex specific wages (Method 1), to costing all disability days at average full-time working male daily earnings (Method 2). MSD was defined as either arthritis/rheumatism or back problems. RESULTS : Based on the GSS results, 6.8% of Canadians of working age declared being limited in their activities mainly because of MSD, and 8.8% mainly because of other CD. Valuation of MSD-related limitations using method 1 yielded IC of 9 billion CAN$, i.e. more than a third of all IC due to illness. When incorporating the value of unpaid labour in calculations (Method 1a), MSD-related IC was surprisingly lower. Based on the NPHS data, 23.6% of Canadians of working age reported being diagnosed for either arthritis or back problems, while 33.6% report other CD, and no MSD. However the former accounted for 49% of all disability days, including those of respondents without any CD. Method 2 estimates (based on NPHS data) indicate IC for MSD could be as high as 26.5 billion CAN$. Methods 2a and 2b estimate excess IC relative to a comparison group. Based on a comparison to healthy individuals (Method 2a), two-thirds of IC due to chronic illness are those of MSD patients. Finally, comparing to all non-MSD respondents, IC specifically related to MSD (Method 2b) is estimated at 16.2 billion CAN$. IC in billions of CAN$

Arthritis Back problem Other CD Total % of total from MSD

Method 1

Method 1a

Method 2

Method 2a

Method2b

2.7 6.3 15.9 24.8 36

2.1 5.6 16.9 24.6 31

17.1 9.4 18.4 54.4 49

13.3 7.2 10.4 30.9 66

10.4 5.8 16.2 -

CONCLUSION: Methodological issues as well as data limitations still impair our capacity to provide precise estimates of the true burden of illness on society. Nonetheless, MSD is responsible for a large portion of IC across all methods.

Statement of purpose The aim of this study was to calculate the indirect costs for patients with early rheumatoid arthritis within the COBRA trial. Analysis of the efficacy and direct costs of the treatments have already been reported; this abstract reports the impact of the indirect costs on the cost-effectiveness of both therapies. Methods 56-week multicenter randomized trial among early RA patients comparing intensive combination therapy (COBRA, n⫽76)): sulfasalazine, methotrexate (stopped at 40 weeks) and prednisolone (initially 60 mg/day, tapered in 6 weekly steps to 7.5 mg/day and stopped after 28 weeks) to sulfasalazine alone (n⫽ 79). COBRA had improved disease activity and physical function more effectively in the first 28 weeks, and persistently slowed radiographic progression (up to 4.5 years). Direct and indirect costs were measured (from a societal perspective) by means of cost diaries and interviews completed by patients during the combined treatment phase and the follow up phase of the trial, each lasting 28 weeks. Differences in costs between groups were evaluated by applying non-parametric bootstrapping techniques. Summary of Results In the first 28 weeks mean indirect costs per patient were US K$ 2.6 and K$ 3.6 for combined therapy and sulfasalazine therapy, respectively (p⫽0.09); Total (direct ⫹ indirect costs) costs were K$ 5.9 and K$ 7.9 (p⫽0.04). These differences disappeared in the second 28 weeks. For the total trial period the mean total costs per patient were K$ 10.3 and K$ 12.8, respectively (p⫽0.11). Sensitivity analyses confirmed robustness of the analyses Conclusions reached COBRA Combined therapy offered additional disease control (improvements in disease activity, physical function and rate of damage progression) and lower costs over and above that of sulfasalazine alone. Disclosure:

Disclosure: This work was supported by the Canadian Arthritis Network.

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THE DIRECT MEDICAL COSTS OF RHEUMATOID ARTHRITIS IN 1999-2000. Frederick Wolfe, Jodi Messer Wichita

A MULTICENTRIC STUDY OF ANNUAL COSTS OF KNEE OSTEOARTHRITIS IN ITALY. Gianni Leardini, Fausto Salaffi, Roberta Montanelli, Simone Gerzeli, Irene Colangelo, Bianca Canesi Venice, Ancona and Milan Italy

Variable

Women (95% CI)

Men (95% CI)

Hospitalization OP Visits Procedures Rheum visits Hand x-rays Drug costs

.13 [.12, .14] 7.55 [7.38, 6.63] 1.69 [1.62, 1.76] 2.60 [2.55, 2.65] .22 [.21, .24] $1764 [1700, 1828]

.14 [.13, .16] 6.37 [6.14, 6.60] 1.56 [1.43, 1.69] 2.40 [2.31, 2.48] .20 [.17, .22] $1673 [1556, 1790]

Disclosure: This project has received grant support from Amgen, Aventis, Bayer, Centocor, Merck, Pfizer, Pharmacia, Roche, Wyeth(Aus), and private donors.

Osteoarthritis (OA) is the most common joint disorder in Italy and throughout the world. Knee OA is especially common, it is a major cause of functional impairment, and results in extensive utilization of health care resources. In order to estimate the economic and social effects of the knee OA in Italian population, we studied retrospectively a cohort of 254 patients over a period of 12 months in 2000-2001. Twenty-nine rheumatological insitutes, from all over the country, took part in the study. It was used a bottom-up approach, analytically measuring pro capita consumptions. As direct costs we considered medical (diagnosis, therapies, and hospitalization) and non-medical costs (transport and paid aids). As indirect costs we calculated losses of productivity borne by patients and caregivers, and informal care provided by caregivers. With the aim to facilitate understanding of currencies, costs were valued in Euro. The deterioration of quality of life - intangible costs - was measured using MOS 36-items short-form Health Status Survey (SF-36), Lequesne Index, Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index, and Rating Scale (100⫽perfect health; 0⫽worst immaginable health state). Direct costs came to 934 Euro per patient a year: 209 Euro were spent on diagnostic proceedings (56% on visits and 44% on instrumental and laboratory tests), 146 Euro on therapies (58% on physiotherapy and 42% on drugs), 233 Euro on hospitalization, and 346 Euro on out of pocket costs (73% on salaries to assistants, 14% on trasport, and 13% on assistive devices). It is interesting to point out that at least 37% of cost are directly charged to patients. Indirect costs ensued almost 30% higher and came to 1236 Euro per patient a year: 31% due to production losses by patients, 60% due to informal care provided by primary caregivers, and 9% by other caregivers. Overall, intangible costs ensued high and total costs correlate with the worsening of quality of life and greatest disease severity. Disclosure: Work reported in this abstract was supported by the Rottapahrm Group

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Background: Estimation of the lifetime costs of RA depends on contemporary data, particularly in view of newer expensive therapies. In addition, the understanding of costs requires knowledge of the extent to which they are altered by disease duration and age. There are no studies that fulfill these requirements. In this project we use a large cohort to address such contemporary cost issues. Methods: 6,227 RA patients were surveyed during 4 consecutive 6 months periods over 8,688 patient years of observation. Hospitalization and utilization rates were determined from Poisson and linear generalized estimating equations. Rates are per patient per 6 month of observation. Results: The median age and disease duration was 60.8 and 10.5 years, respectively. Controlling for age and duration (Table), men had significantly more hospitalizations, but fewer overall outpatient visits, rheumatology visits, medical procedures, hand x-rays, and had lower drug costs. The percent of observations associated with treatments was MTX 53.6%, prednisone 39.5%, hydroxychloroquine 27.5%, leflunomide 11.3 %, etanercept 10.9%, sulfasalazine 8.2%, and infliximab 2.2%. Drug costs were $1,764 for women and $1,673 for men. Overall costs were $4,746 [4,607, 4,885]. Utilization and costs were associated with RA duration and age. For hospitalization, the incidence rate ratio (IRR) per 10 years for age and disease duration was 1.11 (1.05, 1.16) and 1.12 (1.05, 1.64) for women, and 1.31 (1.19, 1.45) and 1.13 (1.04, 1.24) for men. For medical visits each 10 year increase in age and duration was associated with a visit increase of 0.2 (-.08, .13) and .30 (.16, .44) for women, and .26 (.05, .48) and .22 (.003, .44) for men, respectively. Controlling for age and sex, RA costs increase by $670 (529, 810) for each 10 years of illness. Summary: RA costs increase only modestly ($60 per year) with duration of disease, and are similar for men and women. Costs are explained far better by functional loss and comorbidity than by age and duration.

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THERAPEUTIC DECISION-MAKING IN RHEUMATOID ARTHRITIS: FACTORS EMPLOYED BY PHYSICIANS IN TREATMENT CHOICES. Doruk Erkan, Yusuf Yazici, Theodore Fields, Melanie J Harrison, Stephen A Paget NY

GLUCOCORTICOID-INDUCED OSTEOPOROSIS - DEMOGRAPHIC ANALYSIS OF AT RISK PATIENTS, DIAGNOSIS, AND TREATMENT IN A RURAL PRIMARY CARE POPULATION. Eric D Newman, Craig Wood Danville, PA

Background: Physicians consider many factors in their choice of medications for RA. Despite a drug’s perceived effectiveness and safety, it may be avoided for financial or other reasons. Objective: To determine the level of importance physicians ascribe to different variables in the decision-making process regarding RA therapy. Methods: Physician members of the ACR were notified by mail of a survey regarding medication preferences for RA that was posted on our website. Using a 5-point numerical rating scale (1⫽not to 5⫽extremely important), physicians were asked to assign a level of importance to 7 variables(table) that may play a role in their medication selection. Mean values for the variables were calculated for each medication and compared for each of the following pairs: NSAIDs/Cox2 inhibitors; MTX/leflunomide(LEF); and etanercept(ETC)/infliximab(INF)(table). Results: 328 responses returned from 3/13/01-5/07/01 qualified for analysis. Each variable except AB scored ⬎2.5 for each medication. Mean scores for AB were higher for anti-TNFs, only. SEP was assigned the greatest importance for NSAIDs and Cox-2, and was significantly more important for NSAIDs(p⬍0.0001). Cost and SEP scored highest in importance for selection of LEF. SEP similarly scored the highest in importance for MTX, which was significantly less compared with LEF(p⬍0.05). Cost and reimbursement were both very and equally important in the selection of either anti-TNF agent. While availability was assigned the greatest level of importance for ETC, AB and SEP were significantly less important for ETC compared with INF.

Patients on chronic glucocorticoids (GC) are at significant risk for osteoporosis and fracture (glucocorticoid-induced osteoporosis or GIOP). Many of these patients are cared for by both specialists and primary care physicians (PCPs), and therefore the opportunity for routine testing and treatment should be higher. The purpose of this study was to examine the demographics, the use of bone density testing and the use of prescription osteoporosis medications in patients taking chronic GC who were seen in our rural health care system. Utilizing the electronic medical record, all patients 18 or older seen at 37 primary care sites between 1/1/98 and 12/31/99 were analyzed for chronic GC use (defined as ⬎ 6 months of GC during the sample time frame). Variables examined included basic demographics, concomitant diagnoses, type and dose of GC used, use of bone mineral density testing (BMD), prescription therapy, and specialty care. 1291 patients on chronic GC were seen with a mean age of 53⫾17 years (20-95). 58% were female. 80% of patients used prednisone as their GC. An ICD-9 code of osteoporosis was listed in 11%. The most common diagnoses were solid organ transplant (18%), COPD/bronchitis (18%), asthma (17%), dermatitis (15%), rheumatoid arthritis (7%), and polymyalgia rheumatica (5%). Only 15% of patients had a BMD before or after the start of GC during the sample time frame (4% before GC, 11% after GC). Patients seen by a specialist were more likely to have had a BMD (25% vs. 6%, p⬍0.001). Patients seen by nephrology, rheumatology, and dermatology had the highest BMD rates compared with patients seen by PCPs only (27% vs. 6%, p⬍0.001; 21% vs. 6%, p⬍0.001; 15% vs. 6%, p⫽0.048) Treatment included hormone replacement therapy (11%), bisphosphonates (6%), and calcitonin (2%). Patients who were seen by any specialist were not more likely to be on prescription osteoporosis therapy than those patients who had only seen a PCP (23% vs. 20%, p⫽.252). However, patients seen by rheumatology and dermatology had higher rates of prescription therapy use compared to patients seen by PCPs only (42% vs. 20%, p⬍0.001; 34% vs. 20%, p⫽0.032). The use of diagnostic testing in patients at risk for GIOP is low. Likelihood of treatment is highest in patients seen by rheumatology. Significant opportunity exists for continued focus on patients at risk for GIOP, to address the “ownership” of responsibility, understand the barriers, and improve awareness.

Variables

NSAIDs vs COX2

Cost of the medication (Cost) Administrative Burdens (AB) Availability Patient Preference Reimbursement Route of administration Side effect profile (SEP)

3.5 vs 3.8* 2.4 vs 2.5 3.9 vs 3.5* 3.8 vs 3.5* 3.6 vs 3.7 2.8 vs 2.6* 4.3 vs 4.1*

MTX vs LEF

ETC vs INF

2.7 vs 4.1* 2.0 vs 2.4* 3.0 vs 3.3 3.6 vs 3.5 2.6 vs 3.8* 3.0 vs 2.8* 3.9 vs 4.0*

4.6 vs 4.6 2.9 vs 3.4* 4.7 vs 3.7* 3.9 vs 3.9 4.5 vs 4.5 3.6 vs 4.0* 3.7 vs 3.9*

*:p⬍0.05 Conclusion: The final treatment choice in RA is based on a complex equation of factors, only one of which is effectiveness. Issues other than efficacy may dominate the decision-making process and lead to suboptimal prescribing of the most effective medications.

Disclosure: This project was supported by an unrestricted grant from Merck & Co., Inc.

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DIRECT COSTS IN DUTCH RHEUMATOID ARTHRITIS PATIENTS. S MM Verstappen, J WG Jacobs, E Buskens, H Verkleij, J WJ Bijlsma, On behalf of the Utrecht Arthritis Cohort Study Group Utrecht and Bilthoven, The Netherlands

DEVELOPMENT OF A PREDICTIVE MODEL AND A RISK ASSESMENT INSTRUMENT TO FACILITATE DIAGNOSIS OF LOW BONE MASS IN POSTMENOPAUSAL WOMEN. Wafa Ben Sedrine, Thierry Chevalier, Marie-Christine Micheletti, Bertrand Gelas, Jean-Yves Reginster Liege, Belgium; Montpellier, France; Monaco, Monaco and Liege, Belgium

The objective was to estimate total direct costs in Dutch rheumatoid arthritis (RA) patients. Economic consequences of a chronic disease like RA play an important role in decisions regarding new drug-therapies and changes in social security. To get an insight into these economic consequences we estimated the annual total direct costs. 615 RA-patients in the Utrecht region, the Netherlands, filled out a questionnaire. Costs were estimated for groups of patients with different disease duration (0-2, 2-6, 6-10, ⬎10 yr.). Total direct costs were the sum of seven predefined categories of costs. Demographic characteristics and mean (SD) total direct costs per year in Euro for the four groups with increasing disease duration are shown in the table.

mean age (SD) % female mean HAQ (SD) mean costs / yr. (SD)

0-2 (n⫽96)

2-6 (n⫽261)

6-10 (n⫽152)

⬎10 (n⫽106)

54 (15) 72 1.1 (0.7) 4943 (9638)

60 (14) 74 1.0 (0.7) 3760 (6706)

63 (13) 66 1.2 (0.8) 4484 (10937)

62 (12) 79 1.6 (0.8) 8098 (11841)

High mean direct costs in the first two yr. of the disease were mainly due to (expensive) admissions in care facilities and to (frequent) contacts with health care workers (29% and 30% resp. of total direct costs). High costs in the last group of patients (⬎ 10 yr.) were mainly due to hospitalisation, including operations, and purchase of devices (24% and 41% resp.). Dividing the total patient population into two groups, patients with high ⬎ [euro]4765 vs. low ⬍ [euro]4765 costs, revealed that disease duration and several clinical and psychological variables were significantly (p ⬍ 0.05) worse in the group of patients with high direct costs. These results show that economic consequences of RA are high during the whole course of the disease. However, per category the percentage of total direct costs is different within each stage of the disease.

Background : Although mass screening for osteoporosis is not recommended among postmenopausal women, its seems necessary to detect post menopausal women at risk of low bone density. Two predictive scales have been developped : the SCORE1 (Simple Calculated Osteoporosis Risk Estimation) and the ORAI2 (Osteoporosis Risk Assessment Instrument). We propose in this study a new predictive model and a new risk assessment instrument to facilitate diagnosis of low bone mass in postmenopausal women. Methods : Using a retrospective data base from the Department of Epidemiology and Public Health, University of Liege, Belgium, we identified post menopausal women by 65 dependants variables. All these women had a Bone Mineral Densitometry (BMD), by dual-energy x-ray absorptiometry (DXA) on 3 sites : hip, femoral neck and lumbar spine. The main outcome mesure was low BMD (T score of 2.5 or more standard deviations below the mean for young Belgian women) at either one of the 3 sites measured. Logistic regression analysis and receiver operating characteristic (ROC) analysis were used to identify the simplest algorithm that would identify women with low bone mass and to built a risk assessment instrument. Results : The study population comprised 1304 women (848 with a normal BMD, 456 with a low BMD). A simple algorithm based on age, weight, current HRT use, current or past HRT use, past medical history of minimal trauma fracture, was developped. A risk assessment instrument was then developped from these 5 variables. This instrument, with a cut off point fixed at 3, showed a sensitivity at 93.8% (95% CI 91.1%-95.8%), and a specificity at 38.5% (95% CI 35.6% - 41.4%). Conclusion : This instrument with 5 variables identifies the majority of women likely to have low bone mineral density at either one of the 3 following sites : hip, femoral neck and lumbar spine. These results have to be validated by a prospective study. References 1 - Lydick E and al. - Am J Manag Care 1998 ; 4 : 37-48 2 -Cadarette SM and al. - CMAJ 2000 ; 2 : 162-9

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TOTAL HIP AND KNEE JOINT REPLACEMENT SURGERY FOR RHEUMATOID ARTHRITIS - IS IT WORTH THE COST? Lyn M March, Kate L Tribe, Helen M Lapsley, Marita J Cross, Brett G Courtenay, Clarissa Bachmeier, Peter M Brooks St Leonards, Kensington, Darlinghurst, NSW, Australia; Brisbane, QLD, Australia and Switzerland

PREVENTIVE TREATMENT OPTIONS FOR OSTEOPOROSIS IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). Elizabeth C Hsia, Katrina Armstrong, Daniel A Albert Philadelphia, PA

This study aimed to determine out-of-pocket costs and health outcomes among RA patients having total hip (THR) and total knee (TKR) surgery. Patients completed questionnaires pre-op to record expenses in the previous 3 months and health status immediately prior to surgery. Patients kept detailed cost diaries and completed SF-36 and HAQ each 3 months for the first post-op year. 12 month follow-up data were available from 37 patients (78% TKR), 78% F, mean age 59 yrs, disease duration 21 yrs. This was 66% of possible RA surgical patients despite 4 years of recruitment. 3 months pre-op average out-of-pocket costs were AUS$314 with prescription medication, special equipment & community services the main components (each ⬃20%). Mean out-of-pocket costs did not decline over the post-op period with prescription medication the main ongoing cost (60%). Multiple regression identified female gender, poor physical function (higher HAQ), high pre-op expenditure and poor SF-36 emotional scores as significant determinants of these costs. There was a significant improvement from baseline in all SF-36 scores apart from general health over the first 12 months post-op however RA patients still fell well below age matched population values. This is in contrast with our previously reported OA patients who reported a decline in out-of-pocket costs and a highly significant improvement in health status post-op to match population levels. There was a statistically significant improvement in total HAQ score(pre-op: 1.60 vs post-op: 1.38 ;p⬍0.01) but the clinical significance may be questioned. while components of the HAQ (walking and rising) did improve by a clinically important amount (⬎0.3), these RA patients remained significantly more disabled than a community dwelling elderly population a decade older. When surveyed two years post-op, 96% were satisfied, 92% said they would have it again and 22% were willing to pay ⬎AUS$20,000 for the surgery. CONCLUSIONS: Frequency of joint replacement surgery for RA appears low. Costs to RA patients after surgery remain considerable and health status is generally poor, however satisfaction levels are high. Disclosure:

INTRODUCTION: Osteoporosis is a significant cause of morbidity and mortality in SLE patients. Treatment choice for prevention is affected by the risk of coronary heart disease (CHD), fracture (Fx), thromboembolism, and SLE disease activity. OBJECTIVE: To compare various treatments to prevent osteoporosis. METHODS: We constructed, using DATA 3.5 (TreeAge), a time-dependent Markov model that compared life expectancy (LE) from 4 preventive Rx options: 1) HRT; 2) raloxifene (RAL); 3) alendronate (ALEN); and 4) no Rx. Health outcomes included hip Fx, vertebral Fx, CHD, breast cancer (BrCa), thromboembolism, SLE flare, and death. The model simulated the life span of a hypothetical cohort of 50 year-old postmenopausal women with SLE. In the base-case analysis, we assumed that HRT and RAL had no effect on the risk of SLE flare, however we modeled propensity to cause SLE flare in sensitivity analyses. Further base-case assumptions were HRT reduced the risk of CHD (relative risk RR⫽0.56) and HRT increased BrCa risk (RR 1.35). HRT and ALEN were considered equally effective in reducing the risk of hip Fx (RR⫽0.53). RAL had only modest effects on CHD and hip Fx incidence (approximate RR⫽0.9), but reduced BrCa risk (RR⫽0.24). Both HRT and RAL increased the risk for thrombosis 2-3 fold. RESULTS: Results in LE (years). Optimal choice is bolded.

Base-case Sensitivity Analyses: HRT/RAL Effect on SLE flare RR⫽2.0 HRT/RAL Effect on CHD RR⫽1.0 HRT/RAL Effect on CHD RR⫽1.2 HRT/RAL Effect on Hip Fracture RR⫽1.0 HRT/RAL Effect on Thrombosis RR⫽8.0 Lifetime Breast Cancer Risk (base-case 10%) 20% Lifetime Breast Cancer Risk 80%

No Rx

HRT

Raloxifene

23.81

24.82

24.06

Alendronate 24.07

23.81 23.81 23.81 23.81 23.81 23.50 22.07

23.98 24.04 23.72 24.52 24.60 24.45 22.70

23.28 23.84 23.54 24.01 23.87 23.84 22.76

24.07 24.07 24.07 24.07 24.07 23.76 22.30

DISCUSSION: The gains in LE are equal, or greater than, gains from mammography and Pap smears. Under base-case conditions, HRT is most effective in extending LE, but this depends on whether HRT increases the risk of SLE flare and whether it reduces the risk of CHD. We will incorporate quality of life and cost-effectiveness to define the optimal choice. Disclosure:

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DOES HELICOBACTER PYLORI (Hp) INFLUENCE THE GASTRO-INTESTINAL(GI) TOLERABILITY OF NON STEROIDAL ANTI-INFLAMMATORY DRUGS(NSAIDs) ? Thierry Schaeverbeke, Nathalie Broutet, Franck Zerbib, Bernard Combe, Philippe Bertin, Francis Perie, M Joubert-Collin, Herve Lamouliatte, Francis Megraud Bordeaux, Montpellier, Limoges and Puteaux, France, Metropolitan

REPRESNTATIONAL CAPABILITY OF SNOMED-RT™FOR RHEUMATOLOGIC TERMS AND CONCEPTS ENCOUNTERED DURING INPATIENT RHEUMATOLOGY CONSULTATION SERVICES AT TERTIARY CARE TEACHING INSTITUTION. Alexander P Ruggieri, Dirk M Nuenninghoff, Kenneth J Warrington, Christopher G Chute Rochester, Minnesota

Hp has been identified as a major cause of gastritis and peptic ulcer. The bad GI tolerability of conventional NSAIDs is also widely documented. However, The influence of the infection by Hp on theGI tolerability of NSAIDs remains unknown. Objectives: 1) to determine the prevalence of GI symptoms in patients treated with NSAIDs whether or not they are infected by Hp, 2) to estimate the impact of Hp eradication on these symptoms. Methods : Multicentric, community based case-controlled study and randomised, placebo-controlled study. Inclusion of patients presenting with a degenerative articular disease motivating the prescription of an NSAID for at least 2 weeks. Stratification of the patients in two groups (Hp-positive and Hp-negative) by a serological rapid digital test. Randomisation of Hp-positive patients in two subgroups, receiving an eradication treatment (group 1), or a placebo (group 2). Patients not infected by Hp were included in group 3. Main outcome mesure: Prevalence of GI symptoms at week 2, 6 and 12. Results : 327 patients; group 1: n ⫽ 90; group 2: n ⫽ 92; group 3: n ⫽ 145. No significant differences were observed between group 3 and group 2 at W2 and W6, and only pyrosis was significantly more frequent in group 3 at W12 (p⫽0.02). Overall GI symptoms were significantly more frequent in group 1 than in group 2 and group 3 at W2 (p⫽0.02) ; this was mainly related to diarrhoea, main side effect of antibiotics (21.8 % vs 4.6 % in the group 2 and 5.6 % in the group 3). At W12, the prevalence of overall GI symptoms and pyrosis was the same in group 1 and group 3, and respectively the half and the quarter than that in group 2, although these differences did not reach the significance. Conclusion : 1) The GI tolerance of conventional NSAIDs does not differ whether or not patients were infected by Hp. 2) Systematic eradication of H. pylori does not seem to improve GI tolerance of NSAIDs for the short-term. 3) Medium-term observed tendency needs to be confirmed. 4) Such study should now be done for long-term treatments with Coxibs.

In an electronic medical record environment controlled healthcare terminologies can provide a consistent and standardized means to automatically link specific and detailed patient problems to clinical decision support systems, diagnosis-specific knowledge resources, and to index and aggregate specific clinical cases of interest for research and outcomes study. SNOMED-RT™(Standardized Nomenclature of Medicine)is a proprietary healthcare terminology proposed to serve these purposes. Comprehensive, complete, and robust conceptual content coverage for the rheumatology domain however must be demonstrated before a standardized terminology can be applied in rheumatic disease research or clinical care. Our purpose was to examine and test the representational capability of SNOMED-RT for rheumatologic terms and concepts. METHODS: Rheumatological terms from “working” clinical problem lists for inpatients on a Rheumatology consultation service in a teaching tertiary care hospital were recorded in an Excel™spreadsheet, and then electronically processed using the Mayo Vocabulary Processor, an automated tool that generates candidate matching terms using a beta version (June 2000)of SNOMED-RT. Conceptual matching by the candidate SNOMED terms was judged for each rheumatologically relevant clinical term by full concensus opinion of three rheumatologists. RESULTS: 325 unique clinical rheumatological terms were generated from 51 individual patient consultations. The clinical rheumatolgical terms covered categories of clinical symptoms, clinical signs, lab and radiographic findings, diagnoses, histopathology characterizations, procedures, and pathophysiologic processes. A candidate matching term was generated for every rheumatolgical term submitted. Rheumatological conceptual content capture was judged sufficient for 280 (86% p⬍.01)of the original 325 terms generated from the rheumatology-relevant clinical problem lists. CONCLUSIONS: For a corpus of clincal rheumatological terms generated from rheumatology consultations during a specified period in a tertiary care teaching hospital, SNOMED-RT™ was judged to provide considerable but less than complete conceptual content coverage. With appropriate augmentation SNOMED-RT™could become a promising standardized controlled clinical terminology to represent and index clinical information in the rheumatology domain. Disclosure:

Disclosure: This study was financially supported by Takeda

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DO RA PATIENTS IN REMISSION STILL HAVE SYNOVITIS ? - AN ULTRASOUND ASSESSMENT. Andrew K Brown, Mark A Quinn, Zunaid Karim, Richard J Wakefield, Philip G Conaghan, Philip J O’Connor, Paul Emery Leeds, West Yorkshire, United Kingdom

VALUES AND USEFULNESS OF SERUM PROCALCITONIN LEVEL IN INFLAMMATORY DISEASES. Isabelle Delevaux, Marc Andre, Michele Colombier, Rene-Jean Begue, Jean-Charles Piette, Olivier Aumaitre Clermont-Ferrand and Paris, France

Background: Treatment of RA is directed at suppressing inflammation with the aim of eliminating synovitis and establishing a state of remission. Remission is usually defined by the ACR Remission Criteria which is a composite measure containing several subjective and objective components, many of which can be influenced by other factors. Ultrasonography (US) is a safe, non-invasive technique which can provide a direct multi-planar assessment of the rheumatoid synovium and has been validated as a specific tool for the detection of synovitis. Aim: To evaluate patients with RA in ’remission’ as assessed by consultant rheumatologists, using the ACR Remission Criteria and US. Method: Consecutive patients with a diagnosis of RA and deemed to be in ‘remission’ by the assessing consultant rheumatologist were recruited. Each patient underwent a standard assessment including full metrology, measurement of RF and acute phase markers, and x-rays of hands and feet. Remission was calculated using the ACR Remission Criteria. US of the dominant hand MCP joints was recorded by a single, blinded sonographer using an ATL HDI 3000 machine with a 10-5 MHz linear array ‘hockey-stick’ transducer. For the purposes of this study, only the presence or absence of synovitis was recorded. Results: 41 patients have been recruited: mean age 59 years (range 23-87), 64% RF positive, 67% erosive on x-ray, mean disease duration 8 and a quarter years, mean number of DMARDs 2.2 and female:male ratio 2.9:1. 56% of patients deemed to be in ‘remission’ satisfied the ACR Remission Criteria. 71% of patients in ACR Remission had synovitis detectable on US whereas 82% of patients not in ACR Remission had US evidence of synovitis. Summary: US identified synovitis in the majority of patients who satisfied the ACR Remission Criteria. This may explain the discrepancy between clinical improvement and progression of joint damage in patients with RA. ‘Remission’ as assessed by a consultant rheumatologist may not be associated with ACR Remission. Whilst the ACR Remission Criteria select a population with a lower frequency of synovitis, they do not exclusively identify an absence of inflammation. Conclusion: Patients in remission as assessed by a consultant rheumatologist and the ACR Remission Criteria do still have synovitis. US assessment of the synovium may improve the accuracy of disease activity assessment in RA patients. Longitudinal studies are required to fully evaluate the clinical importance of US-detected synovitis in this patient group.

Procalcitonin (PCT) is now a well known tool for detection of systemic bacterial infections. On the opposite, PCT is generally not induced or slightly increased in autoimmune disorders. The values of serum PCT level remain unknown in many others inflammatory diseases. Objective: to determine if PCT may discriminate bacterial infections from various active inflammatory diseases. Methods: From October 1999 to January 2001, serum PCT (immunoluminometric assay, normal values ⬍ 0.5 ng/ml) was determined in 140 patients with fever and/or increased serum inflammatory markers. A flare of the inflammatory disease (either initial or relapsing) was diagnosed in 35 cases, and bacterial infections was diagnosed in 56 others. Results: - Inflammatory diseases (n⫽35), PCT ⬍ 0.5ng/ml: Still’s disease (n⫽4; CRP:78-313 mg/l), ulcerative colitis (n⫽9; CRP:0.9-77.5 mg/l), cristal arthritis (n⫽3; CRP:80-278 mg/l), sarcoidosis (n⫽1; CRP: 6.7 mg/l), connective tissue diseases (n⫽2 ; CRP: 5.4-10 mg/l), vasculitis (n⫽14; CRP:7.5-260 mg/l), amyloidosis (n⫽1; CRP: 8.5 mg/l), TRAPS (n⫽1, CRP:40.9 mg/l). - Bacterial infections (n⫽56, PCT:0.1-160.7 ng/ml, CRP:3-460 mg/l). Herein, a serum PCT level above 0.5ng/ml seems to be a good marker for bacterial infection, with a specificity of 98% and a sensibility of 67%. None patient with inflammatory disease had an increase of serum PCT in the absence of concomitant infection. Two patients, one with vasculitis and the other with rheumatoid arthritis, who both had pulmonary infection had an increase of PCT (0.9 and 1.7ng/ml). PCT appeared mostly useful to differentiate inflammation from infection in patients with Still’s disease or crystal arthritis, two conditions where CRP may be higher than 150 mg/l. Conclusion: in daily practice, PCT seems to be a useful marker both for the differential diagnosis between bacterial infection and inflammation, and for the detection of superimposed bacterial infection in patients with systemic inflammatory diseases.

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LONGITUDINAL EVALUATION OF MRI SCORING IN RHEUMATOID ARTHRITIS - AN INTERNATIONAL MULTICENTER STUDY OF INTERREADER AGREEMENT. Philip J O’Connor, Mikkel Ostergaard, Mette Klarlund, Charles Peterfy, Paul Astin, Desiree van der Heijde, Paul Emery Leeds, West Yorkshire, United Kingdom; Copenhagen, Denmark; San Francisco and Netherlands

RISK OF OVARIAN FAILURE AND FERTILITY AFTER INTRAVENOUS CYCLOPHOSPHAMIDE ARE INDEPENDENT OF THE UNDERLYING DISEASE. A RETROSPECTIVE STUDY IN 85 PATIENTS. Du Le Thi Huong, Zahir Amoura, Nathalie Costedoat-Chalumeau, Abdallah Sbai, Pierre Duhaut, Bertrand Wechsler, Jean-Charles Piette Paris, France

Background. The OMERACT MRI group have produced a possible scoring method for MRI abnormalities of synovium and bone in the small joints of rheumatoid arthritis (RA) patients. We wished to study the ability of this scoring method to reflect change in scores over time. Methods. Four readers from 3 international centres scored a total of 40 sets of MRI images of 4 metacarpophalangeal joints (MCPJ) of RA patients (20 sets of images pre- and post-treatment, read in a random order to avoid bias). Sequences: axial T1 pre- and post-gadolinium (Gd); coronal T1, T1FS post-Gd and T2FS. Scoring: synovitis global 0-3, bone erosions and bone edema were scored for the proximal and distal joint halves, using 10% volume estimates. Agreement between the scorers was assessed using multi-rater (non-weighted) kappa statistics. Results. Agreement on difference-over-time scores: kappa values for scoring synovitis ranged from 0.20 - 0.44 (fair to moderate range); for proximal bone erosions, good agreement was seen for the 2nd MCPJ but overall poor agreement (0.05 - 0.34); agreement on distal erosions was difficult due to low incidence of damage in the cohort. Agreement on extent of bone edema change was poor but there was good agreement on the direction of change (0.36-0.85). For analysis of images from the 2 time points, good agreement was seen for synovitis and bone erosions but not for bone edema. Conclusions. Synovitis scoring over time seems reproducible but the good agreement on direction of change suggests further evolution is required for scoring bone abnormalities. The improved cross-sectional statistics (compared with previous exercises) reflects increased training which should also have beneficial effects on future longitudinal scoring exercises.

Objective: To compare risk of ovarian failure and fertility in women treated with intravenous cyclophosphamide (IVCY) according to the underlying disease. Patients and methods: Retrospective review of the data of 85 consecutive women (56 with SLE, 29 with other diseases, mainly Wegener granulomatosis and diverse systemic vasculitides). Results: Mean age at IVCY initiation was 29 ⫾ 10 years (range: 13 to 53 years). Mean dosage was 0.9 ⫾ 0.14 g per pulse (range: 0.5 to 1) and mean number of pulses 13 ⫾ 6.5 (range: 3 to 42). With a mean follow-up of 5.1 ⫾ 3.7 years, 23 women developed amenorrhoea within 1 to 12 months, with a mean duration of 4 ⫾ 3.6 months between IVCY initiation and amenorrhoea. Amenorrhoea was sustained in 19 women (13 with SLE and 6 with other diseases, NS). Risk of ovarian failure correlated with age at IVCY institution (p⬍0.0001) and there was a non-significant trend towards an increased risk with higher number of pulses. Eighteen women (13 with SLE and 5 with other diseases) became pregnant under or after cyclophosphamide therapy totaling 22 pregnancies. The mean age at IVCY and the mean number of IVCY (maximum 40 pulses) before pregnancy were similar in women with SLE and with other diseases. Six pregnancies occurred during IVCY which ended in induced abortion (n⫽4), spontaneous abortion (n⫽1) and normal pregnancy after IVCY withdrawal in 2 women wishing to keep their pregnancy despite the risk of teratogenicity. Sixteen pregnancies occurred 2.9 ⫾ 2.1 years (range: 1 to 9) after IVCY withdrawal. Three ended in induced abortion for severe morphological anomaly (n⫽2) or SLE relapse (n⫽1), 3 in spontaneous miscarriages and 10 in livebirths of healthy newborns. Conclusion: Risk of ovarian failure depends essentially on age at IVCY initiation. Pregnancy may occur during IVCY therapy, therefore efficient contraception is mandatory. After IVCY withdrawal, pregnancy is possible with a favorable outcome in two-thirds of cases.

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RHEUMATOLOGICAL NEEDLE ARTHROSCOPY: A 5-YEAR FOLLOW-UP STUDY OF SAFETY AND EFFICACY. Rebecca Batcheleor, Karen Henshaw, Paul Astin, Paul Emery, Richard Reece, Douglas J Veale Leeds, United Kingdom

PATTERN OF USE OF COX-2 INHIBITORS AND GASTROPROTECTIVE AGENTS COPRESCRIPTION COMPARED TO THAT OF NSAIDS IN AN ELDERLY POPUALTION. Elham Rahme, Michael Marentette, Sheldon Kong, Jacques Lelorier Montreal, QC, Canada; Whitehouse Station, NJ

Introduction: Needle arthroscopy under local anaesthesia allows direct visualisation of the synovium and cartilage, it also allows extensive joint lavage and guaranteed delivery of intraarticular therapy. We report the results of a 5-year follow-up postal questionnaire of the first 506 patients who undertwent arthroscopy at Leeds between May 1995 and September 1999. Methods: A total of 506 patients underwent 654 rheumatological arthroscopies between 1995 and 1999 in the Rheumatology Rehabilitation Research Unit at Leeds. Questions related to safety, tolerability, efficacy and complications, results were merged with the original database to analyse outcome according to indication, diagnosis and intervention (lavage vs lavage ⫹ steroids). Results: 352 questionnaires (54%) were returned following two mailings. The diagnoses included RA (n⫽143); Seronegative arthritis (n⫽42); OA (n⫽116) and others (n⫽51). The indications for arthroscopy were diagnostic (n⫽107); therapeutic (n⫽55); persistent effusion (n⫽40); research (n⫽117) and other (n⫽23). The mean (range) of follow-up was 40.7 (12-60) months, the mean age was 54 years. Steroid was instilled after the procedure in 56% of patients. The joint was better or the same in 73% of patients, improvement was most marked in RA (52%) than in OA (42%) patients, however subsequent deterioration was reported in 40% of RA/SN and in 60% of OA patients. Intra-articular steroid was required during follow-up in 20% of patients. Pain remained a problem in over 40% and 15% underwent a further arthroscopy. 295 (84%) of patients had no post-operative complications, 7 (1%) patients reported experiences of wound infection or delayed healing, 3 (0.8%) reported infection requiring antibiotic treatment and 1 (0.2 %) reported haemarthosis. Joint replacement surgery was undertaken in 23 (7%) of patients, only 1 patient had this earlier than planned while it was deferred in 5 (1%). Conclusions: This is the largest follow-up study of 352 patients, for up to 5 years after needle arthroscopy. It shows that the procedure is safe and well-tolerated. It shows some therapeutic benefit, mainly in the short-term, however this was sustained in a significant number.

Introduction: Coxibs (rofecoxib and celecoxib) have a low potential for upper gastrointestinal (GI) toxicity compared to dual COX-1 / COX-2 inhibiting non-aspirin nonsteroidal antiinflammatory drugs (hereafter NSAIDs). Although advantageous for the patients, the widespread usage of these drugs has caused concern among drug plan managers due to the economic burden involved. One of the key cost-drivers is the co-prescription of gastroprotective agents (GPA) to prevent/treat the GI adverse events associated with NSAIDs. Objective: (1) To identify the determinants of coxib versus NSAID utilization in seniors. (2) To compare GPA co-prescription associated with either therapy. Methods: This was a retrospective cohort study of all seniors (65⫹) in Quebec, Canada, who filled a prescription for a coxib or an NSAID between April and November 2000. Subjects had at least one full year of observation in the database prior to study enrollment; the first filled prescription for coxib or NSAID after that year constituted the index date. Risk factors for GI events were identified based on pharmaceutical and medical records. Two logistic regression models were used (1) to identify determinants of coxib versus NSAID utilization;(2) to examine GPA coprescription with either therapy controlling for GI risk factors. Results: We identified 42,267 patients in the coxib cohort and 8,235 patients in the NSAID cohort. Determinants of coxib versus NSAID utilization included (odds ratio, 95% confidence intervals): female gender (1.44; [1.36, 1.52]); concomitant anticoagulants (1.28; [1.07, 1.52]); age ⬎ 85 (1.26; [1.12, 1.41]); diagnosis of musculoskeletal disease (1.91; [1.79, 2.04]); and prior GI hospitalization (1.85; [1.19, 2.86]). Patients with hypertension (0.80; [0.75, 0.85]) and diabetic patients (0.78; [0.70, 0.86]) were more likely to be prescribed NSAIDs. Incidence of GPA co-prescription adjusted for GI risk factors, were lower for patients in the coxib cohort than the NSAID cohort (0.49; [0.45, 0.54]).Conclusions: Coxib utilization was more common in patients at higher risk of GI events. When adjusted for risk susceptibility, coxibs were associated with a 50% reduction in GPA co-prescriptions compared to NSAIDs.

Disclosure:

Disclosure: This study was supported by Merck Frosst Canada Ltd.

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LEFLUNOMIDE FOR THE TREATMENT OF RHEUMATOID ARTHRITIS: A SYSTEMATIC REVIEW AND META-ANALYSIS. Manathip Osiri, Beverley Shea, Maria Suarez-Almazor, Vibeke Strand, George Wells, Peter Tugwell Bangkok, Thailand; Ottawa, Canada; Houston, TX; San Francisco, CA

DOES INCOME AFFECT THE ACCESSIBILITY OF COX-2 INHIBITORS: A DRUG UTILIZATION REVIEW STUDY WITH THE QUEBEC GOVERNMENT DATABASE. Elham Rahme, Michael Marentette, Sheldon Kong, Jacques Lelorier Montreal, QC, Canada; Whitehouse Station, NJ

Objectives: To systematically review the evidence from clinical trials on the efficacy and toxicity of leflunomide, a novel disease-modifying antirheumatic drug (DMARD), for the treatment of patients with active rheumatoid arthritis (RA). Methods: We conducted a search in MEDLINE, EMBASE, Current Contents and the Cochrane Controlled Trial Register using the Cochrane Musculoskeletal Group search strategy for human clinical trials up to December 2000. We also hand-searched reference lists, conference proceedings and consulted content experts. Two independent reviewers selected the randomized controlled trials (RCTs) and controlled clinical trials (CCTs) that met predetermined inclusion criteria. Then the data was extracted the quality of RCTs was assessed. Relative risks (RR), weighted mean differences (WMD) or standardized mean difference (SMD) of the values change from baseline was used for presentation. Results: Five trials met the inclusion criteria and were included in this review. Using specific criteria, 4 of the 5 trials were considered to be of high methodological quality. RA patients in the leflunomide group were twice as likely to meet ACR20 response criteria than patients on placebo both at 6 months (RR ⫽ 1.93, 95% CI: 1.51, 2.47) and 12 months (RR ⫽ 1.99, 95% CI: 1.42, 2.77). Other clinical outcomes, including disease activity measures, functional status and radiological scores were also significantly better for leflunomide patients compared to those who received placebo. No significant differences were observed between leflunomide and sulphasalazine (SSZ) or methotrexate (MTX) for most of the outcomes. Adverse events were more common in the leflunomide group compared to placebo, but in total, more patients withdrew from placebo than from leflunomide. Overall withdrawal rate and adverse events in the leflunomide group were not significantly different from SSZ or MTX. Conclusions: Leflunomide appears to improve all clinical outcomes and delays radiographic progression at both 6 and 12 months of treatment compared to placebo. Its short-term efficacy and adverse events are comparable to SSZ and MTX. Long-term efficacy and toxicity remains to be established.

Introduction: In Quebec, Canada a co-payment of up to 25% of the prescription drug cost is required. This co-payment may influence treatment choice in low-income patients including those receiving social assistance (welfare patients). Objectives: (1) To identify the determinants of coxib versus NSAID utilization in low income patients 18 - 64 years of age; (2) to assess the role of patient socioeconomic status in therapy selection. Methods: This was a retrospective cohort study of all patients 18 - 64 years of age who were covered by the Ministry of Health for their prescription drugs (all citizens who do not have a drug insurance otherwise) and who received a prescription for a coxib or an NSAID between April and November 2000. Subjects were required to have at least one full year of observation in the database prior to study enrollment; the first filled prescription for coxib or NSAID after that year constituted the index prescription. Risk factors for GI events were identified based on pharmaceutical and medical records. A logistic regression model was used to determine the effect of socioeconomic status (welfare recipients versus non-welfare) on therapy choice controlling for GI risk factors. Results: The study included 38,619 patients in the coxib cohort and 22,036 patients in the NSAID cohort. Determinants of coxib utilization compared to conventional NSAIDs included (odds ratio, 95% confidence intervals): age ⱖ 45 (2.58 [2.44, 2.73]); diagnosis of a musculoskeletal disease (1.90; [1.83, 1.98]); female gender (1.16 [1.12, 1.22]); Number of concomitant drugs (1.27 [1.25, 1.28]); history of prior GPA utilization (1.28; [1.20, 1.35]). Patients with hypertension (0.85; [0.80, 0.89]) and diabetic patients (0.63; [0.57, 0.70]) were less likely to be prescribed coxib compared to NSAIDs. Compared to non-welfare patients, patients receiving welfare were less likely to be prescribed coxib compared to NSAIDs (0.85; [0.81, 0.88]). Conclusions: Coxib utilization was more common in patients with higher levels of comorbidity. When adjusted for risk susceptibility, patients receiving welfare were about 15% less likely to receive a coxib rather than an NSAID compared with non-welfare patients.

Disclosure:

Disclosure: This study was supported by Merck Frosst Canada Ltd.

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RHEUMATOLOGISTS’ PERCEPTIONS AND ATTITUDES OF PSORIATIC ARTHRITIS COMPARED WITH RHEUMATOID ARTHRITIS. David J Chang Philadelphia, PA

COMPARISON OF CARDIOVASCULAR (CV) ADVERSE DRUG REACTIONS (ADRs) BETWEEN ROFECOXIB AND CELECOXIB BASED ON THE WHO/UMC SAFETY DATABASE. Sean Zhao, Matthew Reynolds, James B Lefkowith, Andrew Whelton, Felix M Arellano Skokie, IL; Peapack, NJ and Hunt Valley, MD

A survey was conducted to study rheumatologists’ perceptions of psoriatic arthritis (PsA) as compared with rheumatoid arthritis (RA). 105 ACR members practicing adult rheumatology, identified from a local rheumatism society, were mailed a survey. Instructed to consider the typical adult patient with peripheral PsA or RA, rheumatologists were queried about the diseases regarding severity, disability, comfort level in treating the diseases, and effectiveness and use of second line agents. 59 (56%) completed responses were received. Rheumatologists who rated PsA in the various categories are as follows: Disease severity–very severe 7%, moderately severe 69%, mildly severe 24%; Disability–very disabling 9%, moderately disabling 62%, mildly disabling 29%; Comfort in treating–very comfortable 61%, comfortable 31%, neutral 5%; Effectiveness of second line drugs–very effective 9%, somewhat effective 79%, neutral 5%; Proportion of patients using second line drugs–.21 to .40 13%, .41 to .60 34%, .61 to .80 39%, .81 to 1.0 12%. The following represents the percentage of rheumatologists rating their various perceptions and attitudes of PsA compared with RA:


RHEUMATOLOGISTS’ PERCEPTIONS AND ATTITUDES OF PsA vs. RA CATEGORY Disease severity Disabling Comfort in treating Effectiveness of 2nd line drugs % of patients on 2nd line drugs

PsA⬍⬍RA

PsA⬍RA

PsA⫽RA

PsA⬎RA

PsA⬎⬎RA

3% 9% 0% 3% 5%

42% 53% 17% 54% 68%

49% 36% 83% 37% 25%

5% 2% 0% 5% 2%

0% 0% 0% 0% 0%

All respondents indicated RA was more common than PsA: ⱖ20x more common (23%), 15-19x (14%), 10-14x (19%), 5-9x (30%), ⬍5x (14%). Rheumatologists followed MTX liver biopsy guidelines for RA (58%), psoriasis (23%), or neither (19%); only 2% routinely performed pretreatment liver biopsies. In conclusion, rheumatologists consider PsA to be less common, less severe, and less disabling than RA. They feel as comfortable treating PsA as RA. Second line drugs are not as effective and used less frequently in PsA compared with RA. Most rheumatologists follow RA guidelines for MTX liver safety monitoring; almost none perform pretreatment liver biopsies.

Objective: To compare spontaneous reports of CV ADRs between rofecoxib and celecoxib. Methods: The study is based on the WHO/UMC safety database that includes nearly 2 million spontaneous ADR reports from 57 countries. We used a Bayesian Confidence Propagation Neural Network (BCPNN) to evaluate the reports, which allows to: (1) detect unexpected patterns in data; (2) examine how such patterns vary over time; and (3) measure disproportionality by using a statistical parameter, IC-value, for each drug-ADR combination. An IC-value significantly ⬎0 for a specific drug-ADR pair implies that the association of the pair is much stronger than background. The higher the IC-value, the more the combination stands out from background. The comparison was conducted in individual WHO-ART preferred ADR terms and combined ADR terms. Results: In the analyses for combined CV-related ADRs, IC-values for rofecoxib were significantly higher than for celecoxib for: hypertensive events (2.15 for rofecoxib vs 1.33 for celecoxib), cardiac failure (2.39 vs 0.48), cardiac event (0.98 vs 0.41), and cerebrovascular event (1.48 vs 0.03). Thrombotic events for both drugs were not significantly different from background, nor from each other (0.46 vs -0.20). Analysis based on individual WHO-ART preferred terms indicated that 16 CV-ADR categories were significantly associated with either rofecoxib or celecoxib. Among these, rofecoxib was strongly associated with all 16 categories. Celecoxib was associated with 6. The magnitude of the IC-values for rofecoxib was greater than those for celecoxib in 15 of the 16 categories and 6 of the 16 comparisons were statistically significant (p⬍0.05). The significant comparisons were cardiac failure (3.61 for rofecoxib vs 1.67 for celecoxib), cardiomegaly (2.39 vs -0.48), cerebrovascular disorders (1.82 vs 0.63), hypertension (1.93 vs 0.68), myocardial infarction (1.44 vs 0.37), and atrial fibrillation (1.67 vs 0.35). Conclusions: The analysis indicates that rofecoxib has a significantly higher association with CV-ADRs than celecoxib. COX-2 agents may not be directly associated with thrombotic events. The stronger association between CV-related ADRs and rofecoxib may be explained as a consequence of hypertensive events. Further pharmacoepidemiological studies should be conducted to confirm the results. Disclosure: Sponsored by Pharmacia Corporation and Pfizer Inc.

Disclosure:

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EDEMA AND EDEMA ASSOCIATED ADVERSE EVENTS (AEs) AMONG 6,673 CELECOXIB, ROFECOXIB, NON-SPECIFIC (NS) NSAID, AND NON-NSAID USERS RECEIVING ORDINARY CLINICAL CARE. Frederick Wolfe, Matthew Reynolds, Thomas A Burke, Sean Zhao Wichita, KS; Peapack, NJ; Skokie, IL

PRESCRIPTION HABITS FOR OSTEOARTHRITIS: RESULTS OF A NATION-WIDE PHARMACOEPIDEMIOLOGICAL SURVEY ON 10,000 PATIENTS. Bruno Fautrel, Pascal Hilliquin, Francois-Andre Allaert, Sylvie Rozenberg, Philippe Coste, Michel Rossignol Paris, Corbeil, Dijon and Courbevoie, France and Montreal, Qc, Canada

Background: Although all classes of NSAIDs are associated with edema via renal effects, recent trials have suggested that rofecoxib is associated with a greater incidence than celecoxib. Objectives: To compare edema-related events among celecoxib, rofecoxib, and NS NSAID users in a clinical practice setting. Methods: 6673 arthritis patients, participating in a long-term outcome study, completed a questionnaire concerning edema. A questionnaire on drug usage and adverse events (AEs) occurring during the previous 6 months was also mailed biannually. The first analyses were restricted to patients who exclusively used an NS NSAID, rofecoxib, or celecoxib, to measure pure drug effect rather than one possibly contaminated by multiple drug use. Patients with overlapping NSAID use were excluded as were patients who discontinued rofecoxib or celecoxib. Results: The celecoxib, rofecoxib, NSAID, and non-user groups were similar in age, gender, and functional status as measured by HAQ disability index. Mean age was 62.1 years, 79.5% were females, and 75% had RA. 764 patients used rofecoxib only, 1233 used celecoxib only, 1784 used NS NSAIDs only, and 2345 did not use any NSAID. Six-month rates of edema were 26.8% for rofecoxib, 19.6% for celecoxib, 18.9% for NSAID, and 19.9% for non-users. After controlling for age, gender, and RA, compared to non-users, odds ratios (OR) for edema were 0.95 (95% CI⫽0.79, 1.14) for celecoxib, 0.95 (95% CI⫽0.81, 1.12) for NSAIDs, and 1.35 (95% CI⫽1.10, 1.65) for rofecoxib. OR for rofecoxib was significantly greater than that for celecoxib (p⬍0.01). Results were essentially unchanged when COX-2 patients who discontinued drug for AEs were added to the analyses. There were no significant differences in hospitalization for congestive heart failure or renal disease between groups. In an additional analysis, we compared the proportion of all patients who had used rofecoxib or celecoxib to an AE attributed to either drug. 2.5% of rofecoxib users and 1.0% of celecoxib users reported edema as an AE related to the drug (p⬍0.001). Attributable risk of reported edema for rofecoxib relative to celecoxib was 7.2%. Attributable risk when using reported edema AEs was 1.5%. Conclusion: Patients on celecoxib had rates of edema that were significantly lower than rofecoxib, but not different from other NS NSAIDs, or from non-NSAID users.

Osteoarthritis (OA) in real life settings is often difficult to study, especially in France where no reliable database exists to explore prescription habits of physicians. Methods: In 2000, a nation-wide survey in more than 5,000 physicians, either general practitioners (90.3%) or rheumatologists (9.7%), representative of the French medical demography, was conducted. Each recruited the first 2 patients consulting for hip, knee or hand OA after the onset of the survey. Results: Medical information was available for 10412 patients, whose OA was diagnosed on both clinical and radiological findings in 84.5%. Their mean age was 66.2 ⫾10.2 years with a sex ratio F:M of 1.96.

Disclosure: Sponsored by Pharmacia Corporation and Pfizer Inc.

Prescriptions

Frequency

Analgesics NSAID Anti-OA agents* Topical NSAID Steroid joint injection in past year Hyaluronic acid injection in past year Physiotherapy

94.4% 77.7% 89.8% 60.2% 19.7% 1.5% 28.4%

Drug combinations 2 drugs: 40% - 3 drugs: 55.1% 2 drugs: 25.3% - 3 drugs: 72.2% 2 drugs: 38.3% - 3 drugs: 58.1%

Renewal 93.1% 71.1 65.4

New 6.9% 28.9% 34.6%

(Mean number 1.7 ⫾0.9) Massage: 77.7% Muscle strengthening: 50.6%

*such as glucosamine sulfate. Both analgesics and NSAIDs were prescribed as needed vs daily in the majority of patients (67.4 and 70.4%). There was no major difference in the use of these medications according to the frequency of pain, the type of symptoms and the site of OA. Moreover, 28.8% of OA patients reported use of complementary and alternative medicine. The most commonly used were mesotherapy 48.4% (subcutaneous injections of medication), acupuncture 23.4%, herbal remedies 15.6%, homeopathy 14.8%. Conclusion: This study provides a representative overview of the prescription habits of French physicians for OA. It emphasizes the great frequency of therapeutic combinations, with minor differences according to the symptoms or the site of OA. Local therapy, especially steroid injections, and physiotherapy were also commonly used. Disclosure: This study has been sponsored by Laboratoires Pharmascience, 92400 Courbevoie, France.

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HYPERTENSION AND HYPERTENSION ASSOCIATED ADVERSE EVENTS (AEs) AMONG 6,673 CELECOXIB, ROFECOXIB, NON-SPECIFIC (NS) NSAID, AND NON-NSAID USERS RECEIVING ORDINARY CLINICAL CARE. Frederick Wolfe, Matthew Reynolds, Thomas A Burke, Sean Zhao Wichita, Kansas; Peapack, NJ; Skokie, IL

FEWER GI-RELATED TREATMENT DISCONTINUATIONS WITH ETORICOXIB COMPARED WITH NONSELECTIVE CYCLOOXYGENASE INHIBITORS (NSAIDs). Sean Harper, James Bolognese, Michael Lee, Douglas J Watson, Sean Curtis West Point, PA

Background: Clinical trials suggest that rofecoxib is associated with increased hypertension risk. The extent to which this exists in ordinary rheumatic disease patients is unknown. Objectives: To compare self-reported hypertensive events among celecoxib, rofecoxib, and NS NSAIDs in rheumatic disease patients receiving ordinary care. Methods: 6,673 arthritis patients participating in a long-term outcome study completed a questionnaire concerning hypertension and a questionnaire on drug usage and adverse events (AEs) that occurred during the previous 6 months. The first analyses were restricted to patients who exclusively used an NS NSAID, rofecoxib, or celecoxib, to measure pure drug effect. Patients with overlapping NSAID use were excluded as were those who discontinued rofecoxib or celecoxib so that edema could be evaluated without possible contamination by AEs. Results: There were no significant differences in age, sex, ethnicity, or health and functional status among the groups. Mean age was 62.1 years, 79.5% were women, 75% had RA, and 44% had a history of hypertension. After controlling for age, gender, and arthritis diagnosis, compared to non-users, odds ratios (OR) for BP increase were 1.14 (95% CI 0.94, 1.37) for celecoxib, 0.98 (0.82, 1.16) for NSAIDs, and 1.44 (1.16, 1.79) for rofecoxib (P⫽0.050). 764 patients used rofecoxib only, 1,233 used celecoxib only, 1,784 used NS NSAIDs only, and 2,345 did not use any NSAID. Among patients with a history of hypertension, ORs for BP increase were 1.47 (1.10, 1.97) for rofecoxib, 1.15 (0.89, 1.49) for celecoxib, and 1.12 (0.88, 1.42) for NS NSAIDs (p⫽0.108). For rofecoxib vs celecoxib, increased BP was reported by 37.6% and 32.1% of patients with a history of hypertension, and 9.8% and 6.5% of those without. In those with a history of hypertension, rofecoxib patients were more likely to switch to a stronger anti-hypertensive medication (OR⫽1.27; 0.72, 2.23) than celecoxib users (OR⫽0.71; 0.40, 1.27), NS NSAID users (0.70; 0.42, 1.17), and non-users (OR⫽1.0). Discussion: Increases in BP are common in rheumatism patients. In this study, rofecoxib, but not celecoxib, was associated with significant increases in BP and hypertensive AEs. Celecoxib did not significantly differ from NS NSAIDs nor from those receiving no treatment.

Purpose: NSAIDs are associated with GI symptoms like acid reflux, dyspepsia, epigastric discomfort, heartburn, nausea, vomiting and abdominal pain (NSAID-type GI symptoms) which often result in treatment discontinuation (d/c). We examined d/c due to NSAID-type GI symptoms specifically and symptoms referable to the GI tract in general in osteoarthritis (OA), rheumatoid arthritis (RA) and chronic low back pain (CLBP) patients treated with etoricoxib (60, 90 or 120 mg daily combined), a selective cyclooxygenase (COX)-2 inhibitor to those taking nonselective COX-1/-2 inhibitors (i.e. NSAIDs: diclofenac or naproxen combined)in a prospectively defined combined analysis of Phase IIb-III trials. Methods: Blindly coded investigator-reported symptoms that mapped to the GI system (and abdominal pain which maps to “whole body”) and led to treatment d/c were tabulated from all 8 randomized, double-blind, phase II-III efficacy trials of etoricoxib in OA, RA and CLBP. Some trials included both placebo- and active-comparator controlled periods and treatment switches from one period to the next; this report describes results of a combined analysis using data from the active comparator-controlled treatment periods (maximum 792 days). Time to treatment d/c due to NSAID-type GI symptoms and to any GI symptom or abdominal pain was evaluated using a survival approach. Cumulative incidence was computed via the Kaplan-Meyer estimate. The relative risks (RR; etoricoxib vs NSAIDs) of treatment d/c were estimated with the Cox proportional hazards model. Results: 2651 patients were treated with once daily etoricoxib (60 mg only [N⫽906], 60 and 90 mg [N⫽332], 90 mg only [N⫽950], 90 and 120 mg [N⫽343] or 120 mg only [N⫽120]) and 1329 were treated with NSAID (diclofenac 150 mg daily [N⫽232] or naproxen 1000 mg daily [N⫽1097]). The rate/100 patient-years (py) for treatment d/c due to NSAID-type GI symptoms with etoricoxib was 3.0 vs 5.6 for NSAIDs (RR⫽0.57; 95% confidence interval [CI] 0.37, 0.87; p⫽0.01). The rate/100 py for treatment d/c due to any GI symptom or abdominal pain with etoricoxib was 5.9 vs 11.3 for NSAIDs (RR⫽0.57; 95% CI 0.42, 0.77; p⬍0.001). Conclusions: In this combined analysis of all 8 randomized, double-blind, Phase II/III clinical efficacy trials of patients with OA, RA or CLBP, treatment with etoricoxib significantly reduced by 43% treatment discontinuations due to NSAID-type GI symptoms, or that due to GI symptoms in general, compared with treatment with nonselective cyclooxygenase inhibitors (i.e. NSAIDs).


Disclosure: Research sponsored by a grant from Merck & Co., Inc.

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ANTI-INFLAMMATORY DRUG DISCONTINUATION IN ARTHRITIS CLINICAL PRACTICE: A COMPARISON OF NSAIDS, CELECOXIB, AND ROFECOXIB. Frederick Wolfe, Matthew Reynolds, Thomas A Burke, Sean Zhao, Dan Pettit Wichita, KS; Peapack, NJ; Skokie, IL and New York, NY

A COMPARISON OF THE CLINICAL IMPACT OF ROFECOXIB VS. CELECOXIB ON NEW ONSET HYPERTENSION. Walter L Straus, Suna Barlas, Gary J Stever, D Craig Brater West Point, PA and Indianapolis, IN

Background: Drug discontinuation is common among patients using anti-inflammatory drugs (NSAID and COX-2). The effect of the tolerability differences on discontinuation rates, however, has not been evaluated using clinical practice data. Objectives: To investigate the discontinuation rates of COX-2 and non-selective NSAID and factors associated with discontinuation in patients with arthritis. Methods: A total of 14,394 patients completed up to 3 surveys mailed at 6-month intervals. At each survey, patients reported on the previous 6 months, including reasons for treatment discontinuations. Endpoints were discontinuation due to any cause and to adverse event. Discontinuation rates are reported as percent of patients within the six-month period. Results: Drug use was celecoxib (n⫽6,706, 27.0), rofecoxib (n⫽2,685 18.7), non-selective NSAIDs (n⫽6,619 46.0), and no NSAID (n⫽3,142 31.4).The rate of discontinuation was 3-fold higher among those who initiated therapy within the previous six-months, compared to those who were continuing users. In addition, rates of discontinuation fell over the 18 months of observation for COX-2 NSAIDs, reflecting decreasing channeling bias. Discontinuation was significantly associated with osteoarthritis, HAQ, SF-36, age and comorbidity, but not sex. Among patients using COX-2 agents, rofecoxib patients were more likely to discontinue treatment for any cause than those on celecoxib, (OR⫽1.35; 95% CI 1.04 to 1.75; p⫽0.03). The rate of discontinuation for AEs was higher for rofecoxib (11.8%) than celecoxib (6.4%). Discussion: The discontinuation rates are high among those initiating NSAID therapy, but decease as a function of the time following drug release. Among those continuing NSAIDs beyond the initiation phase, attrition rates are at least 9% per 6-month period.

Background: Rofecoxib (VIOXX) and celecoxib (CELEBREX) are the first available cyclooxygenase-2 selective inhibitors. Clinical trial data have indicated that like nonselective NSAIDs these drugs occasionally cause modest elevations of blood pressure, presumably through the effect of COX-2 inhibition upon the kidney. Head to head clinical trial comparisons of the two coxibs have provided conflicting results. To assess the clinical impact of coxibs on new-onset hypertension, we examined the experiences of patients in a large managed care population. Methods: Retrospective cohort study of patients aged 18 and over, who received at least one prescription for rofecoxib or celecoxib, and who did not receive concurrent therapy with any other NSAID during the 18 months starting January, 1999. We compared continuously enrolled patients according to their respective rate of new onset hypertension (defined as an initial prescription for an anti-hypertensive drug following receipt and up to 30 days after completion of their final coxib prescription), among persons without evidence of hypertension during the preceding 12 months. Propensity scoring was employed to adjust for potentially important differences in baseline demographic and comorbidity characteristics of patients prescribed the two drugs, as well as for coxib usage patterns. Results: Among 249,117 persons, 1116 fulfilled entry criteria (rofecoxib, n⫽183; celecoxib, n⫽933). Compared with patients receiving celecoxib, rofecoxib patients were younger (mean 61 vs. 65 years, p⫽0.0004) and more likely to have received a higher coxib dosage (p⫽0.001). The adjusted model found new onset hypertension in 3.83% of patients receiving rofecoxib compared with 3.43% of those who received celecoxib (p⫽0.790). Conclusions: In a large managed care population, the rates of new onset hypertension were similar for rofecoxib and celecoxib. Since hypertension also occurs independently of coxibs, these reported rates likely overestimate the true incidence of coxib-attributable new onset hypertension.

Rates of Discontiuation (%), January 2001

All Usage New Starts Continuing Use

Celecoxib n⫽2002

Rofecoxib n⫽1434

Naproxen n⫽601

Nambutone n⫽300

13.8 29.8 9.1

18.5 34.7 10.6

11.2 28.0 8.8

23.0 56.1 17.8


Disclosure: Three authors are from Merck & Co., and Craig Brater is a consultant from Indiana University.

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A LOWER INCIDENCE OF UPPER-GI PERFORATIONS, ULCERS AND BLEEDS (PUBs) IN PATIENTS TREATED WITH ETORICOXIB VS. NONSELECTIVE CYCLOOXYGENASE INHIBITORS (NSAIDs). Sean Harper, Michael Lee, Sean Curtis, Jennifer Ng, Douglas J Watson, James Bolognese West Point, PA

THE EFFECT OF CYCLOOXYGENASE-2 INHIBITORS ON BLOOD PRESSURE. M Jeannie Cho, Catherine E Cooke Baltimore and Ellicott City, MD

Purpose: Etoricoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that in clinical studies improved signs and symptoms of osteoarthritis (OA), rheumatoid arthritis (RA) and chronic low back pain (CLBP), and with reduced potential for GI injury. We compared investigator-reported and confirmed PUBs in patients taking etoricoxib (60, 90, or 120 mg daily) with NSAIDs (naproxen, ibuprofen, or diclofenac, combined) in a combined analysis of Phase II-III trials. Methods: Investigators in all 10 Phase II/III clinical trials in OA, RA, and CLBP reported potential PUBs for adjudication by external, blinded committee that used prespecified criteria to confirm events. A combined analysis of all PUBs during active-treatment periods (max 792 days) or w/in 14 days of stopping treatment was performed. It included events during unscheduled procedures, but excluded those diagnosed during planned surveillance procedures in the endoscopy trials. Some of the trials included treatment switches from one period to the next. Time to first PUB was evaluated using survival analysis. Cumulative incidence was computed with Kaplan-Meyer. Relative risk (RR) were estimated with the Cox proportional hazards model. Results: 3123 patients were treated with once daily etoricoxib (60 mg only [N⫽906], 60 and 90 mg [N⫽332], 90 mg only [N⫽950], 90 and 120 mg [N⫽343], or 120 mg only [N⫽592]), and 1799 were treated with NSAID (diclofenac 150 mg daily [N⫽232], ibuprofen 2400 mg daily [N⫽226], or naproxen 1000 mg daily [N⫽1341]). Fifty-seven patients with investigator-reported PUBs (48 confirmed) were eligible for the analysis. The rate/100 patient-years for investigatorreported PUBs was 1.42 vs 3.39 for NSAIDs (RR⫽0.53; 95% CI 0.31, 0.91; p⫽0.02), while that for confirmed PUBs was 1.20 vs 2.85 (RR⫽0.52; 95% CI 0.28, 0.94; p⫽0.03) with etoricoxib vs. NSAIDs, respectively. The results were driven mainly by comparisons to naproxen. There were two few complicated PUBs, and too few events during placebocontrolled treatment periods for meaningful analysis. Analysis by drug dose was not possible due to sparsity of data and confounding by type of study. Conclusion: In this combined analysis of all ten Phase II/III clinical trials of patients with OA, RA, or chronic low back pain, treatment with etoricoxib significantly reduced the incidence of investigator-reported and confirmed PUBs by approximately 50% compared with treatment with nonselective cyclooxygenase inhibitors (i.e. NSAIDs).

Objective: The purpose of this study was to compare the effect of the cyclooxygenase-2 (COX-2) inhibitors, celecoxib and rofecoxib on blood pressure. The goal was to determine if these changes were a COX-2 inhibitor class effect, or if celecoxib and rofecoxib exhibit distinct blood pressure effects. Method: Patients were identified through pharmacy claims data, and a retrospective review of medical records was conducted. Patients were included for analysis if blood pressure readings were present within 90 days before and after start of the COX-2 inhibitor, and no changes were made in medications affecting blood pressure. The average blood pressure before the start of the COX-2 inhibitor was compared to the average blood pressure after initiation and during continuous use. Paired t-tests were used for within group comparisons, and two sample t-tests for comparisons between groups (alpha⫽0.05). Results: Data was abstracted from 249 patient records. 109 patients were included for analysis. 140 patients were excluded due to an absence of blood pressure readings 90 days before and after start of the COX-2 inhibitor, and/or changes were made in medications affecting blood pressure.

Systolic BP (mmHg) Diastolic BP (mmHg)

Table 1. Comparison of average blood pressure at baseline and during COX-2 inhibitor therapy.

Table 2. Comparison of average blood pressure at baseline and during COX-2 inhibitor therapy for patients 65 years and older.

Celecoxib (n⫽52)

Rofecoxib (n⫽57)

Celecoxib (n⫽41)

Pre-BP

Post-BP

Pre-BP

Post-BP

Pre-BP

Post-BP

Pre-BP

Post-BP

134.14 74.13

132.99 74.69

134.0 74.8

138.76 (p⬍0.05) 75.34

134.8 73.29

132.86 74.5

134.5 74.38

141.87 (p⬍0.05) 75.6

Rofecoxib (n⫽42)

There was also a statistically significant difference in mean systolic blood pressure change between celecoxib and rofecoxib in patients 65 years and older (p⬍0.05). The average total daily dose of COX-2 inhibitor during post blood pressure evaluation was 219.2 mg for celecoxib and 25.23 mg for rofecoxib. Conclusions: The effect of COX-2 inhibitors on blood pressure does not appear to be a class effect. Celecoxib did not appear to affect blood pressure, while rofecoxib significantly increased systolic blood pressure. Furthermore, rofecoxib demonstrated a statistically greater increase in systolic blood pressure in comparison to celecoxib in patients 65 years and older. Blood pressure should be frequently monitored in patients receiving rofecoxib with particular caution in elderly patients. Further randomized, controlled trials are needed to determine the effect of COX-2 inhibitors on cardiovascular events. Disclosure: The co-author, Catherine E. Cooke, is employed by Pfizer U.S. Pharmaceuticals.


Disclosure: Research sponsored by a grant by Merck & Co., Inc.

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THE USE OF GASTROPROTECTION IN PATIENTS USING NON-STEROIDAL ANTI-INFLAMMATORY DRUGS IN A GENERAL RHEUMATOLOGY CLINIC: GUIDELINES AND AN AUDIT OF OUR PRACTICE. Jo Rachel MacGowan, Nisar Pandit

COMPARISON OF COX-2 SPECIFIC NONSTEROIDAL ANTI-INFLAMMATORY DRUGS (NSAIDs) TO OTHER AGENTS IN THE TREATMENT OF ARTHRITIS IN THE CLINICAL SETTING. David E Yocum, Kathryn A Nordensson Tucson, AZ

Introduction: Non-steroidal anti-inflammatory drugs (NSAIDs) cause approximately 2000 deaths due to gastrointestinal toxicity per year in the United Kingdom. The role of proton-pump inhibitors (PPIs), histamine-2 receptor antagonists (H2RAs) and prostaglandin analogues (misoprostol) in protecting against these adverse events has now been studied extensively, and the place of COX-2 selective NSAIDs (sNSAID) is also slowly emerging. We have devised evidence-based guidelines for the use of gastroprotective agents in patients requiring NSAIDs and have audited our practice. Method: 90 case-notes of patients attending for general rheumatology follow-up were analysed serially. Age of patient, indication for, and type of NSAID, concomitant medication with aspirin, corticosteroid and warfarin, and history of peptic ulcer disease or upper gastro-intestinal bleed were recorded. Patients on non cyclo-oxygenase selective NSAIDs (nsNSAID) were categorised according to whether gastroprotection was a) indicated but not prescribed, (InP), b) indicated and prescribed (IP); c) not indicated but prescribed (nIP), and d) not indicated and not prescribed (nInP). Results: 50 (55.5%) of all patients attending the rheumatology clinic were taking a NSAID. 17 (34%) were on a COX-2 selective NSAID (sNSAID), (i.e. rofecoxib, celecoxib or meloxicam), and 33 (66%) were taking nsNSAIDs. Only 3/15 of those on nsNSAIDs who were eligible for gastroprotection were actually receiving any. Patients were stratified into the aforementioned groups, and this broke down as follows: Group InP: 11 (33%); Group IP: 3 (9%); Group nIP: 0; Group nInP: 19 (58%). 3/17 patients receiving COX-2 selective NSAIDs were also taking aspirin for cardioprotection. Conclusion:The majority of patients who were eligible for gastroprotection were receiving none. A minority of patients were inappropriately prescribed gastroprophylaxis where none was required. The commonest reason for use of a COX-2 selective NSAID was intolerance of conventional non-selective agents and only in a minority was the indication based on age, concomitant corticosteroid therapy or history of peptic ulcer disease. A number of patients on aspirin for cardioprotection were prescribed COX-2 selective agents, thus losing gastroprotection. The most popular choice of gastroprotective drug was a PPI. Only one patient received a standard dose H2RA (and thus was unlikely to be sufficiently protected against NSAID-associated gastric ulceration) and none received misoprostol.

Discontinuations for adverse events and lack of efficacy were equal for all agents. CONCLUSIONS:COX-2 agents are being used in over 40% of patients receiving NSAID. While the dropout rate is very similar between agents, the COX-2 agents are more likely to be used in an older population where prophylactic ASA and GI protective agents are more commonly used.

Disclosure:

Disclosure: Funded by an unrestricted educational grant from Boehringer-Ingelheim

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COMPARISON OF RENAL ADVERSE DRUG REACTIONS (ADRs) BETWEEN ROFECOXIB AND CELECOXIB BASED ON THE WHO/UMC SAFETY DATABASE. Sean Zhao, Matthew Reynolds, James B Lefkowith, Andrew Whelton, Felix M Arellano Skokie, IL; Peapack, NJ and Hunt Valley, MD

HEALTHCARE RESOURCE UTILIZATION FOR PERFORATIONS, ULCERS AND BLEEDS IN THE VIGOR (VIOXX GI OUTCOMES RESEARCH) STUDY. C Bombardier, L Laine, G Carides, J Pellissier, D Ramey, D Watson, A Reicin Toronto, ON, Canada; Los Angeles, CA and Rahway, NJ

Objective: To compare spontaneous reports of renal ADRs between two COX-2 agents, rofecoxib and celecoxib. Methods: The study is based on the WHO/UMC safety database that includes nearly 2 million spontaneous ADR reports from physicians and other health professionals in 57 countries. We used a Bayesian Confidence Propagation Neural Network (BCPNN) method to evaluate these spontaneous reports. This method allows to: (1) detect unexpected patterns in the data; (2) examine how such patterns vary over time; and (3) measure disproportionality by using a statistical parameter, IC-value, for each drug-ADR combination. An IC value significantly greater than 0 for a specific drug-ADR pair implies that the association of the drug-ADR pair is much stronger than background. The higher the value of the IC, the more the combination stands out from the background. The comparison was conducted in two types of ADR terms, individual WHO-ART preferred ADR term and combined ADR terms. Results: In the analyses for the combined renal-related ADRs, the study found that IC-values for rofecoxib were significantly higher than celecoxib in 5 of 6 compared renal-related ADR categories: water retention (1.97 for rofecoxib vs 1.18 for celecoxib; P⬍0.01), abnormal renal function (2.38 vs 0.70; p⬍0.01), renal failure (2.22 vs 1.09; p⬍0.01), cardiac failure (2.39 vs 0.48; p⬍0.01), and hypertension (2.15 vs 1.33; p⬍0.01). In the analyses based on individual WHO-ART preferred terms, the study found that a total of 20 renal-ADR terms were significantly associated with either rofecoxib or celecoxib. Among these, rofecoxib was strongly associated with 19 identified renal-ADR terms. Celecoxib was associated with 11. In addition, the magnitude of IC-values in 19 of 20 ADRs compared for rofecoxib was greater than that for celecoxib. Among them, 11 were statistically significant. Conclusions: The analysis indicates that rofecoxib has significantly higher renal toxicity than celecoxib. The adverse impact on the renal system may result in a serious cardio-and/or cerebrovascular event. Further pharmacoepidemiological studies should be conducted to confirm the results.

Background: The VIGOR Study was a randomized, double-blind, clinical trial assessing the GI safety of rofecoxib 50 mg. once daily (n⫽ 4047) versus naproxen 500 mg twice daily (n⫽4029) in patients with rheumatoid arthritis. The treatment groups were not different with respect to prior GI events (8%), prior NSAID use (82%) and baseline dyspepsia (21%). The median duration of follow-up was 9.0 months, maximum 13 months. Purpose: To compare the rates of hospitalization and mean number of outpatient healthcare contacts associated with suspected, confirmed and complicated upper GI events in patients treated with rofecoxib, a highly selective COX-2 inhibitor, vs. naproxen. Methods: All investigator-reported upper GI perforation, ulcers and bleeds (PUBs) were adjudicated by an independent committee using prespecified criteria. The proportions of patients with hospitalizations for confirmed PUB, and the rates of PUB hospitalization (per 100 patient-years, and based on the Kaplan-Meier method) in the two treatment groups were compared for all randomized patients. Additional analyses compared proportions of PUB patients hospitalized and the mean number of outpatient contacts given a PUB event. Results: The incidence of confirmed PUBs, the primary endpoint, was significantly lower with rofecoxib compared to naproxen (2.1 vs 4.5 % per year; p⬍0.001, relative risk 0.46). Compared to patients treated with naproxen, patients treated with rofecoxib were hospitalized less often for PUBs (crude rate: 0.4% vs 0.8%, 95% CI of difference -0.7% to -0.1%) and for complicated PUBs (0.2% vs 0.6%, 95% CI of difference-0.6% to -0.1%). PUB hospitalization rates per 100 patient years were lower for rofecoxib than for naproxen (0.63 vs. 1.26, risk ratio 0.50, 95% CI of risk ratio 0.28 to 0.89). Analyses of the one-year hospitalization incidence rates for PUBs based on the Kaplan-Meier method produced analogous results. Patients treated with rofecoxib had significantly fewer outpatient contacts (p⬍0.05) for PUBs than patients treated with naproxen. As expected, in those patients who experienced a PUB, rates of outpatient contacts and hospitalizations were not different between treatment groups. Conclusions: In the VIGOR Study, RA patients treated with the highly selective COX-2 inhibitor rofecoxib for up to 13 months had significantly fewer PUBs and required fewer hospitalizations and outpatient contacts for PUBs than patients treated with naproxen.


PURPOSE: COX-2 specific agents have been shown to have lower incidence of GI events, do not affect platelet aggregation and are felt to be safer in older individuals. To examine this in the outpatient setting, we looked into our database of over 8,000 patients seen over the last 3 years in an internal medicine and rheumatology clinic, comparing the use of the 2 COX-2 specific agents, rofecoxib and celecoxib to other NSAIDs. METHOD: The database of over 8,000 patients was examined for patients taking either rofecoxib, celecoxib, naproxen, ibuprofen or meloxicam. Demographics, concomitant prophylactic aspirin, concomitant GI protective agents and discontinuations with reasons were collected. RESULTS: A total of 1,752 patients were found who had taken these agents (500 celecoxib, 226 rofecoxib, 226 naproxen, 681 ibuprofen and 69 meloxicam). The following table lists some of our results: DRUG

D/C’d

ASA

Cele Rofe Nap Ibu Mel

32% 33.6% 33.6% 28% 26%

17.8% 14.7% 6.7% 12.5% 27.5%

GI AGENT

⬎65 yrs

28.4% 26.7% 10.7% 13.3% 25.5%

50.6% 44% 23.4% 26.6% 47%

Disclosure: This study was funded by Merck. Drs Bombardier and Laine are members of the VIGOR steering committee. Drs. Carides, Pellissier, Ramey, Watson and Reicin are employees of Merck.

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ONSET OF DISEASE-MODIFYING ANTIRRHEUMATIC DRUG (DMARD) TREATMENT IN MINORITY AND DISADVANTAGED PATIENTS WITH RHEUMATOID ARTHRITIS (RA). Maria E Suarez-Almazor, Wednesday Foster, Donald M Marcus, Namieta M Janssen, Sandra Sessoms

VARIATION BY RACE/ETHNICITY AND GENDER IN TOTAL JOINT REPLACEMENT DECISIONS. Huan J Chang, Priya S Mehta, Salimah Boghani, Joshua Jacobs, Susan C Scrimshaw Chicago, IL

Objective: Early treatment of RA has been shown to delay joint damage and improve outcomes The aim of this study was to determine the onset of DMARD therapy in RA patients initially seen at one of two different rheumatology settings (public vs. private) Methods: All medical records of patients with RA initially seen at two rheumatology clinics from 1996 onward were reviewed to determine the duration of disease at the time of initiating DMARD therapy. The first clinic is located in a public hospital within the County Hospital District, and provides care primarily to minority, disadvantaged and uninsured patients. The second clinic is private, located within one mile of the County Hospital and provides care to patients with health insurance coverage. Both clinics are affiliated to the same medical school. Results: From 1996 on, 118 new patients with RA were seen in the public clinic and 167 in the private one; 84% of the patients in the public clinic were Hispanic, Black or Asian. Mean disease duration at the time of the initial visit was 7 and 9 years respectively (p⫽0.10, NS). Fifty percent of the patients seen at the public clinic had previously received a DMARD compared to 72% in the private clinic (p⬍0.001); 46% in the public system had received oral steroids compared to 79% in the private one (p⬍0.001). For those patients who had received DMARDs, the mean disease duration at onset of therapy was similar in both settings (3.96 vs.3.66 years; p⬎0.05). In the public clinic one third of those who had previously received DMARDs had discontinued treatment; 75% of the White patients had received DMARDs compared to 45% of non-Whites. Treatment rates were not significantly different across years (1996-2000), although small trends were noticed with patients attending the clinic earlier in the course of the disease in later years. No significant differences were observed between clinics for patients with a disease duration of 5 or more years: 28% in the public clinic and 22% in the private one had never received DMARDs. However, in the County Hospital, White patients with longer disease duration were more likely to have received therapy than non-Whites (p⫽0.02). Conclusions: Our findings suggest that there are differences in the onset of DMARD therapy in patients initially attending public vs. private rheumatology settings, with a significant delay in uninsured and disadvantaged patients, which is more marked for ethnic minorities.

Knee osteoarthritis (OA) affects 6 million American adults. While total joint replacement surgery (TJR) is considered an extremely valuable intervention for knee OA, utilization of TJR in the United States varies widely by both gender and race/ethnicity. For instance, women have significantly worse preoperative functional status at time of TJR for OA, suggesting they choose TJR at a more advanced disease stage, possibly at the expense of quality of life. The reason for this disparity is unclear. Purpose: We conducted this study to examine which factors are most important to patients considering TJR and whether these factors differ by race/ethnicity or gender. Methods: Six focus groups consisting of patients actively considering TJR were conducted. Participants were asked 2 questions: 1) the most important personal factors for TJR, and 2) the most important personal factors against TJR. Conversations were recorded for data analysis purposes, and transcriptions were analyzed for thematic content by 2 independent evaluators. ATLAS.ti was used to tabulate theme frequency by gender and race/ethnicity. Results: Preliminary data show all groups agreed on some mediating factors (e.g., pain as a factor for TJR; uncertainty of surgical results as a factor against TJR). Other (modifying) factors appear weighted differently by different groups. For instance, technology was more important to African-Americans and social support was more important to women. As OA severity increased, modifying factors became less important. Conclusions: In summary, factors against TJR decrease in number as factors for TJR increase in dominance with disease progression. Our results indicate different genders and racial/ethnic subgroups consider the same factors when making decisions regarding TJR, but weight them differently. These differences may contribute to gender and racial/ethnic disparity seen in TJR utilization. Disclosure:

Disclosure:

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ACCESS TO MEDICAL CARE IN RHEUMATOID ARTHRITIS PATIENTS: A POPULATION SURVEY OF SEVEN STATES IN THE USA. Eswar Krishnan Palo Alto, CA

PATIENTS’ CONCERNS REGARDING TOTAL KNEE REPLACEMENT: EFFECTS OF GENDER AND RACE/ETHNICITY. Huan J Chang, Priya S Mehta, Aaron Rosenberg, Susan C Scrimshaw Chicago, IL

There is a growing concern that the newer, more efficacious therapies for Rheumatoid Arthritis (RA) may not be accessible to all the patients who need them. Data from the Behavioral Risk Factor Surveillance System (BRFSS) were analyzed to study some of the parameters of access to health care in RA patients in the general population. In 1999, BRFSS, a state-based annual random-digit-dialed telephone survey of non-institutionalized adults in the USA included an arthritis module as well as questions on details of health insurance. BRFSS is a state-based annual telephone random-digit-dial sample survey of non-institutionalized adults. A special analysis module in STATA was used to calculate means and proportions using appropriate weights to offset the effects of non-response and non-coverage and to standardize the sample. Overall, there were 18,781 people sampled (n) from seven states representing 20 million people (N). The crude prevalence of self-reported, physician diagnosis of RA was 2.7% (95% confidence interval 2.4-3.0 % n⫽ 563, N⫽537,088). The majority of RA patients were women (62%), white (79%) and at least high school level of education (70%). 170,000 RA patients (31%) were over the age 65. The median income range was $20,000 to $24,999. The proportion of patients with no insurance ranged from 13% for whites to 25% for blacks (Overall 15% n⫽80,000; similar to general population rates p⫽0.76). However, Medicare covered a higher proportion of RA patients than the general population (41% vs. 20% p⬍0.00001). Of the survey respondents under the age 65, RA patients were more likely to have Medicare coverage than general population (18% vs. 5%). Seventeen percent of RA patients (n⫽66,000) stated that they were unable to see a doctor in the preceding 12 months because of cost concerns (general population 11%, p⬍0.001). Compared to general population, patients with RA had more days when self-care, recreation or work was interrupted due to poor health (10.5 versus 15.4 days in the preceding 30 days, p⬍0.001). Among patients with RA, the presence or absence of health insurance did not make a difference in the number of days of activity-limitation due to poor health (15.5 versus 15.6 days p⫽0.53). In 1999, about one in every six RA patients in the seven states surveyed did not have any health coverage. Although there was no statistically significant morbidity-gap between the uninsured and the insured, this may no longer be true with the availability of newer agents than can markedly reduce disability in those who have insurance to pay for them.

It is well known that women and racial/ethnic minorities under-utilize high-cost procedures such as total knee replacement (TKR) surgery. For instance, women have significantly worse functional status at time of TKR for OA compared to men; this suggests they choose TKR at a more advanced disease stage, possibly at the expense of quality of life. The reasons for these disparities are unclear. Purpose: This study examines differences in patients’ concerns regarding TKR by gender and race/ethnicity. Methods: Six focus groups consisting of patients actively considering TKR were conducted. Discussion topics included patients’ questions and concerns regarding TKR. Conversations were recorded for data analysis purposes, and transcriptions were analyzed for thematic content by 2 independent evaluators. ATLAS.ti was used to tabulate patients’ concerns by gender and race/ethnicity. Results: Preliminary data show all groups shared similar concerns (e.g., “How long will I be in the hospital”; “How quickly can I walk [after TKR]”). However, some issues were more prevalent among certain gender and racial/ethnic groups. For instance, concerns regarding anesthesia during the procedure were more important to African-Americans, and concerns regarding rate of recovery were more important to women. Conclusions: In summary, our results indicate that different gender and racial/ethnic subgroups focus on different concerns when faced with the option of TKR. These differences may contribute to gender and racial/ethnic disparity seen in TKR utilization. Disclosure:

Disclosure:

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TOWARD AN EFFICIENT STRATEGY FOR OSTEOPOROSIS SCREENING: DIAGNOSTIC ACCURACY AND COST EFFECTIVENESS OF THE PROPOSALS OF THE BELGIAN SOCIAL SECURITY INSTITUTE. Wafa Ben Sedrine, Olivier Ethgen, Jean-Pierre Devogelaer, Jean-Marc Kaufman, Stephane Goemaere, Genevieve Depresseux, Jean-Yves Reginster Liege, Brussels and Gent, Belgium

USE OF COMPLEMENTARY AND ALTERNATIVE MEDICINE (CAM) BY PATIENTS WITH CHRONIC BACK PROBLEMS. Bruno Fautrel, Yvan St Pierre, Sylvie Rozenberg, Michel Rossignol, Pierre Bourgeois, Lawrence Joseph, Viviane Adam, John R Penrod, Ann E Clarke Montreal, Qc, Canada and Paris, France

Objective: The Belgian social security institute (hereafter, INAMI) suggested a list of criteria that would be considered as a prerequisite for the reimbursement of bone densitometry. Our aim is to evaluate the diagnostic accuracy of these criteria and to gauge how useful they could be from a resource allocation perspective. Methods: From a retrospective sample of 3748 consecutive Caucasian women aged 50 years or more, diagnostic accuracy has been evaluated through measures of sensitivity (Sn), specificity (Sp), positive (PPV) and negative predictive values (NPV). Benchmark values for age and Body Mass Index (BMI) that best discriminate between osteoporotic and non osteoporotic patients were identified. They were used alone and in combination with the INAMI test to define different screening strategies and associated diagnostic costs per osteoporotic patient detected were estimated. Results: Applying the INAMI criteria for detecting osteoporosis yielded a Sn of 68.9%, a Sp of 50.7%, a PPV of 42.9% and a NPV of 57.3%. Marginal costs (in Euros) per patient detected of each strategy are as follow:

Nb of scans

INAMI⫹age⬎60⫹BMI⬍25 age⬎60⫹BMI⬍25 INAMI⫹age⬎60 INAMI age⬎60 Mass screening

663 1106 1459 2108 2231 3737

406 576 694 904 932 1295

Incremental patients detected 406 170 118 210 28 363

Mean Cost per patient detected 35,4 41,7 45,6 51.0 52.0 62,6

Total cost

Marginal cost

Cost-Eff. Ratio

14387,1 24000,2 31660,3 45743,6 48369,3 81092,9

14387,1 9613,1 7660,1 14083,3 2625,7 32723,6

35,4 56,5 64,9 67,1 93,7 90,5

Conclusion: Opening BMD coverage to women on basis of the INAMI conditions would be more cost effective than mass screening or applying an age criteria as alone. However the best strategy is the one using age, BMI and INAMI criteria as well. Disclosure:

Odds ratio Age 45 to 54 years 55 to 74 years > 75 years Household income > 30,000 CAD missing data Province of residence Atlantic Prairies British Colombia Current smoker Good general health status Co-morbid conditions Chronic sinusitis Food allergy Antidepressants taken in the past 2 months Number of consultations with MD

0.81 0.55 0.33 1.39 1.07 0.42 1.36 1.41 0.68 1.33 1.43 1.56 1.52 1.02

[95% CI] [0.64; [0.45; [0.20; [1.13; [0.86; [0.31; [1.17; [1.03; [0.57; [1.07; [1.11; [1.20; [1.03; [1.01;

1.01] 0.68] 0.55] 1.71] 1.32] 0.57] 1.60] 1.93] 0.82] 1.65] 1.85] 2.02] 2.26] 1.03]

Conclusion: Users were younger, had higher income and better self-reported health status than non users. However, they were more likely to consume antidepressants which can indicate mood disturbance. Moreover, CAM use was not associated with a reduced use of traditional health care, as shown by a higher number of medical visits. CAM use appears unrelated to CBP severity, since it was not associated with any medical parameters such as pain, analgesic intake or disability indices. Disclosure: This work has been supported by grants of the Canadian Arthritis Network and the French Society for Rheumatology.

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Screening Strategy

Nb of patients detected

Chronically ill patients often use CAM, which is often related to poor health status, pain, depression, dissatisfaction with traditional care and/or the need for additional care. Methods: The 1996 National Population Health Survey is a population-based Canada-wide survey on health status. Respondents indicated if they were suffering from MD-diagnosed chronic conditions, including chronic back problems (CBP), and if they used CAM in the previous year. Among CBP patients, users of CAM were compared to non users with regards to self-reported medical and social characteristics. A multivariate analysis, using the Bayesian Information Criteria, identified the best predictors of CAM use in CBP patients. Results: Among 66271 respondents, 11736 self-reported CBP. 41.91% of them used CAM in the previous year, which greatly exceeds rates observed in patients with none or other chronic diseases (11.61% and 15.16%, respectively - p⬍0,0001, chi-2). Predictors of use are indicated below:

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USE OF COMPLEMENTARY AND ALTERNATIVE MEDICINE (CAM) AMONG PATIENTS WITH EARLY RHEUMATOID ARTHRITIS (RA): PRELIMINARY RESULTS FROM THE SONORA STUDY. Nancy A Shadick, Amanda Boening, Claire Bombardier, Elena M Massaroti, Peter K Gregersen, Carmelo Formica, Michael Weisman Boston, MA; Abbott Park, IL; Toronto, Ontario, Canada; Manhasset, NY and Los Angeles, CA

MISSED PREVENTION OPPORTUNITIES AFTER UPPER GI BLEEDING. Paul M Peloso, Ahkash Saxena, Dharmapaul Raju Iowa City, IA and Canada

Purpose: The majority of patients (60-90%) with longstanding rheumatoid arthritis (RA) have used CAM at some time in their illness. However, little is known about the factors associated with CAM use in early onset RA. We surveyed subjects enrolled in the SONORA (Study of New Onset Rheumatoid Arthritis) study to determine these factors. Methods: A total of 254 subjects were surveyed. Early RA was defined as having signs and symptoms of RA as determined by a board-certified rheumatologist for ⬎3 months but ⬍12 months. Data were collected directly from both patients and physicians participating in the study and will be collected yearly for the duration of the 5-year study. Results Patients were mostly female (71%) and had a mean age of 52.3 yrs (SD: 15.6). They had a mean RA disease duration of 2.9 months; 36.7% had incomes of ⬎$50,000 per year and had a mean HAQ score of 0.97 (SD: 0.73). Sixty-one subjects (24%) took an herbal supplement or sought care from an alternative practitioner. Thirty-six patients took an herbal supplement, 26 of whom consumed supplements with glucosamine. In logistic regression analyses including age, sex, education, income, disease duration and HAQ score, only higher income predicted CAM use (OR 2.63, 95% CI [1.28, 5.42]). Conclusions: In early onset RA, a minority of patients use CAM for disease management. Individuals with higher income rather than more severe disease use CAM in early RA. Disclosure: This study is supported by a grant from Abbott Laboratories.

There are well established risk factors for upper GI bleeding (UGIB), including NSAIDs, ASA, H. Pylori, coumadin and comorbid medical conditions. Their identification is needed for appropriate 2ndary prevention recommendations. We assessed reporting of risk factors for UGIB in a tertiary hospital setting for patients admitted to medical and surgical services. Methods: We identified all admissions for UGIB, using ICD-9 discharge codes, in a defined population, during 1998 and reviewed all patient records for those admitted to a University Hospital. Using a pretested data extraction form, we captured reporting of demographic features, comorbid illnesses, outcomes and planned secondary prevention strategies for all patients. We used descriptive statistics to examine completeness of risk factor recording and to compare medical and surgical services. Results: There were 214 patients admitted to 3 hospitals for UGI bleeding in 1998 with 83 to the University hospital. Males comprised 72%, mean age 63 years (min-max⫽19-92) with 9 deaths (11%) and principle care provided by surgeons in 27%. Risk factor reporting was often missed. Alcohol use was absent from 76% of charts, ASA use from 60%, NSAID use from 71%, prior peptic ulcer disease from 56%, H. Pylori assessment from 57% and important comorbidities were frequently not commented upon. For instance, presence or absence of coagulopathies was not commented on 90%, diabetes not in 52%, coronary disease not in 59%, high blood pressure not commented on in 67% and presence or absence of arthritis not commented on in 83%. Given this lack of risk factor reporting, not surprisingly, strategies for managing risk factors post UGIB resusitation were rarely presented in written follow-up plans. For instance, stopping NSAIDs was commonly recommended, where use of a gastroprotective agent or a safer NSAID were never mentioned. Only 35% of those who may have had H. Pylori were tested, and only 6% of those who may have been using alcohol were referred for further alcohol management. There were few differences between admissions managed by surgeons vs. Family Practice/Internists, with surgeons more likely to test for H. Pylori (p⬍0.004), and patients admitted to surgeons more likely to undergo surgery than medical patient admissions (33% vs. 17%). Conclude: While this study describes what is recorded in the chart and not what was discussed with patients, the findings are worrisome. The absence of reasonable recognition and recording of risk factors for UGI bleeding mitigates against sensible preventative recommendations. Disclosure:

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2000 vs 2001: IS THERE ANY CHANGE IN RHEUMATOLOGISTS’ OPTIMAL CHOICE OF FIRST-LINE RHEUMATOID ARTHRITIS(RA) THERAPY? Doruk Erkan, Yusuf Yazici, Theodore Fields, Melanie J Harrison, Stephen A Paget New York, NY

PAIN SCORE INJECTION EVALUATION FORM - A MECHANISM TO COMMUNICATE OUTCOMES OF OFFICE BASED PROCEDURES. John A Goldman

Background: The last few years have seen the introduction of several new treatments for RA. Objective: To identify the physicians’ choice of optimal first-line therapy for RA and evaluate how these choices have changed since last year. Methods: Physician members of the ACR were notified by mail of a survey regarding medication preferences for RA that was posted on our rheumatology website. The questionnaire contained 3 RA histories with an initial presentation of mild, moderate or severe disease activity. Physicians were asked to identify their choice(s) of first-line therapy if the cases represented themselves or a family member to determine what physicians perceived to be the optimal treatment. Responses were compared those for a similar mail-in survey using the same cases, which was completed in 2000. Results: 329 responses returned between 3/13/01-5/07/01 qualified for analysis, and were compared to the 336 responses analyzed last year(3/12-4/25/00). There was no difference in the physicians’ choice of sulfasalazine. MTX and hydroxychloroquine selection was unchanged except for mild disease in which their frequencies increased. Selection of NSAIDs decreased, while Cox-2 inhibitors increased significantly for all 3 cases. Among new medications(table), LEF and INF selection increased significantly from 2000 to 2001 for all cases. ETC selection increased only for the mild case. When examining selection of any anti-TNF agent, selection increased significantly for all 3 cases. %, 2000 vs 2001

Mild

Moderate

Severe

Leflunomide(LEF) Etanercept (ETC) Infliximab (INF) ETC or INF Total TNF

2 vs 10* 5 vs 14* 0 vs 3* 2 vs 5 7 vs 22*

5 vs 12* 27 vs 28 2 vs 11* 5 vs 10* 34 vs 49*

8 vs 15* 42 vs 43 5 vs 23* 13 vs 13 60 vs 79*

*:pⱕ0.002 Conclusion: MTX remains the most preferred medication for the initial treatment of RA. However, the greater preference for new medications is likely due to the increasing familiarity with these drugs, and widened acceptance of earlier and aggressive therapy. The overall increase in anti-TNF preference is mostly attributable to increased selection of INF. Only once the ETC supply is replenished will it become evident if this increase in INF has occurred as a result of true physician preference or drug availability.

Rheumatologists daily perform local injections using local anesthetics, corticosteroids, synthetic hyaluronate, cytotoxic drugs and other substances into joints, bursas, tendons and myofascial sites. This paper analyzes an injection evaluation form to assist in the outcomes evaluation of the benefits or lack of benefits of local injections. McGory (Am J Orth, 7:2000) first published a similar evaluation form and this paper evaluates it from the rheumatology point of view. 77 people who had 110 injections and returned their injection forms were evaluated. There were 70 women and 7 men, with an average age of 61 years (range 40-83 years)who answered a visual analog scale of 0-10 for four questions. Injection groups evaluated included bursitis (36), myofascial sites (24), osteoarthritis (19), rheumatoid arthritis (9) and a variety of others less frequently. Some of the data is confounded by the fact that multiple sites and mixed groups i.e. osteoarthritis and bursitis, were injected. The following table demonstrates the questions and indicates the average responses and range for some selected groups of patients.

Question # 1. The worst pain they had had before the injection 2. The pain one hour after the injection 3. The pain one day after the injection 4. The pain one week after the injection Total Improvement

Average for all (range)

Average for 36 with bursitis (range)

Average for 24 myofascial sites (range)

Average for 19 osteoarthritis (range)

Average for RA 9 (range)

8.1 (2-10)

8.1 (3-10)

8.4 (6-10)

7.47 (0-10)

7.3 (2-10)

5.4 (0-10)

5.2 (0-10)

6 (2-10)

4.2 (0-10)

5.6 (1-9)

3.9 (0-10) 2.6 (0-8) 5.4 (1-10)*

4.0 (0-10) 2.9 (0-9) 5.3 (1-10)

4.1 (1-9.5) 2.6 (0-10) 5.77 (0-9)

3 (0-7) 2.6 (0-8) 4.9 (-2-10)

3 (0-7) 1.9 (0-8) 5.5 (0-9)

There was an average improvement of 5.4 (1-10)[p⬍0.05*] for all. Four people recorded lack of success of the injections and 2 got worse. Conversely 104 people did show a positive response with an average one-week improvement of 5.4 and 18 had no pain at the end of the week. The return rate of the questionnaires was 77 percent, which then became a part of the permanent record and facilitated follow-up care and patient recall. The questionnaire only recorded data for seven days in hopes that this short duration would facilitate a greater return rate of the questionnaire and more data to tabulate. Longer-term data would give more complete information on the patient response. This study shows that patients can easily record a simple injection questionnaire and this questionnaire can show success as seen here or failure of the injection(s). This type of data can easily be collected and would benefit any rheumatologist trying to document response to therapy. We need such data to show what a rheumatologist can do and do well. Disclosure: I have given talks or attended meetings sponsored by Pfizer, Searle, Merck, Proctor and Gamble, Aventis, McNeil, Immunex and Centocor. There is no disclosure regarding this abstract, which was done independently.


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THE GEISINGER HEALTH SYSTEM OSTEOPOROSIS DISEASE MANAGEMENT PROGRAM: SIGNIFICANT IMPROVEMENT IN DIAGNOSIS, TREATMENT, AND FRACTURE RATE AND REDUCTION IN COST AFTER 4 YEARS. Eric D Newman, William T Ayoub, Ralph H Starkey, Craig Wood Danville and State College, PA

A COMPARISON OF PATIENT SATISFACTION WITH CARE PROVIDED BY A CLINICAL NURSE SPECIALIST, INPATIENT TEAM CARE AND DAY PATIENT TEAM CARE IN RHEUMATOID ARTHRITIS. G J Tijhuis, C G Kooiman, A H Zwinderman, J MW Hazes, F C Breedveld, T PM Vliet Vlieland Leiden, The Netherlands

In 1996, our health system developed a disease management program whose purpose was to improve the outcome of patients with osteoporosis. The program consisted of development and implementation of provider guidelines, patient education and empowerment for better bone health behavior, and bone density testing using heel ultrasound and DXA. Outcome measures were followed yearly in our health plan population between 1996-2000 (157,000 person-years of observation in women ⬎55 and 25,000 person-years of observation in men ⬎75). Osteoporosis guideline booklets were distributed to 1500 physicians in our system, followed by discussions with over 600 providers. Patient educational programs were implemented at community pharmacies and primary care sites by trained allied health care personnel. Twenty-five primary care sites were trained in the use and interpretation of heel ultrasound. Since 1996, the diagnosis of osteoporosis rose 400% in women ⬎55 and 350% in men ⬎75. Use of bone density testing increased 1000% (7,663 scans in year 2000). Use of prescription osteoporosis treatment (bisphosphonate) rose 600% in women and 450% in men. Hip fracture rate fell by 36% in women ⬎65 (p⫽0.001). Cost analysis of fracture, testing, and medication demonstrated a reduction of $7.8 million from the anticipated spending of $62.3 between 1996-2000. This program demonstrates that a multifaceted approach to osteoporosis targeting providers, patients, and technology paralleled a significant improvement in outcomes and cost in a large health system population.

Aim The role of clinical nurse specialists in the management of patients with rheumatoid arthritis (RA) is evolving. How patient satisfaction with care provided by nurse specialists compares to other types of intensive multidisciplinary care remains unclear. The aim of this study was to compare patient satisfaction between care provided by a clinical nurse specialist (12 weeks), inpatient (2 weeks) and day patient (3 weeks) multidisciplinary team care in RA. Methods The satisfaction study was part of a multi-center, randomized controlled trial on the cost-effectiveness of 3 forms of multidisciplinary care, in which consecutive outpatients with RA and increasing functional limitations participated. Basic sociodemographic and disease characteristics were recorded. Endpoint measures (physical ability, quality of life, disease activity) were determined at weeks 6, 12, 52, 72 and 104. Patients’ satisfaction with care was measured within 3 weeks of termination of the care programs, by means of a questionnaire (28 questions; 8 domains; range 28⫽not satisfied to 140⫽very satisfied), including a visual analog scale (VAS;0-100). Between group differences were tested with analysis of variance. Results 210 patients (median age 58 years, disease duration 1.8 years and HAQ score 1.25) were included in the study. No sustained differences in clinical effectiveness were seen between the 3 groups over time. The total satisfaction scores were 108 (SD 10), 113 (SD 16), and 116 (SD 12), in the nurse specialist, inpatient and day patient groups, respectively (p⫽0.01; nurse specialist vs day patient). Significant differences between nurse specialists and day patients were seen in the domains duration of treatment, efficiency, co-ordination, office environment and non-financial access (all p⬍0.05) Patients’ satisfaction measured on a VAS was significantly lower in the nurse specialist group (73;SD 23) than in both the inpatient (86;SD 20) and day patient care (92; SD 10) groups (p⬍ 0.001). Conclusion RA patients with increasing functional disability were less satisfied with care provided by a clinical nurse specialist than with inpatient or day patient team care. It has to be further examined how specific aspects of care of the clinical nurse specialist can be improved.

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CAN TRIAGE IMPROVE THE APPROPRIATENESS OF REFERRALS TO AN EARLY ARTHRITIS CLINIC? Gerard Gormley, Keith Steele, Drew Gilliland, Paul Leggett, Clare Matthews, Gary Meenagh, Allister Taggart, Aubrey Bell, Gary Wright, Ruth Milligan, Elaine Wylie, Mike Stevenson, Roly Mc Kane, Dermot O’Reilly

AN EVALUATION OF SYSTOLIC BLOOD PRESSURE CHANGES WITH CELECOXIB, ROFECOXIB, AND TRADITIONAL NONSTEROIDAL ANTIINFLAMMATORY DRUGS IN A RHEUMATOLOGY PRACTICE SETTING. Richard Pittsley, Rick Dettloff East Lansing, MI

There is mounting evidence that early treatment with disease modifying agents improves outcome in patients with rheumatoid arthritis. Early Arthritis Clinics (EACs) facilitate the fast track assessment of referrals considered, by family physicians, to exhibit features of early inflammatory arthritis. However a large proportion of these referrals may be inappropriate (eg. degenerative arthritis, fibromylgia)1 . OBJECTIVES To determine whether trained rheumatology nurse specialists (RNS) and trained family physicians (TFP) can improve the quality (positive predictive value) of referrals to EACs. METHODS Patients who had been referred by their own family practitioner with a possible diagnosis of inflammatory arthritis, to one of three EACs, were recruited to the study. Each patient was independently assessed by one of 2 RNS and one of 4 TFP, who had received a 3 session training course in the diagnosis of inflammatory arthritis. Patients were then assessed by one of 6 hospital rheumatologists (HR), who had been shown in a previous study to have a high level of diagnostic agreement. Each assessor determined the appropriateness of the referral according to predefined criteria. The level of agreement was calculated using the kappa statistic (k), where a value of 1 represents total agreement. RESULTS All of the 96 patients who were referred, consented to participate. 49(51.0%)of the referrals were deemed appropriate by the assessing HR. 43(87.8%)of the 49 were also identified by the RNS as being appropriate referrals, compared to 44(89.8%) by the TFP. 47(49.0%)referrals were deemed inappropriate by the assessing HR. 43(91.5%)of the 47 were identified by the RNS compared to 41(87.3%) by the TFP, as being inappropriate referrals to an EAC. The k value for the RNS & HR was 0.79 compared to 0.77 for the TFP & HR and 0.81 for the RNS & TFP. CONCLUSION These results demonstrate that triage by either TFP or RNS is both feasible and effective, improving the quality (positive predictive value) of referrals to EACs from approx. 50% to 90%. This effect could permit better use of secondary care services enabling more specialist time to be devoted to patients with inflammatory arthritis. The results also have implications for health service management and for postgraduate medical and nursing education. 1. Gough et al(1992) Objectives and outcome of running an early inflammatory arthrtis clinic. Bailliere’s Clinical Rheumatology 6(2): 261-279.

The objective of this project was to review the incidence of systolic blood pressure changes associated with the cyclooxygenase-1 (COX-1) sparing and traditional nonsteroidal antiinflammatory drugs (NSAIDS). An eighteen-month retrospective review (January 1999 thru October 2000) of one hundred forty-five patient charts was performed. Consecutive patients over the age of sixty years with a diagnosis of osteoarthritis or rheumatoid arthritis who were receiving celecoxib, rofecoxib, or traditional NSAIDS, for a minimum of two consecutive weeks were evaluated. Blood pressure evaluations taken from the office visit prior to the NSAID start and also the subsequent office visit were used to calculate blood pressure changes. One hundred seven (74%) of the patients reviewed were female. The mean age was 74.7 years ⫾ 6 years (SD). A diagnosis of osteoarthritis was found in eighty-eight (60%) of the patients. Cox-1 sparing prescriptions included celecoxib (n⫽107) and rofecoxib (n⫽113) and traditional NSAIDS (n⫽103) included naburnetone, salsalate, diclofenac, and etodolac. Significantly fewer prescriptions for rofecoxib (n⫽51 (45%)) resulted in either no change or a decrease in systolic blood pressure compared to celecoxib (n⫽60 (56%)) or traditional NSAIDS (n⫽66 (64%)), p⬍0.03. Significantly more prescriptions for rofecoxib (n⫽25 (23%)) resulted in a systolic blood pressure increase of 20 mmHg or greater compared to traditional NSAIDS (n⫽10 (10%)) or celecoxib (n⫽5 (5%)), p⬍0.001. The average systolic blood pressure prior to the initiation of celecoxib, rofecoxib, and traditional NSAIDS was 139.45 mmHg, 138.24 mmHg, and 140.82 mmHg, respectively. Only rofecoxib was associated with a significant change from baseline to an average of 144.85 mmHg, p⫽0.008. This retrospective review demonstrated a significantly greater likelihood of systolic blood pressure elevation with rofecoxib compared to celecoxib or traditional NSAIDS. Blood pressure should be monitored routinely in all patients receiving COX-1 sparing or traditional NSAIDS. Disclosure: Rick Dettloff is Aphid consultant to Pfizer, Inc.

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PATIENT SATISFACTION WITH PRIMARY CARE PHYSICIANS AMONG PATIENTS WITH RHEUMATOID ARTHRITIS (RA) COMPARED TO THOSE WITH ANOTHER CHRONIC ILLNESS. Michelle Bidaut-Russell, Christopher G Scott, Alan R Zinsmeister, Harvinder S Luthra, Barbara P Yawn, Sherine E Gabriel Rochester, MN

FOLLOW-UP ACTIONS OF USERS OF A LOCAL ARTHRITIS FOUNDATION HOTLINE. Lynn B Overman, Richard S Maisiak, Kenneth Saag, Jorge Nunez, Sandra Falkenhagen, Linda Austin Birmingham and Montgomery, AL

Objective: To determine whether primary care provided by generalists vs. subspecialists resulted in different levels of patient satisfaction among persons with chronic illness. Methods: A postal survey containing the Primary Care Provider Questionnaire (PCPQ) and the Health Status Questionnaire (HSQ) was mailed to samples from 2 population based cohorts of patients with RA and Diabetes Mellitus (DM). RA was defined according to the ACR diagnostic criteria and DM was defined according to the National Diabetes Data Group criteria. All cases were at least 35 years of age and Rochester, Minnesota, residents at RA or DM incidence or prevalence date. Descriptive statistics, Pearson correlation coefficients and multiple regression models were used to describe and compare the determinants of patient satisfaction. Results: A total of 86 cases (74% female) with RA and 208 cases (56% male) with DM responded to the survey. Cases were between 41 and 95 years old. Mean disease duration was 11.5 years (RA) and 15 years (DM). Most patients described their health as fair or good. Even after adjusting for gender differences, RA cases were more likely than DM cases to have a specialist (i.e. rheumatologist) as their primary care doctor. RA patients, whether seeing a specialist or a generalist, had comparable HSQ physical health, mental health, social functioning, vitality, and bodily pain scores. DM patients seeing a specialist had more bodily pain (p ⫽ 0.003) and poorer physical functioning (p ⫽ 0.03) than those seeing a generalist. Across both chronic illnesses and physician specialties, median scores for patient satisfaction ranged from 17 to 18 for overall satisfaction (maximum 20); 30 to 33 for interpersonal skills (maximum 35); 23 to 26 for technical quality (maximum 30); and 20 for access to care (maximum 25). Multiple linear regression models revealed that only 6.8% to 7.3% of the variation in satisfaction is explained by HSQ scores, patient demographics, and physician specialty. Conclusions: Both RA and DM patients were highly satisfied with their care, regardless of the specialty of the provider. All together, physician specialty, patient demographics, and HSQ scores explained only a small proportion in the variation in satisfaction. These findings point to the need for additional research to further elucidate the determinants of patient satisfaction.

Although local community arthritis organizations, such as the Arthritis Foundation (AF), have long provided advice and assistance to hotline callers, little is known about the impact of local AF hotlines on subsequent behavior. The purpose of this study was to determine who called, what actions callers took after a hotline call, and their level of satisfaction with and understanding of arthritis information they received. We conducted a follow-up mail and telephone survey of callers to a local AF chapter at a mean of 12 ⫹/- 2 months after their initial call. We investigated 18 pre-defined potential actions that callers took as a result of their hotline encounter. We were able to contact 215 (45%) from a sample of 482 callers. Respondents were demographically similar to callers who tended to be mostly female(87%), middle-aged (mean⫽45 yrs), and white(85%). The primary types of self-reported arthritis for which the respondents sought assistance were predominately OA(28%), RA(19%), and fibromyalgia(25%).The most frequent(29%) request was for a list of arthritis doctors. The most frequently reported post-call actions were: asking a physician more questions(64%), returning to their regular MD(63%), taking medications properly(54%), exercising more often(55%), learning the exact name of their diagnosis(53%)and eating a balanced diet(53%). At least 20% took five other helpful actions. Respondents gave high ratings (scale 0⫽ dissatisfied/poor understanding to 10 ⫽ fully satisfied/good understanding) for understanding materials (8.4 ⫹/- 2.5) and satisfaction (8.3 ⫹/- 2.5) with materials. 53% felt helped by the service. In summary, hotline callers tended to be disproportionately women and those with RA and fibromyalgia. Our findings also suggest that the hotline interaction encouraged a majority of generally satisfied callers to take actions that might help them to better cope with their arthritis. Disclosure:

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PREDICTORS OF CYCO-OXYGENASE (COX)-2 NONSTEROIDAL ANTI-INFLAMMATORY DRUG (NSAID) USE AMONG MEMBERS OF A LARGE MANAGED CARE ORGANIZATION. Fausto Patino, Amy Mudano, Jeroan Allison, Ted R Mikuls, Qing Li, Larry W Moreland, Sharina Person, Kenneth G Saag Birmingham, Al

NIAMS COMMUNITY HEALTH CENTER MODEL TO ADDRESS CROSS-CULTURAL RESEARCH. Janet S Austin, Kelli L Carrington, Tamara Y Oyola-Santiago, Julie L Townshend, Barbara Mittleman Bethesda, MD

Purpose: Despite the extremely common use of NSAIDs, patient safety is a significant concern for frequent users. Increasingly, the use of COX-2 selective NSAIDs has been advocated for certain high-risk patients. We identified patient and provider characteristics associated with the use of COX-2 selective agents in a retrospective cohort of frequent NSAID users. Methods: Using administrative and claims data from a regional managed care organization, we identified patients with 3 consecutive NSAID prescriptions from June 1998 to January 2000. We then established a random cohort of 136 physicians, each with a minimum of 5 previously identified patients. Medical records were abstracted by trained reviewers using a customized version of the MedQuest computer software. Multivariable logistic regression was used to determine the association between COX-2 use and selected patient/provider/health plan characteristics (bivariate p ⬍ 0.25 for entry in the final model). Results: 462 charts were abstracted from 104 physicians (43% internal medicine, 41% generalist/family practice, and 16% rheumatology). The mean patient age was 61.7 ⫾ 12.1 years, 67% were female, and 80% were Caucasian. Subjects received a median of 2 different NSAIDs (range 1 to 10) over the study period. Patient age and gender were not associated with COX-2 administration. In multivariable models, referent to patients seeing generalists, patients seeing rheumatologists (OR ⫽ 5.4; 95% CI 2.4-11.9) and internists (OR ⫽ 2.7; 95% CI 1.6-4.5) were significantly more likely to receive a COX-2 NSAID. Patients with a history of gastrointestinal (GI) disease (OR ⫽ 2.6: 95% CI 1.1-6.1) and osteoarthritis (OA) (OR ⫽ 2.9; 95% CI 1.8-4.8) were also more likely to receive a COX-2 agent. In contrast, a history of hypertension (OR ⫽ 0.5; 95% CI 0.3-0.97) was negatively associated with COX-2 use. Conclusion: In this retrospective managed care cohort, GI disease and OA were significantly and positively associated with the use of COX-2 selective agents while a history of hypertension was negatively associated with the use of COX-2 agents. Even after multivariable adjustment for a number of potential confounders, patients seeing rheumatologists and internists were more likely to receive COX-2 agents than patients seeing generalists. Further analysis is planned to determine factors associated with the transition from non-selective NSAIDs to COX-2 selective agents.

The NIAMS Community Health Center (CHC) will facilitate understanding the pathogenesis and natural history of rheumatic diseases through a research protocol directed toward issues of health disparities in the Washington, DC area. In order to develop a shared agenda with the community and to generate mutual trust, the NIAMS Health Partnership Program (HPP) has undertaken a process of community outreach through a series of community meetings and discussions. An African American and a Hispanic/Latino Core Group of community leaders and members act as partners to provide process evaluation with guidance and feedback toward this effort. The CHC is an outgrowth of the dialogue with the community, which resulted in a collaborative health promotion plan entitled the NIAMS Health Partnership Program: A Diversity Initiative. The goals are to increase awareness of rheumatic diseases; increase awareness of the importance of early detection and treatment of rheumatic diseases; evaluate the impact of rheumatology care for patients; increase awareness of and access to clinical investigations designed to understand, treat and prevent complications and chronic disabilities associated with rheumatic diseases; and increase the number of investigators in the biomedical research fields related to rheumatic diseases. At the CHC, the natural history protocol is designed to survey the nature of rheumatic diseases in the community and to assess the demographic, epidemiologic, and clinical characteristics of these disorders in this setting. The CHC will also serve as a venue in which focused studies regarding aspects of health disparities in the rheumatic diseases can be undertaken. In conclusion, this partnership with the community from concept to reality, has shown marked progress toward building trust in participation of minority communities in biomedical research. The CHC will offer a direct benefit to the community by providing medical diagnosis, consultation, and care by rheumatologists with a public health benefit to research by providing an opportunity to increase understanding of health disparities in this population. Disclosure:

Disclosure: Dr. Saag has served as a consultant and speaker to Merck and Co. and has received unrestricted grants from Merck and Pharmacia.

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ARTHRITIS SELF HELP COURSE IN CHINESE. Lei-Chun Fung, Kate R Lorig San Francisco and Stanford, CA

COST-EFFECTIVENESS ANALYSIS OF ETANERCEPT MONOTHERAPY VERSUS INFLIXIMAB PLUS METHOTREXATE IN THE TREATMENT OF RHEUMATOID ARTHRITIS. Daniel C Malone Tucson, AZ

The Arthritis Self Help Course in Chinese is adapted from the original Arthritis Self-Management Course developed by the Stanford Patient Education Center. Culturally appropriate modifications were made for conducting the course in Chinese. Forty-eight Cantonese speaking individuals participated in the course. Eighty-one percent completed pretest and 4-month post-test questionnaires. Participants demonstrated a significant (p⬍.05)improvement in self-rated health, pain and frequency of range of motion exercise. They had a decrease of 1.3 visits to physicians over 4 months (p⬍.05). Participants also completed a process evaluation at the end of the course. They found the course very helpful. Specifically, it assisted them in meeting new people with arthritis, in learning new skills and encouraged them to take a more active role in the daily care of their disease. The Arthritis Self Help Course in Chinese has demonstrated effectiveness in helping Chinese speakers to become better self-managers of their disease. Disclosure:

Purpose: To compare the cost-effectiveness of etanercept monotherapy versus infliximab plus methotrexate (MTX) for DMARD resistant rheumatoid arthritis (RA). Methods: A cost-effectiveness analysis was conducted comparing etanercept 25mg monotherapy versus infliximab 3mg/kg or 10mg/kg every 4 or 8 weeks plus MTX. A decision model was constructed with a 1 year time horizon. The primary measure was clinical effectiveness based upon the American College of Rheumatology 20% criteria (ACR20), as reported in the literature for both products. The model incorporated minor and serious infectious events. Medication costs were based upon average wholesale price and an assumed patient weight of 74kg. Infliximab infusion administration costs were included ($54 per dose). Adverse event costs were estimated using national fee schedules or published literature. Results: The ACR20 at year 1 was 69% for etanercept 25mg twice weekly, compared to 42% to 59% for infliximab 3mg/kg every 8 weeks or 10mg/kg every 4 or 8 weeks with MTX. The average first year cost was $12,336 for etanercept, compared to $13,958 to $71,041 for infliximab 3mg/kg every 8 weeks to 10mg/kg every 4 weeks with MTX. The average costeffectiveness per successfully treated patient was $17,906 for etanercept, compared to $33,233 to $120,409 for infliximab 3mg/kg administered every 8 weeks to 10mg/kg every 4 weeks with MTX. The model was most sensitive to the cost or effectiveness of etanercept, but this did not change the relative cost-effectiveness ranking. For infliximab 3mg/kg every 8 weeks to be as cost-effective as etanercept 25mg, the efficacy of etanercept would have to decrease to 37% achieving an ACR20, or the cost of infliximab treatment (infliximab ⫹ MTX ⫹ infusion costs) would have to decrease $7052. Alternatively, the efficacy of infliximab plus MTX would have to increase to 82% achieving an ACR20. Conclusion: This analysis suggests etanercept is a dominant therapy (less costly and more effective) compared to infliximab ⫹ MTX. For DMARD resistant RA, etanercept monotherapy is more cost-effective than infliximab ⫹ MTX regardless of the dose of infliximab. Disclosure: Daniel Malone was a paid consultant to Immunex

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RATES OF IMMUNIZATION AGAINST PNEUMOCOCCAL DISEASE AND INFLUENZA IN A RHEUMATOLOGY CLINIC BEFORE AND AFTER AN INTERVENTION. Emily C Somers, Michael Rabinowitz, Mona M Riskalla, W Joseph McCune Ann Arbor, MI

SATISFACTION AND SITE PREFERENCE AMONG PATIENTS RECEIVING INFLIXIMAB (REMICADE) INFUSIONS. Karen Boutin, Bonita Libman, Kathryn Cartularo, Lisa Evans, James Vecchio Burlington, VT

The Advisory Committee on Immunization Practices recommends vaccination for influenza and pneumococcal disease for adults with chronic illnesses. We measured influenza (flu) and pneumococcal polysaccharide (PPS)vaccination coverage in adult rheumatology patients, most of whom had chronic illness, and intervened to improve coverage rates in this population utilizing a quality assurance questionnaire. Methods: During the 2000-2001 influenza season, clinic patients completed a questionnaire regarding flu and PPS vaccinations during the current and previous seasons, and immunosuppressive use (prednisone, methotrexate, azathioprine, cyclophosphamide, and mycophenolate mofetil). The questionnaire served as an intervention since it was reviewed the same day by the rheumatologist, and patients were offered vaccinations when relevant. Data were analyzed using SAS v8 (Cary, NC). Results: Of 282 adult rheumatology patients, 234 had confirmed chronic illnesses. The mean age was 40.3 years. The number of immunosuppressive drugs being taken was 0 in 87 (32.5%), 1 in 90 (32.6%), 2 in 91 (33%), and 3 in 8 (2.9%) patients. Influenza vaccine: 158 patients (57.45%) received influenza vaccination during the previous season vs. 199 (72.36%) during the current “intervention” season (p⫽0.0001, c2). 81/199 patients were vaccinated the day of the intervention. Among immunosuppressive-treated patients, 115 (61.83%) were vaccinated the previous season vs. 147 (79.03%) during the current season (p⬍0.0001, c2). Pneumococcal vaccine: 105 (38.6%) patients had PPS prior to the current season, vs. 184 (67.65%) post-intervention (p⬍0.0001, c2). Among immunosuppressed patients, 88 (47.57%) were previously vaccinated vs. 150 (81.08%) post-intervention (p⬍0.0001, c2). Summary: Compliance with established guidelines for immunization of patients with chronic illnesses was significantly improved by administration of a standardized questionnaire that alerted rheumatologists to the patients’ immunization status.

Purpose: To evaluate satisfaction and site preference of patients receiving infliximab infusions in their physician’s office (office-based) and/or at an infusion center other than at their physician’s office (IV center). Patients and Methods: 51 patients who had received infliximab infusions for psoriatic arthritis, rheumatoid arthritis, and/or inflammatory bowel disease were identified. Surveys inquiring about satisfaction in several areas of infliximab therapy were returned from 37 patients. Patients indicated the location of their infusions and whether they preferred office-based or IV center therapy. Of those who returned surveys, 11 received their infusions at an IV center only, 14 received office-based infusions only, and 8 received infusions at both sites. Answers were graded on a 1 to 5 scale, with 1 being very unsatisfied, and 5 being very satisfied. 3 patients reported unsatisfied or very unsatisfied answers: 1 patient due to disease symptoms, length of time of doctor visit and length of time of infusion; 1 patient due to disease symptoms; 1 patient due to not receiving a post infusion telephone call. 11 of the 14 patients who received office-based infusions only had a preference, and 9 preferred office-based infusions. 5 of the 12 patients who received IV center infusions only had a preference, and 4 preferred IV center infusions. 5 of the 8 patients who received infliximab infusions at both sites had a preference, and all preferred receiving office-based infusions. Conclusion: Most patients expressed satisfaction with their infliximab infusion services. The majority of those patients who experienced both office-based and IV center infliximab infusions preferred receiving their infusions in the office. The majority of those patients who received office-based infusions only also preferred receiving them in the office. This indicates that patients prefer receiving their infusions in an office-based setting and supports the continuation of this practice. Disclosure:


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EARLY REFERRAL OF RHEUMATOLOGY PATIENTS BY INTERNAL MEDICINE RESIDENTS. Jennifer L Johnson, Dorothy K Johnson, Francisco P Quismorio, Lori Port, Mireya Pena Los Angeles, CA

POST-INFUSION REACTIONS ASSOCIATED WITH INFLIXIMAB THERAPY. Nancy Daley, Carrie Smith, Sue Shott, Robert Katz Chicago, IL

We examined the attitude, knowledge and behaviors of internal medicine residents(IMR) towards early referral of patients with musculoskeletal(M/S)symtoms to a rheumatologist and rheumatology health professionals for diagnosis and management. We conducted a qualitative interview of IMR in a university medical center training program, 13 physicians (7 residents in the 3rd year of training,4 in the 2nd year and 1 intern) were recruited. Nine have completed a 4-week rotation in clinical rheumatology sometime during their residency. All except two were graduates of American medical schools. Every IMR attends a weekly ambulatory clinic during their 3-year training where they serve as the primary health care provider for a predominantly ethnic minority and urban poor population. They were asked to describe in detail their plans for diagnosis and treatment of patients with arthritis and/or other rheumatic problems. All 12 IMR stated that they frequently see patients with M/S complaints in their clinics. They would perform the initial evaluation, order diagnostic tests indicated and prescribe a non-steroidal anti-inflammatory. Nine said that they would refer only the most complicated patients at the initial visit (including fibromyalgia). All felt that IM and other primary care physicians could take care of uncomplicated SLE and RA patients and early referral of patients to the rheumatologist was not part of their practice plan. Most residents were unaware of the community services provided by the Arthritis Foundation, including self-help programs, and patient education materials. The attitude of residents who have undergone a rheumatology rotation was not different from those who have not. Our study suggests that the current curriculum of IMR do not adequately define the role of the rheumatologist and rheumatology health professionals when working with the primary care physician in the early management of patients with inflammatory disease. Disclosure:

Introduction: Infusion reactions (IR) are known to occur with infliximab therapy. Post-infusion reactions (PIR) are less common and occur hours to days after the completion of the infusion when the patient has left the office. Results: In a group of 76 patients with inflammatory arthritis who received infliximab therapy, 7 patients experienced PIR. Three had sero-positive rheumatoid arthritis (RA) and 4 had systemic lupus erythematosus (SLE). Patients were not routinely pretreated with steroids, diphenhydramine and acetaminophen. However, those patients who experienced an IR or PIR were subsequently treated with prophylactic medication. Post-infusion reactions: 1 patient with RA had a fever of 103 degrees, a widespread intense maculopapular rash, marked increase in joint pain, and an elevation of CRP from 3.5 to 134. The PIR occurred 14 days after the first infliximab infusion. The patient experienced a similar PIR with fever, intense pain and a diffuse rash 5 days after the second infusion despite premedication. A second RA patient reported developing mild urticarial rash two weeks after each of 3 infliximab infusions. A third RA patient experienced an urticarial rash 11 days following the first infliximab treatment. The patient was premedicated for subsequent treatments and has not had other IR or PIR. Four other patients had PIR, all of whom had polyarthritis associated with systemic lupus erythematosus. Three of the 4 patients had fever and chills. Three SLE patients reported an increase in joint pain and three had nausea and vomiting. Two SLE patients had a rash. In addition to PIR, 11 patients had reactions during the infliximab infusion (IR) characterized by flushing, arthralgias, diaphoresis or shortness of breath. Conclusion: Delayed reactions to infliximab can occur. These may result from the development of immune complexes and complement activation. Seven post-infusion reactions are described. Disclosure:

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PATIENT COMPLIANCE WITH ACR LABORATORY MONITORING GUIDELINES FOR METHOTREXATE. Joan Rooney, H Ralph Schumacher Philadelphia, PA The American College of Rheumatology has recommended guidelines for monitoring laboratory tests of patients who are taking methotrexate. The guidelines recommended include: CBC, platelet count, AST, ALT and creatinine monitored every 4-8 weeks. Many patients have prescription plans requiring a 90-day supply of medication. Since patients in our practice are given prescriptions for a 12-24 week supply of medication, we questioned whether these patients would comply with instructions for routine toxicity monitoring at recommended testing intervals of no more than eight weeks. In order to determine patient compliance with ACR guideline recommendations, documentation was made of patient phone calls requesting methotrexate renewal. Patient charts were then checked for laboratory results to determine whether they were completed within the 4-8 week recommended period. If no laboratory results were found the patient was contacted and asked to have their laboratory testing done. Patient medications were not renewed until the recommended tests were monitored. Twenty-two patients requested methotrexate prescription renewal by phone. Despite instructions patient compliance within laboratory guideline monitoring was only 22.7%. If these patients had not called testing might have been further delayed. Many patients spontaneously recognized that testing was late. Others, when confronted with the delay, reported cancelled appointments, lack of transportation, lost insurance benefits and lost laboratory requests as reasons for noncompliance. Interestingly, all patients recognized the importance of regular laboratory monitoring. The remaining 77.2% patients in this study did complete the recommended laboratory testing after prompting. All 22 patients did have laboratory work completed. None of these patients had abnormalities indicating toxicity. Despite the absence of adverse reactions we believe that compliance must be improved. Perhaps computerized programs for tracking can be made available.

REDUCING FALL RISK IN INSTITUTIONALIZED ELDERLY. Robyn Abear, Kathy Lutz, Heidi Clark Minneapolis, MN Loss of balance and the associated increase in fall risk is a prevalent issue in America’s long term care and assisted living facilities. In the county of our institution, falls are the leading cause of hospitalization (76%) and injury death (67%) among seniors, with the 1993-97 senior fall mortality rate of 124 deaths/100,000 among the highest in the nation. Decline in physical condition is one risk factor that contributes to an increase in fall risk in the elderly. To combat this decline, our institution, a long-term care and assisted living facility, created a comprehensive exercise program utilizing our new warm water therapy pool and strengthening center. Participants receive an individualized exercise prescription based on their medical history, personal goals, and results from functional evaluations. The exercise prescriptions consist of a combination of strength training machines, free weights, aquatic exercises, flexibility, and/or balance training based on individual needs. Participants exercise 2-3 times/week for 30-60 minutes while assisted by fitness specialists. Eleven Assisted Living and Nursing Home residents (5 male and 6 female, mean age 89 years, range 84-97 years), served as subjects in this non-experimental study. Nine subjects used walking aids for ambulation and 2 were independent ambulators. Functional reach (FR ⫽ maximal safe standing forward reach) measurement tool was used to measure balance before, mean FR ⫽ 6.91 inches (range 1-14.5 inches), and after, mean FR ⫽ 8.86 inches (range ⫽ 3-15 inches), the 3-month exercise intervention. A mean FR increase of 1.95 inches demonstrated a 28.23% improvement. Our results indicate nearly 30% improvement in balance following a 3-month individualized exercise intervention. A functional reach of less than 7 inches is considered a marker for frailty and dependence in the elderly. Our subjects improved their reach from less than 7 inches to nearly 9 inches, moving them beyond the frailty marker to a level of decreased dependence and fall risk. Whether a decrease in falls and fall-related hospitalizations is observed has yet to be determined. Disclosure:

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DETERMINING THE NEED FOR A PILOT PROGRAM FOR INSTITUTIONALIZED FRAIL ELDERLY WITH ARTHRITIS IN A FACILITY OF 163 SKILLED CARE BEDS AND 57 ASSISTED LIVING UNITS. J B Preble Minneapolis, MN

DIETARY INTAKE AND NUTRITIONAL ADEQUACY OF SUBJECT-SELECTED DIETS IN CHILDREN AND ADOLESCENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). Dorthy L Lee, Jennifer B Soep, Clarie B Hollenbeck, Emily von Scheven San Jose and San Francisco, CA

Purpose: To identify needs and components for arthritis care programs and services for institutionalized frail elderly. Our facility consists of 164 skilled care beds and 57 assisted living units. The average age in skilled care is 88. The average age in assisted living is 89. Methods: Data were gathered from 1997-2000. A community market analysis was conducted along with focus groups and individual interviews. An extensive literature search was done to identify existing programs for this population. The local Arthritis Foundation was contacted to learn of any programs they may have for this population. The market analysis was done in the community of our facility. The market area consisted of 12 zip code (7 urban, 5 suburban) areas surrounding our facility and 224,994 people. People aged 65 and older comprised 14.8% of the market. Focus groups were conducted with facility residents, their family members, seniors in the community, and the board of directors for our facility. Individual interviews included interdisciplinary staff. Results: Skilled care residents average 12 diagnoses/medical conditions each. More than 50% of skilled care residents have a physician diagnosis of arthritis. The market analysis identified approximately 32,000 people with arthritis and substantiated the need and viability for a specialty arthritis program. Focus groups indicated the need for specialty care, for more to be done to motivate residents to get active, and for more recreation staff. Focus group participants also stated the need for an attitude of “rehabilitation” among staff. The literature search revealed no arthritis programs specific for this population and no other nursing homes with specific arthritis programs. The Arthritis Foundation also had no programs specific to this population. A specialty program in arthritis care for the frail elderly appeared needed and viable. Donor interest was demonstrated with a gift of 1.5 million dollars to help develop the pilot project The pilot project began in September, 2000. Based on our assessment, the pilot program has 3 components: arthritis education(for staff, residents, resident family members and the community), changes in the physical activity levels of residents, and changes in the physical environment of residents. Approximately 25% of staff have participated in arthritis education programs. Two education programs have been conducted for residents and their families, and one program has been offered to the general public. New physical activity programs have started with 73% of assisted living residents and 9% of skilled care residents involved in the new program offerings. Resident room adaptations are just beginning. Further evaluation and changes to the program are ongoing.

Purpose: Patients with pediatric SLE demonstrate nutrition-related disease including abnormalities of bone growth and body composition. Our purpose was to evaluate dietary intake and nutritional adequacy for children and adolescents with SLE. Methods: Three-day food records completed by SLE subjects and controls were analyzed with ESHA Food Processor software. Macronutrient intake (total calories; % total, saturated and polyunsaturated fat calories; and protein) was compared to daily values (DV) and vitamin and mineral intake was compared to recommended daily allowances (RDA). Groups were compared using the Wilcoxon rank sum test and results are presented as mean % of recommended allowances. Sample: Twenty-five female SLE subjects (age 7-22y [mean 17y], 3% African-American, 40% Asian, 13% Caucasian, 34% Hispanic and 10% mixed) and 46 female controls (age 7-22y [mean 15y], 10% African-American, 17% Asian, 58% Caucasian, 13% Hispanic and 2% mixed) were evaluated. Results: Mean intake for SLE was within 10% of the DV for most macronutrients, except polyunsaturated fat (43%) and protein (164%). Assessment of fat soluble vitamins revealed reduced % RDA for vitamin E (39%), vitamin A (90%), vitamin K (75%), and vitamin D (75%). Assessment of mineral intake revealed reduced % RDA for calcium (68%), magnesium (65%), and phosphorous (85%). Comparison of SLE and controls demonstrated no significant differences for these nutrients. Conclusion: Pediatric SLE diets are sufficient for most macronutrients however several important reductions for vitamin and mineral intake were observed. Reduction of bone nutrients (calcium, magnesium, phosphorous, and vitamin D) may compromise bone health in this pediatric population. Diminished vitamin E intake may result in important loss of antioxidant protection. Future studies to evaluate the contribution of these dietary abnormalities to clinical complications in SLE are needed to understand their importance and to develop nutritional guidelines for this population.

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CHANGE IN THORACIC CURVATURE IN ELDERLY WOMEN AND MEN: NATURAL HISTORY OF KYPHOSIS IN A POPULATION-BASED LONGITUDINAL STUDY. Elizabeth J Samelson, Harold N Rosen, Ike D Iloputaife, Marian T Hannan, David T Felson, Douglas P Kiel Boston, MA

COMPARISON OF DIAGNOSTIC CRITERIA FOR JUVENILE PAIN SYNDROMES. Christy A Weber, Marilynn Punaro Dallas, TX

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Purpose: To evaluate the applicability of the ACR and Yunus diagnostic criteria for Fibromyalgia in an outpatient pediatric rheumatology clinic population and to determine the proportion of children who meet the ACR criteria, Yunus criteria, or were unable to be classified by either set of criteria. Methods: We reviewed the charts of 21 randomly selected patients with chronic diffuse idiopathic musculoskeletal pain seen in the Texas Scottish Rite Hospital Rheumatology clinic since 1999. These patients were categorized according to the ACR standard Fibromyalgia criteria and the proposed criteria by Yunus for juvenile fibromyalgia. Sample: The 21 patients included 18 girls and 3 boys. Average age of onset was 11 years with a range of 4 -16 years. Seventeen were Caucasian and one Hispanic. Results: A retrospective chart review of 21 randomly selected charts from 1999 - 2001 of patients with a chief complaint of widespread chronic idiopathic musculoskeletal pain revealed 33% fit into the Yunus classification only, 29% fit into the ACR classification (all of these patients also meet the Yunus criteria), and 38% were unable to be classified. See table below for further findings. classification

#

Males

Females

Avg. # of Tender points

Avg. age of onset

ACR ⫹ Yunus Yunus Unclassified

6 7 8

0 1 2

6 6 6

12.5 6.7 1.1

14 11 8.9

Although all unclassifiable patients had sufficient somatic complaints to satisfy the Yunus criteria, none of this group had enough tender points to be classified. Conclusion: Sixty-nine percent of the 21 children met the Yunus criteria, 29% of those met the ACR. Thirty-eight percent are unable to be classified because they do not meet the tender point count for either classification despite the multiple somatic complaints and diffuse pain. The unclassified group also had the lowest average age of onset. Diagnostic criteria for idiopathic musculoskeletal pain syndromes in children needs further investigation. Disclosure:

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This study describes the natural history of kyphosis in a population-based group of older women and men followed for 17 years. Thoracic spine x-rays were obtained 1975-76 and repeated 1992-93 for 193 women and 82 men in the Framingham Study (mean age, 63; range, 50-79). Thoracic vertebrae were classified as normal or fractured (⬃20-25% reduction in any vertebral height). Kyphosis angle (KA°)was determined by the intersection of lines drawn from the superior border of T4 and inferior border of T12. Change in curvature was calculated by subtracting the difference between baseline KA and follow-up KA from 90°, such that increase in KA corresponds to increase in curvature, and decrease in KA corresponds to decrease in curvature. Mean KA at baseline was 39.6°in women and 34.0° in men. Curvature increased an average of 10.9°in women and 6.7°in men. Baseline age did not influence change in KA in women or men. Mean change in KA was similar for women with incident VF (12.5°), prevalent VF (10.8°), and without VF (10.6°). Similarly, mean change in KA did not differ between men with incident VF (12.0°), prevalent VF (7.8°), and those free of VF (5.6°). About 1/2 the women and 1/4 the men, regardless of VF status, increased KA ⬎10°. When women and men were combined, prevalent VF did not increase risk of increasing KA ⬎20°(OR⫽1.7; 95% CI⫽0.6-5.2), although incident VF was associated with ⬎20°increase in KA (OR⫽3.6; 95% CI⫽1.4-9.5). ⬍3% of the variance in KA change was explained by prevalent and incident VF. Baseline KA did not affect KA change in women (trend, p⫽.97), but men with low baseline KA had greater increase in KA change (10°vs. 4°for low vs. high quartile of baseline KA; trend, p⫽.009). Consistent with prior studies, incident VF was associated with kyphosis. However, increases of ⬎10°in KA are common in older women and men with or without VF implicating factors other than fracture in the etiology of kyphosis.

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ARTHRITIS SCHOOL FOR CHILDREN WITH JUVENILE ARTHRITIS AND THEIR PARENTS. Oda Kristensen, Hanne Gyldenlove, Troels Herlin Aarhus N, Denmark

SEXUALITY AND THE RHEUMATOID PATIENT. Jackie Hill, Ruth F Thorpe, Howard A Bird Leeds, West Yorkshire, United Kingdom

Aim: To improve the knowledge, understanding and coping of their disease in children with juvenile idiopathic arthritis (JIA) and their parents. The arthritis school program for children was introduced 1999 at the Pediatric Rheumatology Clinic for Western Denmark as an optional alternative to routine visits in the outpatient clinic. The children were seen in “classes” with 5-6 children in each and with the age range of 6-8, 8-10, and 10-12 years, respectively. The school program was run for one year with three months interval. Multidisciplinary sessions of three hours were held for children and parents, respectively. In addition to the educational program the children were examined by the pediatric rheumatologist and routine blood work were performed when necessary. During the first course the children were talking with the nurse and the psychologist learning about JIA. Each child was presenting itself talking about having arthritis and about managing pain. During the next visits the children were seeing the occupational therapist (OT) in our experimental kitchen trying out different helping facilities (e.g. baking a cake together). They were having classes with the physiotherapist learning about the principles of physiotherapy and making group activities in the gym. Finally, the nurse and OT were having classes about how to manage in a normal school with arthritis. Concurrently, the parents were having classes with the psychologist, physiotherapist, OT, social worker and pediatric rheumatologist. The course was assessed by questionnaires. Most parents gave the meeting with other parents having comparable problems a very high value. All children found the courses most exciting and were eager to meet and talk about their disease, and some were, with help from the psychologist, even learning from each other how to manage painful situations like blood tests. We therefore regard the arthritis school program as an inspiring alternative to routine clinical visits.

Nurses aim to provide holistic care for patients but sexuality remains a topic that many feel unable to discuss. Research has indicated that nurses are not proactive in this area and wait for the patient to raise the subject. As RA is a chronic, painful and potentially disfiguring disease, it seems almost inevitable that it impacts on a patient’s sexual life. This study aims to assess the patients’ perceptions on the effect of RA on their sexuality. A self-report questionnaire was given to 74 RA patients who were included in a RCT of patient outcome. It was returned by freepost by 59 (80%) of the cohort, 2/59 remained uncompleted. Ten of the sample were male and 47 were female. Ages ranged from 36-76 yrs (median 59 yrs) and disease duration 2-40 yrs (median 17 yrs). Marital status showed 41 to be married, 5 single, 7 widowed and 4 divorced. Of the 46 patients who were in a relationship, 30 (65%) said that RA did not strain the patient/partner relationship, but 16 (35%) thought that it did. Eighteen respondents believed that RA had altered their sexual relationship. Reasons given were: reduced sexual interest, lack of spontaneity, ageing, symptoms of RA, and one 55 yr old believed it to be the underlying cause of her failed marriage. Twenty three patients thought RA posed limitations on sexual intercourse; pain (n⫽11) being the greatest limiting factor, followed by difficulty in positioning (6) and fatigue (5). One patient felt too ill and another felt that her husband’s OA hip was the main problem. Sexual ability was cited as being important/very important to 58% of the respondents, to the remainder it was of little or no importance. Twenty of the 57 (35%) patients had been asked by a health professional if their RA had affected their sexual lives; all but one of these enquiries had been during a consultation with the rheumatology nurse practitioner. Nineteen patients who had experienced sexual problems had asked advice from a friend or relative (11), or a health professional (8). Asked if they would consider talking to someone about any sexual problems if they arose, 23 said yes, 21 no, and one didn’t know. Patients with RA do experience sexual problems of various kinds but in this study only 35% were provided with the opportunity to discuss the subject. Truly holistic care necessitates that all the patients problems are addressed, including this important topic. Further work is needed to highlight the patients’ requirements and ways of assessing sexual problems during their routine consultation.


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PERCEPTIONS OF PLAY AND LEISURE IN JUNIOR SCHOOL AGED CHILDREN WITH JUVENILE IDIOPATHIC ARTHRITIS. Janine Hackett

PHYSICIAN COMMUNICATION STYLE AND CORTISOL RESPONSE IN FIBROMYALGIA PATIENTS WITH HIGH AND LOW ALEXITHYMIA. Arnstein Finset, Peter Kjaer Graugaard, Kjersti Holgersen Oslo, Norway

Play is recognised as having an important role both in child development and in physical and emotional well being. It is the dominant occupation in childhood, which provides a medium for fun, creativity and self-expression. Past research has suggested that children with disability experience a variety of barriers to engagement in play. Despite this, play is often not assessed as a specific area of occupational performance, and has largely been used as treatment modality by occupational therapists. The aim of this research was to explore the perceptions of play and leisure in junior school aged children with Juvenile Idiopathic Arthritis. Twelve children between the ages of seven and eleven were interviewed in order to uncover any barriers to engagement in play, and the implications if any, for occupational therapy practice. Results showed that all children in the study, regardless of disease activity, reported difficulty engaging in play and leisure activities. Symptoms of the disease, treatment regimes and their side-effects, as well as psychosocial factors were all reported to affect play and leisure experiences. Fatigue appeared to have a significant impact on play, and children also reported that play behaviours were often restricted by parents, friends, and school personnel. Fear of damage, and unclear communication about the effects of activity also resulted in self-imposed restrictions, which further limited play experiences. Although children adopted a number of coping strategies to deal with these difficulties, they reported more indoor play and engagement in more sedentary activities, which often gave rise to feeling of sadness, loneliness and feelings of being different. These findings may have important consequences for occupational therapy practice and provide an important reminder about the importance of assessing play as a vital and distinct area of occupational performance in children. Disclosure:

Purpose: To investigate if physician communication style could influence the stress responses of fibromyalgia syndrome (FMS) patients, as indicated by levels of salivary cortisol. Subjects and methods: Sixty-five female FMS patients were selected for their high (mean: 66.5; SD: 5.3; N⫽ 34) and low (mean: 33.1, SD: 3.7; N⫽31) level of alexithymia (ability to identify and verbalize emotions), as measured with Toronto Alexithymia Scale. The subjects were offered a medical consultation with one of seven physicians and were asked to discuss the most troublesome aspect of their FMS. The consultations were videotaped and analyzed according to the Roter Interaction Analysis System. Saliva was sampled eight times: 2 and 1 day(s) and 20 and 5 min. before the consultation, 5 and 20 min. and 1 and 2 day(s) after the consultation. Results: Two physician communication elements were associated with cortisol recovery from T3 (arrival to laboratory) to T7 (one day post-consultation): asking open psychosocial questions (OPSQ) and providing confirmation to the patient in terms of agreement, approval, legitimization, and empathic remarks. In a 2-ways MANCOVA with cortisol at T7 as dependent variable, alexithymia and OPSQ as dichotomized factor variables, and controlling for cortisol at T3, medication use and trait anxiety, we found a main effect of OPSQ (F⫽ 7.9; p ⬍ .01) and an OPSQ x alexithymia interaction effect (F⫽ 5.6; p ⬍ .05), indicating that being exposed to OPSQ was associated with less cortisol recovery at T7, in particular in high alexithymia patients. Physician confirmation was also entered as a covariate and contributed significantly to the variance of cortisol at T7 (F⫽ 4.5; p ⬍ .01), but in the opposite direction, in that more confirmation was associated with a better cortisol recovery. Conclusions: This study is the first to specify the relationship between physician communication style and cortisol response in FMS patients. Whereas OPSQ tended to delay cortisol recovery, utterances of confirmation, approval and empathy from the physician promoted recovery.


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MEASURING HEALTH-RELATED QUALITY OF LIFE IN JUVENILE IDIOPATHIC ARTHRITIS: VALIDITY OF THE EQ5D. Heather Epps, Laura Ginnelly, Mike Hurley London and York, United Kingdom

THE ROLE OF THE NURSE PRACTITIONER IN THE NON-PHARMACOLOGICAL MANAGEMENT OF OSTEOARTHRITIS AND RHEUMATOID ARTHRITIS. Sydney C Lineker, Cecilia L Cosma, Zsuzsanna M Zagorszki, Catherine Schooley, Mary J Bell, Richard Glazier, Elizabeth Badley, The Arthritis Community Health Centre Demonstration Project Team Toronto, Hamilton and Windsor, ON, Canada

Introduction Assessing health related quality of life (HRQOL) is becoming increasingly important as it is used to inform us about the clinical and cost-effectiveness of patient management, which are used to decide resource allocation and to inform policy decisions. Therefore it is crucial instruments used to assess HRQOL are reliable, valid and responsive. The EQ5D is a widely used generic health index that can be scored to calculate a “utility” value or be used to describe a health profile. It consists of a visual analogue scale to define health state and a 5-part questionnaire to define health. It was developed for adults but its validity in other patient and age groups is unknown. We investigated the validity of the EQ5D in juvenile idiopathic arthritis (JIA) by comparing it with accepted disease-specific measures of function, the Child Health Assessment Questionnaire (CHAQ) and HRQOL, the Child Health Questionnaire (CHQPF50). Methods The CHAQ, CHQ PF50 and the EQ5D were scored by the parents of 47 patients with JIA (mean age of patients 10; range 5-17 years). Spearman’s Rank Correlation Coefficients were used to assess the association between the individual dimensions of the CHAQ, CHQPF50 & EQ5D. Results The CHQPF50 dimensions for physical function, bodily pain & mental health were inversely related to the corresponding EQ5D dimensions of mobility, pain, anxiety & depression (r ⫽ -0.38, p⬍0.01), physical function was inversely related to self care (r ⫽ -0.40, p⬍ 0.01). The CHAQ dimensions of walking, hygiene, dressing & activities were significantly related (p⬍0.001) to the EQ5D dimensions of mobility, self care & usual activities (walking & mobility, r ⫽ 0.47; hygiene & self care, r ⫽ 0.77; dressing and self care, r ⫽ 0.64; activities and usual activities, r ⫽ 0.57). Conclusion The EQ5D is a valid measure of HRQOL in children with JIA and could be used alongside other measures of cost effectiveness in this patient population.

Purpose: Clinical practice guidelines recommend patient education, exercise and referral to physiotherapists(PT) for patients with osteoarthritis (OA) and rheumatoid arthritis (RA), as well as referral to rheumatology for RA and social support for OA. This study examined the role of the nurse practitioner(NP)in the delivery of evidence-based care to people with OA and RA. Methods: Survey of NPs working in primary care using a standardized questionnaire of 3 case scenarios (moderate OA of the knee, early RA, late RA). Descriptive statistics were used to analyze survey responses. Sample: 20 NPs working in 7 Community Health Centres (mean age: 42 years; 95% female; 80% worked in an urban setting). Results: Survey results were available for 13 NPs (65%). Interventions included support(54%), education(23%), exercise/referral to PT(69%), and referral to rheumatology(54%) for early RA; education(31%), support(69%), exercise/referral to PT(85%), and referral to rheumatology(62%) for late RA; and education (54%), support(7%), and exercise/referral to PT (92%) for the OA knee. Conclusions: In this study we confirmed that NPs have an important role in the delivery of evidence-based care for patients with OA and RA. Social support for OA of the knee and education for RA were underutilized or underreported. Disclosure:

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REDUCED MEDICAL COSTS FOR LUPUS PATIENTS DURING A PATIENT COUNSELING INTERVENTION. Richard S Maisiak, Lynn B Overman Birmingham, AL

OCCUPATIONAL THERAPY IN RHEUMATOID ARTHRITIS: A SYSTEMATIC REVIEW. Cornelia H Van den Ende, Esther M Steultjens, Lex M Bouter, Dirkjan Van Schaardenburg, Marie-Antoinette H Van Kuyk, Joost Dekker Utrecht, Amsterdam and Nijmegen, The Netherlands

The purpose of this study was to assess the effectiveness of a patient intervention for lowering medical costs associated with the management of lupus symptoms. The study was a randomized trial with one treatment condition, Patient Counseling (PC), and one control group, Usual Care (UC). The PC group received up to six 30-min. individual counseling sessions conducted by telephone during a six-month period. The intervention was designed to prevent symptom exacerbation, improve compliance, and improve coping strategy. All 322 study patients with lupus symptoms were females recruited locally and nationally in the US from lists provided by arthritis community organizations. Healthcare utilization data, SF-36 health status data, and demographic data were collected at baseline, six-month, and 12-month follow-up. Analyses were initially performed on the 311 (4% lost to follow-up,PC⫽155,UC⫽156) participants who completed the first six months of the study. Medical costs were based on health care visits, tests, drugs, dialysis, alternative medicines, assistive devices, ER visits, hospitalizations, outpatient surgery, and rehabilitation used in 1998-99. Costs during the intervention period were directly correlated (r⫽.25*) with baseline costs and inversely correlated with SF-36 physical (r⫽-.18*) or mental (r⫽-.20*) summary scores. During the six-month intervention period the total medical costs ($10,419 vs $7435) were significantly (p⬍.05) lower for the PC vs UC group when tested using a regression analysis with adjustment for baseline health status and baseline medical costs. The largest relative cost decrease for the PC group was for hospitalization. Relative medication costs increased for the PC group. The medical costs ($10,186 vs $11,099) during the post-intervention period were similar for the two groups. Health status scores were similar for the two groups at baseline and at follow-ups. The results showed that regular, directed patient contact (⬃$400 cost per patient) can save almost $3,000 in medical costs per SLE patient in a six-month period. *p⬍.01

Objectives: To review the efficacy of occupational therapy (OT) in patients with rheumatoid arthritis. Methods: Seven different occupational therapy interventions were distinghuised: 1) training of motor function, 2) training of skills, 3) instrucion on joint protection, 4) counselling, 5) advice and instruction in the use of assistive devices, 6) provision of splints, and 7) comprehensive occupational therapy. An extensive search in MEDLINE, CINAHL, EMBASE and SCISEARCH was performed. Next to studies with a controlled design (randomised clinical trials, controlled clinical trials) studies with a uncontrolled design(pre-post studies, multiple time series) were included. The methodological quality of controlled studies was rated by a list proposed by Van Tulder et al, for the uncontrolled studies a modified version was used. A qualitative best evidence synthesis was formulated on basis of type of design, methodological quality, and significant findings on outcome and/or process measures. Results: Thirty-seven out of 57 identified occupational therapy studies fulfilled all inclusion criteria. Five out of 21 identified controlled studies had a high methodological quality, 9 out of 16 uncontrolled studies had sufficient methodological quality. No studies were identified concerning the interventions ‘training of skills’ and ‘counselling’. Only two studies were included for the intervention ’advice/instruction on the use ofassistive devices’. The results of the best evidence synthesis show that there is limited evidence that comprehensive occupational therapy and instruction on joint protection are effective on functional ability. Provision of splints was found to be associated with a decrease in pain. Conclusion: There is limited evidence that occupational therapy has a positive effect on functional ability and pain in patients with rheumatoid arthritis. These results are encouraging. However, this review demonstrates that important occupational therapy interventions widely applied in rheumatoid arthritis patients are underresearched. Reference Van Tulder MW, Assendelft WJJ, Koes BW, Bouter LM. Method guidelines for systematic reviews in the Cochrane Collaboration Back Review Group for spinal disorders. Spine 1997;22:2323-30.

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NEITHER WEALTH (LACK OF) NOR POVERTY ASSOCIATE WITH DISEASE ACTIVITY IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). Martha L Sanchez, Gerald McGwin, Jr., Holly M Bastian, Barri J Fessler, Alan W Friedman, John D Reveille, Bruce A Baethge, Graciela S Alarcon Birmingham, AL; Houston and Galveston, TX

EDUCATION, INCOME AND UNMET NEED FOR TOTAL JOINT ARTHROPLASTY. Gillian A Hawker, James G Wright, Elizabeth M Badley, Peter C Coyte, Richard H Glazier Toronto, Ontario, Canada

Purpose/Background: Poverty as defined by the US federal government (income adjusted for the # of people in household) has been used as a proxy for socioeconomic status in outcome studies. However, even with those below the federally defined poverty line, there are different levels of wealth. We sought out to investigate the relationship between poverty, and wealth in disease activity (as an important outcome in lupus). Methods: 205 SLE patients (by ACR criteria) with disease duration ⱕ 5 years at enrollment and participating in a longitudinal outcome study were asked to complete a wealth questionnaire (from the Women’s Health Initiative Study), in addition to other variables during their yearly visits. Disease activity was measured with the SLAM. Variables from the socio-demographic, clinical and behavioral/psychological domains were entered into a regression model with SLAM score as the dependent variable. Results: Patients were predominantly women (90%), and middle age (40 ⫾ 13.1 years), 25 % Hispanics, 42% African-American and 33% Caucasian, 30% were below poverty line. Poverty and wealth (lack of) were not retained in the model. Variables retained are shown below: Variable

F

P

Older age Ethnicity (non-Caucasian) Education (low) Physical function (SF-36)

13.7 4.11 8.94 10.79

0.0006 0.0230 0.0045 0.0020

Conclusions: Ethnicity, education, age, physical function but not wealth (lack of) or poverty appear to impact disease activity in lupus; they may have however impact other outcomes (mortality). Disclosure:

Previous studies have documented a relationship between measures of socioeconomic status and access to care. We estimated potential need for, and willingness to undergo hip/knee arthroplasty in a population-based cohort to determine whether differences exist by level of education and/or annual household income. All 48,218 persons 55 years of age or older in two areas of Ontario, Canada, were surveyed by mail and telephone to determine prior joint arthroplasty and to identify those with current hip or knee problems. In the latter subjects, we assessed highest level of education obtained, annual household income, racial background, severity of arthritis (WOMAC) and the presence of coexisting conditions by questionnaire, documented arthritis by examination and x-ray, and conducted interviews to evaluate subjects’ willingness to undergo arthroplasty. Potential need for arthroplasty was defined by the presence of severe symptoms and disability, the absence of absolute contraindications to surgery, clinical and radiographic evidence of arthritis, and definite willingness to undergo arthroplasty. Overall response rates were at least 72% for all questionnaires and interviews. Adjusting for age and sex, both less education (OR⫽1.45 for ⬍ post-secondary education versus rest; 95% CI 1.17-1.80) and lower income (OR⫽1.34 for ⱕ $20,000 compared with ⬎ $20,000; 95% CI 1.13-1.58) were independently associated with greater likelihood of having potential unmet need for joint arthroplasty. Willingness to undergo joint arthroplasty did not differ according to level of education or income. Thus, adjustment of estimates of potential arthroplasty need for willingness to undergo the procedure did not eliminate the observed education/income gradient. Table: % With ⱖ Moderately Severe Hip/Knee Complaints who Met Criteria for Potential Arthroplasty Need by Level of Education and Income Annual Income Education Level ⬍ post-secondary ⱖ post-secondary

ⱕ 20,000 679/1078 ⫽ 63% 104/151 ⫽ 68.9%

⬎ 20,000 181/486 ⫽ 37.2% 61/224 ⫽ 27.2%

Less education and lower income independently predicted worse arthritis symptoms and disability and greater potential unmet need for hip/knee arthroplasty, but was not associated with willingness to undergo arthroplasty. Disclosure:

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MEDICAL SKEPTICISM IN PATIENTS WITH RHEUMATIC DISEASE. Leigh F Callahan, Shannon S Currey, Janet K Freburger Chapel Hill, NC

OSTEOCLAST PRECURSORS ARE MARKEDLY INCREASED IN THE PERIPHERAL BLOOD OF PSORIATIC ARTHRITIS PATIENTS. Christopher Ritchlin, Sally A Haas-Smith, Edward Schwarz Rochester, New York Extensive bone resorption manifesting radiographically as “pencil-in-cup” erosions, acroosteolysis and large eccentric erosions are associated with psoriatic arthritis (PsA). The mechanisms that underlie this bone loss are unknown, however, we have demonstrated abundant osteoclasts (OC) in PsA but not osteoarthritic bone. We hypothesize that these OC arise from an expansion in the number of circulating OC precursors. Unstimulated peripheral blood mononuclear cells (PBMC) isolated from 19 PsA patients with erosive arthritis and 5 healthy controls were cultured at low density in 10% FBS-DMEM for 2 weeks. OC were identified by the presence of ⱖ3 nuclei per cell and positive tartrate resistant alkaline phosphatase (TRAP) staining. The geometric mean of OC in PsA was 108 */ 2.7 OC per 106 PBMC compared to 3.1 */ 4.8 per 106 PBMC in healthy controls, (p⬍.000002). OC derived from PsA PBMC formed deeper and more numerous resorption pits on bovine bone wafers than control cells. To determine if PsA PBMC release osteoclastogenic factors, PsA PBMC supernatants were added to control PBMC cultures. Addition of supernatants increased both OC number (2 to 51 fold) and degree of bone resorption compared to unstimulated cells. Treatment of 3 PsA patients with etanercept resulted in significant clinical improvement and a decrease in the OC from a geometric mean of 90.4 */ 0.5 to 15.3 */ 0.3 per 10 6 PBMC (p⬍ .05) following 16 to 26 weeks of therapy. Thus, the number of circulating OC precursors is expanded in PsA, and factors released by PsA PBMC drive osteoclastogenesis in vitro. Furthermore, anti-TNF therapy dramatically reduces the number of OC precursors in peripheral blood. This represents a novel mechanism to explain the enhanced bone resorption in PsA. Disclosure: Immunex Corporation, Wyeth-Ayerst Corporation

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PURPOSE: To examine the relationship between medical skepticism (defined as doubts in the ability of conventional medical care to appreciably alter health status) and patient demographics, general health, disease activity measures and physician trust in patients with rheumatic disease. METHODS: Patients with rheumatoid arthritis (RA), osteoarthritis (OA) and fibromyalgia (FM) in a rheumatology database were mailed a self-report questionnaire that included a 4-item measure of medical skepticism. Patients responded on a 5 point scale strongly disagree to strongly agree to the following 4 statements: 1) I can overcome most illness without help from a medically trained professional; 2) Home remedies are often better than drugs prescribed by a doctor; 3) If I get sick, it is my own behavior that determines how soon I get well again; 4) I understand my health better than most doctors do. Measures of general health, disease activity (modified Health Assessment Questionnaire (MHAQ), pain and fatigue visual analog scales (VAS), comorbid conditions, physician trust, and demographics were also included. Internal consistency was assessed, and a generalized linear model that included all the above variables was used to determine independent associations with medical skepticism.. SAMPLE: The analyses were limited to patients with non-missing data (N⫽590 of 741) and included 321 with RA, 125 with OA and 144 with FM. The sample was 77% female and 87% White. Mean yrs of age, education, disease duration, and comorbid conditions were 59.4, 13.6, 10.1, and 1.5, respectively. RESULTS: Mean skepticism was 2.7 (sd⫽.7), with an alpha of 0.61. There were no differences in skepticism among disease diagnoses. In multivariate analysis, greater levels of skepticism were significantly associated with younger age, fewer years of education, less physician trust, greater helplessness, and better self-perceived health. The strongest effects were for physician trust and self-perceived health. CONCLUSION: Medical skepticism, influenced most by physician trust and perceived health, may be an important variable to examine in rheumatic disease outcomes.

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TRANCE/RANKL KNOCKOUT MICE ARE PROTECTED FROM BONE EROSION IN THE K/BxN SERUM TRANSFER MODEL OF ARTHRITIS. Allison R Pettit, Hong Ji, Dietrich von Stechow, Ralph Mueller, Yongwon Choi, Steven R Goldring, Christophe Benoist, Ellen M Gravallese Boston, MA; Philadelphia, PA

LEVELS OF ANTI-TOPOISOMERASE I ANTIBODIES BY ELISA CORRELATE WITH DISEASE SEVERITY IN SYSTEMIC SCLEROSIS. Shinichi Sato, Yasuhito Hamaguchi, Minoru Hasegawa, Kazuhiko Takehara Kanazawa, Ishikawa, Japan

There is considerable evidence that osteoclasts (OC) are involved in focal bone erosion in RA. Tumor necrosis factor-related activation-induced cytokine, also known as receptor activator of NF-␬B ligand (TRANCE/RANKL), is an essential factor for OC differentiation and augments T cell-dendritic cell interactions. To determine the role of OC in focal bone erosion in arthritis, we generated inflammatory arthritis in the TRANCE/RANKL knockout (KO) mouse, characterized by the absence of OC, using the K/BxN serum transfer model. This model provides an opportunity to investigate the role of OC in an arthritis that resembles RA but is not dependent on T cell-dendritic cell interactions. Inflammation was assessed clinically and histologically and was comparable in arthritic TRANCE/RANKL KO mice and arthritic control littermates. Micro-CT and correlated histopathology were used to assess bone erosion. Despite ongoing inflammation, bone erosion in arthritic TRANCE/RANKL KO mice was dramatically reduced compared to that in arthritic control mice. TRAP positive OC-like cells were abundant in areas of bone erosion in arthritic control mice, and were completely absent in arthritic TRANCE/RANKL KO mice, demonstrating the requirement for TRANCE/RANKL in osteoclastogenesis in this model. Cartilage damage was assessed by analysis of areas of direct pannus invasion into cartilage and by evaluation of cartilage matrix degradation. In contrast to the findings in bone erosion, cartilage damage was observed in both arthritic TRANCE/RANKL KO mice and arthritic control mice, demonstrating that TRANCE/ RANKL is not required for cartilage destruction in this animal model. A trend toward milder cartilage damage in the TRANCE/RANKL KO mice was noted. This effect was due in part to the preservation of subchondral bone scaffolding in TRANCE/RANKL KO mice. This study demonstrates the critical role of OC in the pathogenesis of focal bone erosion in arthritis and reveals distinct mechanisms of cartilage destruction and bone erosion. The results reported here support the hypothesis that therapies interfering with osteoclast differentiation or activity, including agents that block TRANCE/RANKL, may prove useful in the prevention of RA bone erosion.

Anti-topoisomerase I (topo I) antibody is highly specific for systemic sclerosis (SSc) and clinically associated with diffuse cutaneous involvement. However, ⬃30% of SSc with this antibody have limited cutaneous involvement, indicating the heterogeneous clinical manifestations of SSc with anti-topo I antibodies. To determine the clinical correlation of anti-topo I antibody levels, antibody levels were determined by ELISA in 30 SSc patients positive for anti-topo I antibody by double immunodiffusion (DID) at their first visit. These patients were relatively active since the mean disease duration was 3.3 years. This ELISA system using the human recombinant topo I protein (amino acids 163-765) had a sensitivity of 100% and a specificity of 98.9% for DID. For longitudinal study, 125 sera from 21 patients were analysed (follow-up: 0.2-4.7 years). Anti-topo I antibody levels were correlated positively with modified Rodnan total skin thickness score (r⫽0.529, p⬍0.01) and renal vascular resistance (r⫽0.486, p⬍0.05), and inversely with %VC (r⫽-0.486, p⬍0.01). Furthermore, anti-topo I antibody levels were significantly elevated in SSc patients with pitting scar/ulcer, arthritis/arthralgia, or esophageal involvement. Thus, anti-topo I antibody levels were correlated with the disease severity in SSc patients with anti-topo I antibody. In a longitudinal study, 5 patients with low levels of anti-topo I antibody (below 100 U/ml) at their first visit did not show any increase in antibody levels during the follow-up period, but rather exhibited stable or decreased change, with relatively stable skin sclerosis. Therefore, low levels of anti-topo I antibody at first visit may identify the milder patient group among SSc patients with anti-topo I antibody. Of 16 patients with high anti-topo I antibody levels (over 150 U/ml) at their first visit, 7 patients showed stable levels, 4 increased levels, and 5 decreased levels. The decreased levels were accompanied by atrophic skin change or the improvement of skin sclerosis during the follow-up while the increased levels were associated with new onset or worsening of organ involvement, including subacute deterioration of interstitial pneumonitis, scleroderma renal crisis with cardiac tamponade, and worsening skin sclerosis. These results suggest a potential clinical significance of anti-topo I antibody levels for evaluating disease severity and predicting the prognosis in SSc.

Disclosure: Dr. Gravallese is a consultant for Amgen, Inc. and a member of their advisory board for RA.

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AN EPIDEMIOLOGIC STUDY OF SCLERODERMA PATIENTS IN SOUTH CAROLINA. Alan N Brown, Yvonne Michel, Holly G Mitchell, Marcy B Bolster

ANTI-DNA TOPOISOMERASE I ANTIBODY IgG TITER CORRELATES WITH DISEASE ACTIVITY IN SYSTEMIC SCLEROSIS. Paul Q Hu, Noreen Fertig, Thomas A Medsger, Jr, Timothy M Wright Pittsburgh, PA

Objective: To examine ethnic and gender differences in the disease type and course of scleroderma patients seen in South Carolina. Methods: 208 sequential patients with scleroderma registered in the Connective Tissue Diseases Database between November 1997 and May 2001 at the Medical University of South Carolina were identified. Disease type, onset, and clinical characteristics were evaluated and differences examined between black and white patients. Results: 33 (16%) white males, 13 (6%) black males, 122 (59%) white females and 40 (19%) black females were identified. Both black and white males were more likely to have diffuse cutaneous disease (62% of cases in each group). Black females were much more likely to have diffuse cutaneous disease than white females (63% of black patients vs. 28% of white patients). Black females had a significantly earlier age of onset of disease (as defined by the date of Non-Raynaud’s onset) than white females (mean age 37 years vs. 48 years, respectively; P⫽0.04). Black males likewise had an earlier age of onset than white males (39 years vs. 50 years, respectively). Black males had a significantly lower level of education than other gender and ethnic groups (P⬍0.001). There were no other discrepancies in the level of education between the other groups. Conclusion: Black patients with scleroderma in South Carolina are more likely to have diffuse cutaneous disease and an earlier age of onset of disease than white patients. These findings are consistent with the demographic features of scleroderma patients reported in other databases. Black male patients in our population are more likely to be less educated than patients of other gender and ethnic groups.

Anti-DNA topoisomerase I (anti-topo I) antibody is commonly detected in sera of systemic sclerosis (SSc) patients with diffuse cutaneous involvement. However, the relationship between anti-topo I antibody and the pathogenesis of SSc is unclear. To examine this possible relationship, we compared the titer of the auto-topo I antibody and clinical features of SSc. We studied 60 SSc patients with varying disease duration and a single serum sample available and 11 SSc patients with serial serum samples. The anti-topo I IgG antibody titer was measured by ELISA using recombinant full-length human topo I as antigen. Antibody titer was defined by using a reference serum. Each patient’s disease activity was judged by physician global assessment, which is the overall evaluation of 9 clinical features of SSc patients. In the 60 SSc patients with single samples analyzed, the mean titer of anti-topo I IgG antibody was 130.5 units in patients with “active disease”, 76.0 units in patients with “intermediate disease” (p⬍0.05, compared to the active group), and 50.8 units in patients with “inactive disease” (p⬍0.001, compared to the active group). As a result, the anti-topo I IgG antibody titer was highly correlated with the disease activity of SSc patients. More interestingly, when serial serum samples from the same SSc patient were analyzed, the antibody titer and the skin thickness score, a major indicator for the disease activity, showed parallel changes in 7 of the 11 SSc patients. In 3 additional SSc patients (out of 11), changes in antibody titer preceded the changes in the skin thickness score. Our results convincingly demonstrate that the titer of the anti-topo I IgG antibody positively correlates with disease activity of SSc patients. Disclosure:

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SOCIODEMOGRAPHIC, BEHAVIORAL, CLINICAL, IMMUNOLOGIC AND GENETIC FACTORS ASSOCIATED WITH DISEASE SEVERITY IN EARLY SYSTEMIC SCLEROSIS (SSc). Peter Shane, Terry A McNearney, Michael Fischbach, Alan W Friedman, Chul W Ahn, John D Reveille Houston, Galveston and San Antonio, TX

TROPONIN T ELEVATION PREDICTS AN ADVERSE OUTCOME IN SYSTEMIC SCLEROSIS (SSC). Dev Mukerjee, Alexandra Balbir, James Moon, John G Coghlan, Carol M Black London, United Kingdom


Objective. To identify factors associated with disease damage/severity in early SSc.Methods. The SSc severity scale (Medsger et al: J Rheumatol. 26:2159-67) was scored for 158 patients in a longitudinal cohort of SSc with early (⬍5 years) disease duration (Genetics versus Environment In Scleroderma Outcome Study [GENISOS]). Sociodemographic (SD) features, organ system manifestations (CLIN), including Rodnan skin score, immunologic (IMM) factors (ANA, anti-topo I, centromere, fibrillarin, RNA pol I-III, Th/To, PM-Scl autoantibodies), immunogenetic (GEN) features (HLA class II genotypes), and behavioral/psychosocial features (PSY), as obtained at enrollment into the study, were analyzed using stepwise linear regression to identify significant independent risk factors for higher damage/severity scores. Results. Among the 80 Caucasians, 25 African Americans, and 53 Hispanics studied, African-Americans had significantly higher damage/severity scores compared to Caucasians (8.48⫾ 3.7 vs. 6.36⫾ 3.0-p⫽0.004) but not compared to Hispanics (7.32⫾3.9, p⫽0.2). Factors independentlyassociated with damage are shown below. Variable

F value

⬍P value

Higher skin score Lower income Anti-U1 RNP ANA, cytoplasmic HLA-DQB1*05 Lower hematocrit Lower DLco Public insurance

56.14 10.59 4.04 5.93 5.46 7.57 33.15 9.39

⬍0.0001 0.0015 0.0466 0.0162 0.0211 0.0068 ⬍0.0001 0.0027

This model was quire robust, accounting for 64% of the overall variance. Significant clinical variables were found that were also components of the overall damage score (skin, hematocrit, and DLco), which were then individually removed from the damage score and the regressions re-examined. Only the Rodnan skin score persisted as an independent clinical risk factor for damage/severity elsewhere (p⫽0.001) in addition to the SD, IMM, and GEN factors. Conclusion. Disease damage/severity early in the course of SSc is influenced by a combination of factors, including lower sociodemographic status, clinical factors (most notably Rodnan skin score,) and serologic and immunogenetic determinants. Disclosure:

Troponin T level have been shown to be elevated in patients with ischaemic heart disease and renal impairment. In both diseases raised levels predict higher mortality .Cardiac involvement in Systemic Sclerosis (SSc) includes coronary artery disease, pulmonary vascular disease with right ventricular dysfunction and myocarditis leading to diastolic dysfunction. It is suspected that SSc myocarditis often follows an insidious course and histological diagnosis is difficult to establish due to the patchy nature of myocardial involvement. Between 1985-1998 serum samples from 380 SSc patients who had been followed up for⬎5 years out of a background population of 483 attending the Connective Tissue Disease Clinic, Royal Free Hospital, London were analysed for troponin T levels using an electroluminescence assay (Alecys 1010, Roche Diagnostics). From these, 135 patients had undergone cardiac assessment, of which 50 patients had hypertension (BP⬎140/90), 14 had CPK levels ⬎200, 65 had abnormal ECGs (16 RBBB, 9 LBBB, 4 paced, 20 septal Q waves, 16 dominantR wave in V1).43 had echo abnormalities (11 PA systolic pressure ⬎30mmHg, 5 RVH,8 valvular abnormalities, 8 LVH, and 11 decreased LVEF).Results: Troponin T positivity (⬎0.01-25 mcgs/l) predicted an adverse outcome for the entire cohort of 380 SSc patients with a 52.3% increased mortality from all causes. Of the 135 patients in whom cardiac investigations were available, only 8 were troponin T positive.Within this group there was no correlation with a variety of assessment criteria.Conclusion: Raised Troponin T levels predict an increased all-cause mortality in SSc. Establishing a causal link requires further investigation. Assessment criteria

Troponin T positive

Troponin T negative

Mortality from a cardiac cause Evidence of active myositis Features of overlap disease with a positive nRNP Abnormal ECG Abnormal ECHO Raised serum creatinine

0 4 1 7 1 0

5 7 4 58 42 5

Correlation of troponin T positivity with abnormal clinical findings in a cohort of 135 SSc patients Disclosure: Dr. Mukerjee is funded by an Entry Level Fellowship from the Wellcome Trust

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CLINICAL AND LABORATORY MEASURES OF DISEASE ACTIVITY IN SYSTEMIC SCLEROSIS. Douglas J Veale, Ursula M Fearon, Karen Henshaw, Hilary Johnston, Naomi Reay, Janet Pope, Daniel E Furst, The Scleroderma Clinical Trials Consortium United Kingdom; ON, Canada and Seattle

MEDICAL PREDICTORS OF DISABILITY, PAIN, AND PSYCHOSOCIAL ADJUSTMENT IN ADULT SSc. Vanessa L Malcarne, Ingunn Hansdottir, Ann McKinney, Renn Upchurch, Helen L Greenbergs, Daniel E Furst, Gretchen H Agee, Philip J Clements, Michael H Weisman San Diego and Los Angeles, CA; Seattle, WA

Objective: This study examines clinical and laboratory parameters of disease activity in Systemic Sclerosis (SSc) patients. Patients & Methods: 96 SSc patients, from 10 centers worldwide, were assessed clinically according to an agreed proforma (Delphi principle) to assess the skin, vascular, lung, heart, renal, gastroenterologic and neurologic systems, including ESR, CRP and scleroderma HAQ (SHAQ). Detailed investigations were available if clinically indicated by the local physician. Serum and plasma were obtained at the same time as clinical assessment following informed, written consent. Levels of TNF␣, OSM, IL1, IL2ra, IL6, IL8, IL10, interferong, TGF␤, VEGF, sICAM-1 and sE-selectin were measured by ELISA (R&D Systems, Oxon). Results: Significant associations were observed using spearmans rho between skin score, swollen joint count and diffuse pattern (r⫽.49, p⬍0.000; r⫽.23, p⫽0.04) respectively, which negatively correlated to anti-centromere antibody (r⫽-.69, p⬍.000). Skin score also correlated to the presence of digital ulceration (r⫽.24, p⫽0.02) and high ESR (r⫽.22, p⫽0.04). SHAQ correlated with CRP (r⫽.28, p⫽0.02) and Raynauds (r⫽.22, p⫽0.04), while CRP was associated with serum creatinine (r⫽.44, p⬍0.000), BUN, GFR (r⫽.34, p⫽0.007), muscle disease (r⫽.25, p⫽0.03), CPK (r⫽.34, p⫽0.02) and swollen joint count (r⫽.29, p⫽0.02). Correlations were noted for many circulating factors, e.g. between IL1, ICAM-1 (r⫽.29, p⫽0.005) and OSM (r⫽.28, p⫽0.007), and IL6 and IL10 with IFN␥ (r⫽.3, p⫽.005; r⫽.36, p⬍.000) respectively. Regression analysis indicated that IL10 predicted skin score (r⫽.24, r2⫽.06, p⫽0.025) , while IL8 and IL2ra levels predicted Raynauds (r⫽.34, r2⫽.11, p⫽0.007). IL2ra also predicted renal crisis (r⫽.44, r2⫽.19, p⬍.000) and BUN (r⫽.4, r2⫽.16, p⫽.001). Raised CK was predicted by ICAM-1 (r⫽.3, r2⫽.09, p⫽0.03) as was ground glass lung on HRCT (r⫽.38, r2⫽.14, p⫽0.01) . OSM predicted swollen joint count (r⫽.34, r2⫽.11, p⫽0.007). Cardiomegaly was strongly predicted by a model including IL6, IL10, VEGF and IL2ra (r⫽.61, r2⫽.33, p⬍0.000). Conclusion: These data demonstrate encouraging relationships between potential clinical and laboratory measures of disease activity. These data provide the basis for more detailed prospective and longitudinal studies of activity measures in SSc. Disclosure:

Objective. To determine the predictive relationship of medical symptoms to disability, pain, and psychosocial adjustment outcomes in patients with SSc. Method. 114 patients with SSc underwent a comprehensive clinical examination (including modified Rodnan skin score). Patients also completed standardized self-report questionnaires assessing disability and pain (the mHAQ) and psychosocial adjustment (the Psychosocial Adjustment to Illness Scale). Hierarchical multiple regression was used to identify significant predictors of disability, pain, and psychosocial adjustment; skin score was entered in the first step and variables that represented current disease symptoms and were significantly related to outcomes in bivariate analysis were entered in the second step. Path analysis was used to test mediational relationships. Results. Skin score was a significant predictor of disability (r2 ⫽ 0.29; p ⬍ .001), pain (r2 ⫽ 0.20; p ⬍ .001), and psychological adjustment (r2 ⫽ 0.08; p ⬍ .01), predicting 29%, 20%, and 8% of the variance, respectively. Significant predictors of disability were joint contracture, total joints tenderness, dependent edema, dyspnea, other extremity ulcers, total tenderpoints, and joint pain on motion, collectively explaining an additional 33% of the variance. Significant predictors of pain were dependent edema and joint tenderness, explaining an additional 21% of the variance. Significant predictors of psychosocial adjustment were postprandial bloating, joint pain on motion, and dependent edema, explaining an additional 34% of the variance. Disability and pain were statistical mediators of the relationship between disease severity and psychosocial adjustment. Conclusion. Skin thickening strongly predicted disability and pain, and moderately predicted psychosocial adjustment. After controlling for skin thickening, dependent edema was a significant predictor of all three outcomes. Joint tenderness, contracture, and pain, as well as postprandial bloating, dyspnea, and ulcers, were important medical predictors. Disability and pain accounted for the relationship between skin thickening and psychosocial adjustment. Disclosure:

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SELECTED CYTOKINES AND SOLUBLE PROTEIN MEDIATORS AS CORRELATES OF DISEASE ACTIVITY IN SYSTEMIC SCLEROSIS WITH DIFFUSE CUTANEOUS INVOLVEMENT (dcSSc). Noreen Fertig, Mary Lucas, Carol A Feghali, Timothy M Wright, Thomas A Medsger, Jr Pittsburgh, PA

RELATIONSHIP OF SKIN SCORE WITH CLINICAL, SOCIODEMOGRAPHIC, SEROLOGIC, BEHAVORIALPSYCHOLOGICAL, AND IMMUNOGENIC DETERMINANTS IN PATIENTS WITH EARLY SYSTEMIC SCLEROSIS. Michael Fischbach, Terry A McNearney, Alan W Friedman, Chul W Ahn, Carol A Feghali, Timothy M Wright, John D Reveille San Antonio, Galveston and Houston, TX and Pittsburgh, PA

A number of laboratory test results have been proposed as correlates of disease activity in systemic sclerosis (SSc). We examined the value of 4 potential markers of activity in dcSSc patients, including endothelin-1 (ET-1), a vascular marker, soluble interleukin-2 receptor (sIL-2R) and interleukin-6 (IL-6), immune system activation markers, and procollagen III peptide breakdown products (P3NP), a proxy for active fibrosis. ELISA assays were used to detect ET-1, sIL-2R, and IL-6 and a radioimmunoassay to identify P3NP. Fifty patients had a single serum sample, 5 had sera from two visits 0.8 - 5.8 years apart, and 5 followed longitudinally had 4 samples apiece over 2.8 - 10.3 years. Disease was judged by one physician as “active” or “inactive” on clinical grounds at the time that the serum was obtained, globally and for each of 9 organ systems. Activity status was judged blinded to the laboratory results. For patients with single serum samples, ET-1, sIL-2R, IL-6 and P3NP all discriminated between active and inactive disease with statistical significance (p ⬍ 0.001, p ⫽ 0.004, p ⬍ 0.001 and p ⬍ 0.001, respectively, Spearman’s rank correlation). In patients with serial samples, in most instances levels of these 4 proteins matched disease activity assessed clinically, i.e. active disease was correlated with higher serum levels and inactive disease with lower serum levels in the same patient. Increased levels of all 4 proteins also were correlated with active skin and heart disease (all p values ⬍ 0.03 to ⬍ 0.001). Increased ET-1 (p ⫽ 0.04) and sIL-2R (p ⫽ 0.022) levels were correlated with active kidney disease, but the latter correlation may be related to renal damage, as the sIL-2R level was correlated with the most recent serum creatinine level (p ⫽ 0.004). Nine of the 22 (41%) inactive disease single specimen patients were receiving an ACE-inhibitor drug at the time that the serum was obtained vs. 4 of 28 (14%) active disease patients (p ⬍ 0.04). Serum levels of cytokines and soluble protein mediators may be useful laboratory markers in the assessment of global and organ system disease activity in dcSSc. Disclosure:

Purpose: To identify factors that predict worsening skin involvment in a multi-ethnic cohort of patients with early scleroderma studied prospectively over 2 1/2 years. Methods: 176 patients (88 Caucasians, 57 Hispanics, 31 African Americans) with scleroderma ⬍ 5 years duration were evaluated bi-annually to determine the clinical, sociodemographic, serologic, behavorial-psychological, and immunogenetic factors that influence disease outcome. Skin thickening was assessed using the modified Rodnan scale. GEE (generalized estimating equation) analysis was used to identify significant predictors of skin score changes over time. Results: Skin score changes during 2 1/2 years of observation were not related to ethnicity, behavorial-psychological factors, or HLA genes. Significant predictors were leukopenia, anti-RNA polymerase 1 antibodies, calcinosis cutis, diabetes and unemployment. Parameter WBC count Anti-pol 1 Diffuse skin Calcinosis cutis Employment Diabetes

Z score

P value

-9.41 3.98 7.43 2.07 -2.12 2.55

⬍.0001 ⬍.0001 ⬍.0001 0.0381 0.0340 0.0108

Significant changes in skin scores during 2 1/2 years were noted in diffuse but not limited scleroderma. In this cohort, skin scores did not correlate with internal organ damage such as pulmonary, GI, or cardiac involvement. Conclusion: The pathogenic mechanisms by which these laboratory, sociodemographic, and co-morbid conditions affect skin involvment is not clear. Nevertheless, identifying these predictors suggests potential areas for intervention and future molecular investigations. Disclosure:

1649

THE PSYCHOLOGICAL IMPACT OF SYSTEMIC SCLEROSIS. Ariane L Herrick, Helen L Richards, Kerry Griffin, Petra Gwilliam Salford and Salford, United Kingdom

PREDICTORS OF SELF-REPORTED PHYSICAL AND MENTAL FUNCTIONING IN SYSTEMIC SCLEROSIS. Alan W Friedman, Terry A McNearney, Michael Fischbach, Randall W Johnson, Carol A Feghali, Timothy M Wright, Chul W Ahn, John D Reveille Houston, Galveston and San Antonio, TX and Pittsburgh, PA

Purpose: Our purpose was to test the hypothesis that patients with systemic sclerosis (SSc) have a high prevalence of anxiety, depression, and dissatisfaction with body image, and to relate these parameters to SSc-related symptoms. Methods: 46 consecutive patients with SSc provided basic demographic information and completed a range of standardised psychological assessments measuring anxiety, depression and attitudes towards the body. Patients also completed a questionnaire designed to evaluate specific SSc-related clinical problems. Spearman’s correlation coefficients, Mann-Whitney U tests and stepwise multiple regressions were employed in the analysis of data. Results: 35.6% and 17.8% of patients sampled were classified as being probable cases for anxiety and depression respectively. Self-rated severity of SSc-related symptoms was not associated with either. Anxiety was significantly related to the extent to which patients considered their body to be a source of shame and disparagement (p⬍ 0.05). Younger patients were more dissatisfied with their bodies (p⬍ 0.01) and younger age was also associated with increased anxiety (p⬍0.05) but not depression. Further, a younger age at recalled diagnosis was associated with greater feelings of shame towards the body (p⬍0.01). Duration of the condition had no impact. Age, age at recalled diagnosis and duration of SSc were not associated with self report of severity of symptoms. Patients who experienced difficulties in relation to eating and toileting had significantly more depression (p⬍0.05), and those with eating problems had significantly more shameful feelings about their body (p⬍0.05). No such differences were apparent for those patients who had experienced changes in hand function, hand appearance or facial appearance. Stepwise multiple regression indicated that age at recalled diagnosis was the most important variable in relation to body dissatisfaction accounting for 28% of the variance (F[1,36] ⫽ 14.1, p⬍0.001). Anxiety contributed a further 13% (F[1,35] ⫽ 7.7, p ⬍0.01) and difficulties relating to eating an additional 5.2% (F[1,34] ⫽5.2, p⬍0.05). Severity of symptoms, current age and depression did not meet the entry criteria for analysis. Conclusion: The results suggest desynchrony between physical and psychological factors in patients with scleroderma. The association between younger age, a younger age at diagnosis and psychological difficulties, and the absence of symptom severity or chronicity in this relationship highlights the importance of paying special attention to the psychological needs of young patients. Holistic assessment of patients including psychosocial as well as clinical assessments may be pertinent in optimising patient management.

PURPOSE: To determine predictors of self-reported physical and mental functioning in Systemic Sclerosis (SSc) from 3 ethnic groups comprising the GENISOS cohort. METHODS: 176 SSc patients of Hispanic (H, n⫽57), African-American (A, n⫽31) and Caucasian (C, n⫽ 88) ethnicity with disease duration ⱕ 5 years were enrolled in a longitudinal study of outcomes. Sociodemographic, Clinical, Immunogenetic, Immunologic and Behavioral variables were ascertained at baseline, and every 6 months thereafter. Self-reported physical and mental functioning were assessed using the physical and mental component summary scores (PCS and MCS, respectively) of the MOS Short Form-36, and physical disability was assessed using mHAQ. Higher PCS and MCS scores indicate better function, while higher mHAQ scores indicate greater disability. Independent predictors of self-reported function over time were identified using Generalized Estimating Equation (GEE) multivariable regression analyses (377 data points each for PCS and MCS; 406 points for mHAQ).RESULTS:

Disclosure:

outcome

predictor

t-statistic

p-value

PCS

creatinine unemployed helplessness fatigue anti-RNA polymerase II sclerodactyly HLA-DQB1*05 musculoskeletal involvement anti-fibrillarin skin score African-American employed social support digital gangrene abnormal illness behaviors anti-RNA polymerase II musculoskeletal involvement

-5.7 -5.1 -4.6 -4.5 -3.6 -3.4 -3.0 -2.8 -2.0 3.9 3.2 -3.0 -2.6 2.5 -9.6 3.5 3.1

⬍ 0.0001 ⬍ 0.0001 ⬍ 0.0001 ⬍ 0.0001 0.0003 0.0007 0.0024 0.0057 0.0445 0.0001 0.0013 0.0024 0.0090 0.0120 ⬍ 0.0001 0.0005 0.0017

mHAQ

MCS

CONCLUSIONS: Self-reported physical and mental functioning over time in early SSc are predicted by a combination of clinical features, autoantibodies, socioeconomic and behavioral variables, and a single immunogenetic factor. Ethnicity was only predictive of disability by mHAQ, with African-Americans developing greater disability over time. Disclosure:

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SEXUAL DYSFUNCTION IN MEN WITH RHEUMATIC DISEASES: IS SCLERODERMA POSITIVELY CORRELATED WITH IMPOTENCE? P Hong, J Pope, J Ouimet London, ON

CLINICAL WEAKNESS AND FATIGUE IN CREST AND SYSTEMIC SCLERODERMA PATIENTS CORRELATE WITH MUSCLE ABNORMALITIES. Jane H Park, Kenneth J Niermann, Lauren B Adams-Gandhi, Rebecca J Meyer, Lloyd E King, Nancy J Olsen Nashville, Tennessee

Prevalence and time of onset of impotence in progressive systemic sclerosis (PSS) is variable in past studies. To ascertain the correlation between sexual dysfunction and PSS, we surveyed men with PSS and 3 age matched controls with rheumatoid arthritis (RA) selected randomly from the same practice. We sent a questionnaire assessing impotence-related factors to 56 men (14 scleroderma, 42 RA). Ten PSS subjects and 27 RA have responded. The mean age of respondents was 52 yrs (range 39 to 72). No statistical differences were found between PSS and RA groups with regards to age, steroid use, smoking, alcohol consumption, height, years of marriage or number of biological children. The prevalence of ever having impotence was 100% (PSS) and 48% (RA), P⬍0.004. When subjects rated quality of sexual relationships, the frequency of difficulty acheiving erections, and satisfaction with intercourse, no differences were found between PSS or RA. When rating their confidence regarding erections, 100% of men with PSS indicated a very low to moderate confidence level, while 62% of RA indicated the same, P⬍0.02. Similarly, 70% of PSS and 28% of RA subjects could rarely maintain their erections until completion of intercourse, P⬍0.02. One-hundred percent of PSS and 56% of RA had noticed a change in their sexual function, P⬍0.01. Of subjects who had impotence, 81% (both PSS and RA) reported it occurring after disease onset. All PSS subjects had Raynaud’s phenomena (RP) compared to 16% RA, P⬍0.0001. Ninety-two percent of subjects with RP had noticed a change in their sexual function, P⬍0.008. Of RA subjects with RP (N⫽4), 75% had experienced impotence, versus 39% of RA without RP, P⬍0.18. Possible confounding factors for impotence were examined; all except nerve damage were insignificant for predicting the likelihood of noticing a change in sexual function. Of the 56% of subjects surveyed who had sustained nerve damage, 95% had also had a change in sexual functioning, P⬍0.0005. PSS subjects were not more likely than RA to have experienced nerve damage (70% PSS; 54% RA, P⬍0.37). Steroid use was considered; 92% of subjects having taken steroids rarely thought about difficulty with erections as compared to 59% of men not on steroids, P⬍0.04. Men having taken steroids were more likely to report erections that were often hard enough for penetration (83% versus 50%), P⬍0.05. Preliminary data suggest that with a larger sample size, the hypothesis that secondary impotence due to vascular factors often occurs in PSS may be supported. RP may be a risk factor for impotence. Disclosure:

Fatigue and weakness are frequently reported as clinical symptoms by scleroderma patients. In order to account for these symptoms, metabolic abnormalities in muscles were characterized using phosphorus-31 magnetic resonance spectroscopy (P-31 MRS). The patient population consisted of 13 patients with a wide range of disease activity from the limited CREST form to severe progressive systemic scleroderma (PSS). Quadricep muscles were examined using a 1.5T magnet to obtain spectra during rest, two levels of graded exercise, and recovery. Eleven normal controls were studied using the same protocol. The levels of ATP and phosphocreatine (PCr), the high energy phosphate compounds required for muscle contraction, were quantitatively determined. In the muscles of scleroderma patients, ATP and PCr levels were 28% lower at rest as compared to normal controls (P⬍0.001). The decreases in ATP and PCr became more pronounced during exercise. Free and ATP-bound magnesium (Mg) levels were also calculated from the spectra according to the method of Gupta. Magnesium is important because it is required for all enzymatic reactions involving the ATP utilized in muscle contraction. During exercise, free magnesium levels in the patients were 30% lower than in controls (P⬍0.014), and the enzymatically active ATP-Mg complex was 38% below normal (P⬍0.0001). Following exercise, PCr recovery rates were determined by fitting to a single exponential equation. In the scleroderma muscles, PCr recovery half-time (Tc) was about twice as long as that of normal muscles (62 vs 113 sec; P⬍0.002), and initial rates of PCr recovery were about one half that of the controls (2.8 vs. 6.1 mmoles/kg/min; P⬍0.004). Impaired PCr recovery reflects defects in mitochondrial oxidative phosphorylation. The low levels of ATP, PCr, and Mg, as well as the delayed PCr recovery rates, were not significantly different in the CREST and PSS subsets. Biochemical defects were present in both groups of patients, suggesting myopathy as a primary abnormality. In summary, metabolic abnormalities in the muscles support a biochemical basis for weakness and fatigue reported by CREST and PSS patients. Disclosure:

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1654

QUALITY OF LIFE AND SEXUAL FUNCTION IN WOMEN WITH SYSTEMIC SCLEROSIS (SSc). Jennifer A Guerriere, Raymond C Rosen, James R Seibold New Brunswick, NJ

RENAL RESERVE STUDIES(RRS),MICROALBUMINURIA(UAE) AND NAIL FOLD CAPILLAROSCOPY(NFC) IN PATIENTS WITH SYSTEMIC SCLEROSIS(SSc) AND WITHOUT OVERT RENAL DISEASE(WRD). Valderilio Feijo Azevedo, Francisco Alves Bezerra Neto, Jose Gastao Rocha de Carvalho, Sebastiao Cesar Radominski, Luis Eduardo Coelho Andrade Curitiba, Parana, Sao Paulo, Sao paulo and Curitiba, Parana, Brazil

Few data are available regarding female sexual function and quality of sexual life in women with SSc. We surveyed 101 consenting outpatients utilizing the validated Index of Female Sexual Function (IFSF); the SF-36 as a more global measure of quality of life; and a survey specific questionnaire about impact of various SSc features on quality of sexual function. Data were correlated with descriptive information on extent and severity of SSc. 60 (59.4%) of 101 women were sexually active. Primary reasons for inactivity were lack of partner (15%), personal choice (13%), and partner health (8%) with only 7% of subjects attributing inactivity to their underlying SSc. IFSF scores were 24.9⫹6.7 which are significantly below healthy controls (30.5⫹5.3) and significantly better than women with FSAD (female sexual arousal disorder)(19.2⫹6.6). All domains of the IFSF were reduced including arousal, lubrication, desire, pain and satisfaction. Quality of sexual life was highly correlated with SF-36 Summary Score (r⫽0.528, P⬍0.001) and the Mental Component Score of the SF-36 (r⫽0.540, P⬍0.001) but was unrelated to the Physical Component Score (r⫽0.194, P⫽0.137). This may in part reflect the absence of sexual function questions in the SF-36. Patients self-identified fatigue, generalized pain, vaginal dryness and vaginal discomfort as leading SSc symptoms affecting IFSF. IFSF scores were unrelated to age, disease classification and duration. Multivariate analysis identified vagina dryness, vaginal discomfort, Raynaud phenomenon and depression as key determinants of reduced IFSF. Women with SSc are sexually active independent of their burden of SSc-related physical difficulties. Complete clinical management should focus on gynecologic health. Our data identify a sexual dysfunction that is predominantly physiologic and which may be amenable to pharmacologic intervention. A followup large-scale trial of a long acting Type V cGMP phosphodiesterase inhibitor for both Raynaud phenomenon and sexual effect is in progress.

Objectives: Considerable evidence indicates that UAE is an intermediate risk marker for renal and cardiovascular prognosis. Although vascular disease is a proeminent finding in SSc, changes in glomerular hemodynamics and UAE induced by amino acids infusion (AAI) are insufficiently known in SSc patients without renal disease. The present study was undertaken to analyse the effects of an four-hour AAI on glomerular filtration rate(GFR) and UAE, compared to baseline studies in the previous 24 hours, and to evaluate NFC in SSc patients and WRD. A control group of 14 normal individuals was also studied.Methods, materials and analytical procedures:an interventional protocol was performed in 20 female SSc patients and WRD, and in 14 normal female individuals, by means of an AAI (500 ml of a 10% solution, without addition of carbohydrate and eletrolytes) administered over a 4-hour period, under moderate water load. Changes in GFR as measured by creatinine clearence and UAE(Euro-DPC‘s Double Antibody Albumin) were evalueted.Capillaroscopy reading evalueted the numbers of loops per millimeter and ectatic loops according Andrade et al. and deletion score according Lee et al.Results:baseline GFR levels were lower in SSc patients(78,9 plusminus 19,8ml/min/1,73m2 x 103,3 plusminus 29,8ml/min/1,73m2 in normals, p ⬍ 0,007) but no differences between groups were observed in baseline and stimulated UAE. AAI induced significant changes in GFR and UAE, from 78,9/ml/min/1,73m2 to 126,4ml/min/1,73m2(p ⬍ 0,001) and 6,5 mg/min to 29,3 mg/min, (p ⬍ 0,001) . The ectatic loops correlate with baseline GFR and stimulated GFR(p ⬍ 0,01 and p ⬍ 0,04, respectively).Conclusions:The most proeminent finding in this population was preserved Renal Reserve in spite of decreased GFR. Basal as well as stimulated UAE levels were similar in both groups. The presence of ectatic loops it seems indicate higher baseline and stimulate GFR.

Disclosure:


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1652

1655

THE RISK OF CARDIAC ARRHYTHMIAS IN PATIENTS WITH SYSTEMIC SCLEROSIS(SSc). S Kontoyiannis, E Gialafos, A Zelides, P Gounopoulos, E Moulopoulou, C G Katsiari, A Nana, M Mavrikakis, Ch Kostopoulos Athens, Greece

INVESTIGATION OF ANORECTAL FUNCTION IN PATIENTS WITH PROGRESSIVE SYSTEMIC SCLEROSIS. Sung Won Lee, Hong Jo Choi, Won Tae Chung Busan, Korea

Cardiac arrhythmias (CA) have been described in patients with SSc having myocardial involvement. Aim of the study was the identification of SSc patients at risk for CA without clinical evidence of cardiac involvement by using the presence of atrial (ALP) and ventricular (VLP) late potentials on signal averaged electrocardiography (SAECG). Twenty SSc patients (16 females, 4 males, aged 52⫾6 years, mean disease duration 7⫾5 years) without clinical evidence of cardiac disease underwent SAECG as well as DopplerEchocardiography (ECHO), thoracic high resolution computed tomography (HRCT) and pulmonary function tests (PFTs) in which spirometry, lung volume and diffusion capacity measurements were included. Forty seven healthy controls were studied by SAECG in parallel. The presence of ALP was defined as P-wave duration (Pd) ⬎120ms and root mean square of the last 20ms of P-wave(RMS20) ⬍3.5 ␮V. The presence of VLP was defined as QRS duration (QRSd)⬎114ms, low amplitude signals (LAS)⬎38ms and root mean square(RMS) ⬍20␮V. ALP were found in 16 of 20 patients (80%) and 1 of 47 controls (2%), whereas VLP were found in 8 patients (40%) and 1 control individual (2%). The mean values⫾SD of the SAECG measurements for patients and controls were: Pd⫽125⫾17 vs 113⫾18 (p⬍0.01), RMS20⫽2.8⫾1.2 vs 5,2⫾2,5␮V (p⬍0,05), QRSd⫽103⫾16 vs 94⫾9 (p⬍0.05), LAS⫽38.5⫾15 vs 25.3⫾9.7 ms (p⬍0.01) and RMS⫽33.4⫾29 vs 54.5⫾39 ␮V (p⬍0.001), respectively. ECHO was abnormal in 12 (60%), HRCT in 13(65%) and PFTs in 10 (50%) patients. There was no significant association between ALP or VLP with ECHO, HRCT, PFTs findings and disease duration. These findings indicate that SSc patients without clinical cardiac disease are prone for arrhythmias. The risk of arrhythmias exists in the absence of possible cardiac or concomitant pulmonary involvement and is not related to disease duration. The prognostic significance of these findings needs further study.

Purpose: The aim of this study was to investigate the anorectal function in patients with progressive systemic sclerosis (PSS), thus to define the clinical role of anorectal manometry in the earlier diagnosis of anorectal involvement of PSS. Methods: Seventeen consecutive patients (all females) with PSS were evaluated with anorectal manometry by the stationary pullthrough technique using the 8-channel hydraulic capillary infusion system for anorectal function. Functional parameters of the manometry were compared between patients with PSS and 20 normal control subjects, matched for age and sex. Results: The mean resting pressure over the high pressure zone (HPZ) in patients with PSS was significantly lower than that in the control group (70.8⫾3.4 mmHg vs. 81.5⫾3.2 mmHg: P⫽0.046). The HPZ in patients with PSS was also significantly reduced compared with that in the control (1.5⫾0.1 cm vs. 2.5⫾0.1 cm: P⫽0002). The rectoanal inhibitory reflex was detected in only 10 patients (59%) in the PSS group, but was present in all except one (95%) in the control (P⫽0.022). More interestingly, RAIR in patients with PSS responded at a higher volume of the air insufflated than that in the control (74% vs. 30% at 20 cc, 21% vs. 30% at 30 cc, and 0% vs. 40% at 50cc, respectively: P⫽0.031). Other functional parameters, including maximal squeeze pressure, minimal sensory and maximal tolerable volume of the rectum, and rectal compliance were not significantly different between two groups. Conclusions: Anorectal involvement reflected by the anorectal manometric dysfunction may be rather an earlier event in patients with PSS. An awareness to perform an anorectal manometric study in every case of PSS may be necessary for earlier subclinical detection of anorectal involvement by the disease. Disclosure:

Disclosure:

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1659

EXOCRINE PANCREATIC FUNCTION IN SYSTEMIC SCLEROSIS: FECAL ELASTASE AS A SPECIFIC, NONIVASIVE TEST TO DETECT EXOCRINE PANCREATIC INSUFFICIENCY. Axel M Marzeion, Dagmar A Meuser, Reinhold G Bretzel, Hans U Kloer, Michael Berliner Giessen, Germany

CIGARETTE SMOKING: A RISK FACTOR FOR DIGITAL VASCULAR COMPLICATIONS IN SYSTEMIC SCLEROSIS. Beverley J Harrison, Alan J Silman, Sam Hider, Ariane L Herrick Manchester and Salford, United Kingdom

Objective: Malabsorption is well known in patients of systemic sclerosis and is regarded as being connected with small bowel bacterial overgrowth secondary to stasis. In addition to this, exocrine pancreatic dysfunction due to pancreatic fibrosis may also be present in a number of patients. The current study is the first to investigate exocrine pancreatic function by means of fecal elastase in patients with systemic sclerosis. Methods: A total of 21 unselected patients with systemic sclerosis were studied. Each patient completed a questionnaire relating to specific gastroenterological complaints. Measurement of serumlipase and -amylase was performed. All patients underwent an ultrasound scanning of the pancreas and determination of the fecal elastase in two independent samples of stool. All patients were also screened for diabetes mellitus using the oral glucose tolerance test. Results: With the exception of one all patients displayed symptoms of gastrointestinal complaints pursuant to the questionnaire. Complaints reported did not indicate any particular bias towards one particular symptom or condition and did not appear initially to be su ¨ pecifically related to exocrine pancreatic function. Measurement of serumlipase and -amylase were in the normal range; only one patient displayed serumamylase of more than twice the normal level. Ultrasound scanning showed in none of the patients atrophy or calcification of the pancreas. Five Patients (23,8%) showed decreased fecal elastase levels indicating a dysfunction of the exocrine pancreas. In the case of one patient the oral glucose tolerance test showed an impairment of glucose tolerance; a further patient had been known to be diabetic (diabetes mellitus type 2) for eight years. Discussion: The study demonstrates that in systemic sclerosis in spite of normal levels of serumlipase and -amylase and unspecific findings in ultrasound scanning of the pancreas symptoms of gastroenterological dysfunction may be due to exocrine pancreatic insufficiency evidenced by reduced fecal elastase. This shows that involvement of the pancreas due to fibrosis could become relevant in the course of systemic sclerosis. Conclusion: Our findings show that reduced fecal elastase due to chronic pancreatitis was present in 23,8% of patients examined in the study. Since a test of fecal elastase is noninvasivly and simple it would appear sensible that all patients with systemic sclerosis should routinely undergo this test; all the more so since such testing provides valuable data for the treatment by means of the replacement or supplementation of pancreatic enzymes.

Background: Patients with systemic sclerosis (SSc) are at high risk of vascular complications, including digital amputation and gangrene. Cigarette smoking is an important risk factor for vascular disease in the general population. Only one study has investigated the effect of smoking on vascular outcome in SSc, and reported that smoking was not associated with digital loss (Arthritis Rheum 1993;36:285-7). Aim: To investigate the influence of cigarette smoking on digital ischaemia in patients with SSc. Patients & methods: 101 patients with SSc (87F 14 M; median age 53 years). The median disease duration (from onset of Raynauds) was 13 years. All patients were assessed annually by a single observer (ALH). Smoking history was classified as (i) current smoking status; and (ii) total no. pack years. Digital ischaemic events were defined as (i) surgical debridement; (ii) admission for intravenous (IV) vasodilators; and (iii) digital amputation. The influence of smoking on digital ischaemic events was explored using logistic regression techniques, adjusting for age, sex and disease duration. Results are expressed as odds ratio (OR) and their 95% confidence intervals (95% CI). The referent group is patients who had never smoked. Results: 17 patients had one or more debridements, 46 were admitted for IV vasodilators and 15 had one or more digital amputations. After adjusting for age, sex and disease duration, current smokers were significantly more likely to have required debridement (OR 4.5; 95% CI [1.1, 18.3]) and admission for IV vasodilators (OR 3.8 [1.1, 12.9]). Smokers were also more likely to have had digital amputation (OR 3.4 [0.8, 15.1]). Smoking had no influence on anticentromere antibody (ACA) status, and adjusting for ACA did not affect the results. Amongst current and ex-smokers, smoking intensity (pack years) was associated with admission for IV vasodilators, but not with debridement or amputation. Conclusion: Amongst patients with SSc, current smokers are 3 to 4 times more likely to incur digital vascular complications. Resources should be directed to supporting smoking cessation in SSc.

Disclosure:

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1660

GASTRIC EMPTYING, MYOELECTRIC ACTIVITY AND UPPER GASTROINTESTINAL SYMPTOMS IN PATIENTS WITH DIFFUSE AND LIMITED SYSTEMIC SCLEROSIS. Karin Franck-Larsson, Hans Hedenstrom, Rolf Dahl, Anders Ronnblom Uppsala, Sweden

TREATMENT OF REFRACTORY DIGITAL ULCERATIONS AND VASCULITIC ULCERATIONS IN SCLERODERMA. Marc R Pritzker, Walter Dorman, Erskine Caperton

Objectives: In this study we investigate the gastric function in patients with diffuse and limited systemic sclerosis (SSc), using gastric emptying with a radiolabeled meal and electrogastrography (EGG). We also determine the correlation between these objective tests and upper gastrointestinal symptoms. Methods: Gastric function was examined in 16 patients with diffuse SSc (dSSc), 14 patients with limited SSc (lSSc) and 15 healthy control subjects with scintigraphic gastric emptying after intake of a radiolabeled meal and simultaneous registration of gastric myoelectric signals by EGG with cutaneous electrodes. The frequency of upper gastrointestinal symptoms was determined by a questionnaire in patients and controls. Results: Out of 30 patients with SSc 20 had normal gastric emptying. Patients with the diffuse form of the disease had more disturbances in their gastric emptying than patients with the limited form. Recordings of EGG did not differ between patients and controls. Seventeen (57 %) of the patients had more subjective complaints from the upper gastrointestinal tract than the control persons but patients with high and low frequency of symptoms did not differ in gastric function as measured with gastric emptying and EGG. There was no correlation between gastric emptying and EGG. Conclusions: Contrary to earlier beliefs we found that a majority of the patients with SSc had normal gastric emptying but a few individual patients had a pronounced delayed gastric emptying. Neither EGG nor a thorough review of upper gastrointestinal symptoms could predict delayed gastric emptying and we thereby concluded that gastric emptying still is the golden standard for detecting gastric dysfunction in patients with SSc. Since there was no correlation among the patients between upper GI-symptoms and gastric emptying, other mechanisms may be responsible for these symptoms, i e a disturbed fundic relaxation or small bowel involvement. The higher prevalence of delayed gastric emptying in dSSc indicates a more severe involvement of the gastrointestinal tract in dSSc than in lSSc.

Digital and extremity ulcerations refractory to “conventional” treatment are a source of morbidity in patients with crest variant scleroderma and may result in amputation or skin grafting. We utilized a regimen of brief infusion of prostacyclin (3-5days) followed by high dose (7-9mgs/kg/day) of persantine to heal ulcerations (10 digital, 4 extremity) in 14 patients with ACR criteria crest variant scleroderma. All patients had failed calcium channel blocker, topical and systemic nitrates and in two cases intra-arterial infusions. Pain relief without narcotics and increased skin temperature were documented in all patients during prostacyclin infusion. Pain relief, increased skin temperature and lesion healing were documented in all patients within 6 weeks of therapy initiation. Episodes of Raynaud’s were diminished by greater than 50% in all patients. Epidsodes were all felt to be of lesser intensity in all patients. No new lesions appeared during the period of therapy. Relapses (new lesions) were documented in 3 patients on therapy greater than 6months following the start of therapy. This novel therapeutic regimen appears to have promise in treating patients with chronic, refractory ulcerations. Disclosure:

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USEFULNESS OF ERYTHROMYCIN (E) VS METOCLOPRAMIDE (M) IN THE GASTRIC EMPTYING OF PATIENTS WITH GASTROPARESIS DUE TO SCLERODERMA (GS). Olga Vera, Luis E Rodriguez, Raul C Ariza, Alejandro Rodriguez, Juan M Miranda, Luis J Jara Mexico, DF, Mexico

EXHALED NO LEVELS IN SSC PATIENTS TREATED WITH PGE1-␣- CYCLODEXTRIN (PROSTAVASIN®) FOR SEVERE RAYNAUD’S PHENOMENON. Maria Rosa Pozzi, Elisabetta Allevi, Luca Ronchi, Marco Gardinali, Maria Grazia Gorgoglione, Stefano Carugo, Luciano D’Angelo, Elisa Banfi, Maria Robuschi, Sebastiano Bianco, Roberto Stabilini Monza, Milano, Italy

Treatment

Erythromycin

Metoclopramide

P value

Gastric Emptying Baseline x⫾ SD (min) Gastric Emptying Final x⫾ (min)

46.66 ⫾ 18.68 29.58 ⫾ 6.41

50.18 ⫾ 23 42.68 ⫾ 18.5

NS ⬍0.01

Disclosure:

Introduction. Recent studies suggest that NO synthesis is impaired in SSc. Patients with active fibrosing alveolitis have increased levels of exhaled NO (e-NO), while those with pulmonary hypertension show reduced levels of e-NO. Inhaled iloprost seems to reduce pulmonary arterial pressure and increase e-NO at the same time. We previously demonstrated that PGE1-␣cyclodextrin (PGE1) improves Raynaud symptoms and reduces parameters of endothelial damage and/or activation in SSc such as sICAM-1, von Willebrand factor and tissue plasminogen activator. We were interested therefore to evaluate whether PGE1 treatment modifies also e-NO levels. Patients and methods.Fifty-two SSc (51 F, 1 M) patients were studied; 30 received PGE1 i.v.(60␮g/3 hrs) for five consecutive days; two additional three-day cycles of infusion were performed at 4 wks interval during winter. We measured e-NO on a chemiluminescence analyzer according to the European Society recommendations. Patients performed a vital capacity test over 20 to 30 sec, with a constant flow of 5 to 15 lt/min, against a resistence (15 cm H20) in order to exclude nasal NO. Results. Mean e-NO levels (⫾SD) of all SSc patients were 10.6 ⫾6.7 ppb. They were not significantly different from the controls. The patients who had a coefficient of diffusion ⬍85% (n⫽4), a vital capacity ⬍95% (n⫽5) and a mean arterial pressure ⬎40 mmHg (n⫽7) apparently did not have different e-NO levels. In the patients treated with PGE1 e-NO levels increased.E-NO mean levels rose from 9.1⫾5.3 to 16.5 ⫾11.2 ppb (Wilcoxon’s signed rank test p⬍0.002) after five-day infusion of PGE1. E-NO returned to basal levels 4 wks later, before the 2nd cycle (9.5⫾7.0 ppb). They rapidly increased again to 16.3⫾13.2 ppb (p⬍0.002) after PGE1 three-day infusion. The same thing was observed before and after the 3rd cycle (7.7⫾6.4 vs 12.9 ⫾ 8.1 ppb; p⬍0.002). Conclusions.These data suggest that PGE1 increases NO synthesis and that NO release contributes to the vasodilating effect of this drug. Disclosure:

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Background: Gastroparesis is an important cause of morbidity in scleroderma. The treatment of GS is based on the use of prokinetic agents, but the results in some cases have not been successful. The erythromycin acts in manner similar to motilin to stimulate gastric emptying. Objective: To investigate the usefulness of erythromycin vs metoclopramide in gastric emptying in patients with GS. Patients and Methods: We studied 27 patients ( 26 women and 1 man) with a mean age of 46⫾ 19 years; all patients met ACR criteria for scleroderma and they had delayed emptying gastric ⬎ 20 min. The patients were randomized in 2 groups: Group 1 received 250 mg of E 20 min preprandial ( tid) and Group 2 took 10 mg of M 20 min preprandial (tid) during four weeks. Gastric emptying was measured by scintigraphy at the onset of the study, and one week after starting the treatment with E and M. A questionnaire was applied to study the frecuency and intensity of early saciety, nausea, vomiting, epigastric pain and pirosis at the start and at the end of the study. Results: A comparison of the gastric emptying between the two groups is shown in the following table.There was a significant decrease in the frecuency of vomiting and in the intensity of pirosis in patients treated with erythromicin in comparison with metoclopramide (p⬍0.05). Conclusion: Erytromycin is well tolerated and more efective than metoclopramide for improving the gastric emptying of patients with GS. Other studies are necessary to confirm our results.[table]

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LONG TERM ILOPROST THERAPY OF SEVERE RAYNAUD’S PHENOMENON IN CONNECTIVE TISSUE DISEASES. Angelo Spano’, Gabriella Loi, Rosa De Luca Bossa, Carlo Venditti, Alfonso Oriente, Michele Cimmino, Antonietta Lupo, Antonio Del Puente, Pasquale Oriente Naples, Italy

A 17 POINT DERMAL ULTRASOUND SCORING SYSTEM - A ROBUST MEASURE OF SKIN INVOLVEMENT IN PATIENTS WITH SYSTEMIC SCLEROSIS. Tonia L Moore, Breda McManus, Mark Lunt, Marina E Anderson, Ariane L Herrick Salford and Manchester, United Kingdom

Objective: To evaluate the efficacy and safety of Prostacyclin-analogue (PGI2) Iloprost in the treatment of severe Raynaud Phenomenon (RP) refractory to oral vasodilatatory treatment in patients affected by Connective Tissue Diseases (CTD). Patients and Method: 20 Patients (16 Female and 4 Male; mean age 46⫾12.13; mean disease duration 6.11⫾4.5), 11 fulfilled the ACR criteria for Systemic Sclerosis (SSC), 3 for localized Scleroderma (SD), 2 for Systemic Lupus Erythematosus (SLE) and 4 for Unclassified Connective Tissue Diseases (UCTD) were enrolled in this study. They received Iloprost (0.5-2 ng/Kg body weight/min) at a constant infusion rate (ranging from 10 ml/h to 40 ml/h) i.v. on 3 consecutive days (12 h/day) for 12 times every 3 weeks. Concomitant immunosoppresive treatment during the period of the study was allowed. Clinical status (pain evaluation; skin colour; number of attacks; symptoms duration; presence of ischaemic lesions; ulcers healing; Visual Analogue Scale of patient assessment: VAS), laboratory parameters and nailfold video capillaroscopy were considered at the baseline and after 3, 6 and 12 infusions. We defined Responders (R) to treatment 3 groups of patients with either one or more of these characteristics: I) ⱖ50% n. of attacks, ⱖ50% symptoms duration,ⱖ75% of VAS (R-1); II) regression of ischaemic lesions and ulcers healing (R-2); III) marked reduction or regression of microhemorrhage in capillaroscopic pattern (R-3). Results: results are shown in Table I. No relevant adverse effects were observed. Mild headache, hot flushing, nausea and joint pain were occasionally referred during the infusions. Conclusions: Severe RP is a critical condition that affects connective tissue disease patients, mainly reducing their quality of life. Traditional therapies are often inadeguate to control this disorder. Our encouraging data suggest to consider long term intermittent i.v. Iloprost therapy as a possible alternative in RP therapeutical management.

Purpose: Skin involvement in systemic sclerosis (SSc) predicts internal organ involvement and mortality, therefore a reliable measure which is sensitive to change would be of enormous benefit in facilitating clinical trials. Although high resolution ultrasound has been advocated as a method of quantifying skin thickness in patients with SSc, previous studies have assessed only a limited number of body sites, potentially reducing the sensitivity of the technique to detect change over time, especially in patients with diffuse cutaneous disease. Our objectives were to (a) measure dermal thickness at multiple sites (17 - corresponding to those assessed using a modified Rodnan skin score) in patients with SSc compared to healthy control subjects and (b) assess intra-and inter-observer variability of the technique. Methods: Dermal thickness (from a 2-dimensional B-mode image obtained using a 20 MHz ultrasound probe) was measured at 17 sites (right and left middle finger, hand, forearm, upper arm, thigh, leg, foot: and forehead, anterior chest and abdomen) in 39 patients with SSc (26 limited cutaneous, 13 diffuse) and 34 healthy controls. Both ‘sum’ (of the 17 sites) and ‘max’ (maximum skin thickness at any site) were documented. To assess inter-observer variability, 35 patients and 33 controls were examined by a second observer. To assess intra-observer variability, 20 patients and 15 controls were studied twice by the first observer. Since the normal skin thickness varies between the different sites assessed, each measurement was converted to a z-score by subtracting the mean thickness in controls at that site and dividing by the standard deviation. Results: The reliabilities are given in the Table. The increased reliability in patients is explained by the greater variation in skin thickness in SSc patients compared to the controls.

TABLE I

After III infusion

After VI infusion

After XII infusion

N. patients/parameter

R-1 R-2 R-3

6 (30%) 2 (25%) 3 (21%)

12 (60%) 6 (75%) 8 (57%)

14 (70%) 7 (88%) 8 (57%)

20 8 14

Dermal z-score (sum) Dermal z-score (max)

Cases Inter 0.80 0.63

Intra 0.96 0.93

Inter 0.97 0.94

Although the maximum z-score was less reliable than the sum, it was a better discriminator between cases and controls (Area under ROC curve ⫽ 0.73 vs 0.47, p ⬍ 0.0001). Conclusion: Our results suggest that the 17 point dermal ultrasound scoring method is extremely reliable, particularly in SSc patients. Therefore the technique has the potential of being able to record small but clinically important changes over time, especially in patients with widespread skin involvement. Prospective studies are now required.

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OPEN LABEL TRIAL OF TAMOXIFEN IN SCLERODERMA. Gary S Hoffman, Colleen K Thomas, William S Wilke, Barri Fessler, Brian F Mandell Cleveland Ohio

NEW METHODS OF ASSESSING SKIN DISEASE IN SCLERODERMA: USE OF DUROMETERS, PHOTONIC DEVICES, AND COMPARISON TO MODIFIED RODNAN SKIN SCORING. Peter A Merkel, Artur Kaluta, Lorenzo Brancaleon, Rondi B Gelbard, Peter McDonald, Vincent Falanga, Jennifer J Anderson, Joseph H Korn Boston, MA; Dundee, United Kingdom

Background:In a preliminary short-term study, Franco et al. (Rev Argentina de Dermatologia 63:168, 1988) reported improved digital temperatures following treatment with the high dose estrogen antagonist tamoxifen in 3 patients with Raynaud’s vasospasm and 8 with scleroderma. Tamoxifen treatment has also been associated with improvement of fibrosing syndromes (retroperitoneal fibrosis) and tumors (desmoid) in some but not all patients. The female predominance in Raynaud’s and scleroderma and the effect of tamoxifen on increasing production of the immunosuppressive cytokine TGFb, raised questions about utility of this agent in scleroderma. Purpose: To perform a pilot study of tamoxifen in scleroderma to determine feasibility of larger phase II and III studies. Methods: Open label prospective study of tamoxifen (10 mg bid) in 15 Caucasian patients (3 M, 12 F) with scleroderma (14 diffuse disease, 1 limited disease). Exclusions: addition of other therapy for scleroderma within prior 6 weeks. Results: Mean age was 55 (34-75) years. Mean duration of scleroderma was 9.3 (1-25) years. Two patients were prematurely dropped: one became tremulous and weak after 3 doses and the other was lost to follow-up at 1 month. For 13 patients, mean duration of treatment was 7 (1.5-32) months. Two of 13 patients treated with tamoxifen experienced unequivocal subjective and objective improvement in cutaneous manifestations and/or Raynaud’s phenomenon. However, 11 other patients either failed to improve or experienced modest improvement, which led to discontinuation of treatment. Patients who improved did not appear to have other clinical features that identified them as a unique subset. Two patients died during the study but it is unclear whether this was related to tamoxifen or the underlying disease. Conclusions: Based on these results we would not recommend tamoxifen for large scale studies in scleroderma.

Purpose: To evaluate new methods to quantify skin disease in patients with systemic sclerosis. The goal is to develop outcome measures complementary to the modified Rodnan skin score (MRSS) that have increased precision and biologic correlates. Methods: Skin thickness was measured semiquantitatively by 17-site (scored 0-3 at each site) modified Rodnan skin scoring (MRSS). Skin hardness was measured using a handheld digital durometer (Rex Gauge Type OO) with a continuous scale; 4 measurements were taken at each skin site. In vivo fluorescence reflectance and emission measurements were made to assess tissue matrix composition using a double monochromatic spectrophotometer linked to a laptop computer. Each methodology measured the same skin sites. Results: 15 patients (11 women/4 men) with diffuse cutaneous sclerosis were studied at 25 visits. Median subject age ⫽ 52 years (range 29-74); median disease duration⫽ 3.5 years (1.5-6.5). 17-site MRSS averaged 19.5 (4-36). Durometer measurements were obtained quickly and painlessly. The durometer was highly reliable with Chronbach alpha coefficients ranging from 0.98-0.99 for four readings and 0.96-0.99 for two readings. The durometer readings were significantly associated with skin scores at all sites tested with correlation coefficients ranging from 0.52 at the calf to 0.80 at the forearm (p⬍0.001 for all sites). The spectrophotometer readings were also easily obtained. The tryptophan:collagen fluorescence ratios were significantly associated with MRSS at the tested sites, ranging from ratio⫽ 0.49 for site score⫽ 0 to 1.36 for site score⫽ 3 (p⬍0.001). The three methods of skin assessment were significantly correlated: MRSS with durometer (r⫽ 0.54, p⬍0.001), durometer with photonics (r⫽ 0.41, p⬍0.01), and MRSS with photonics (r⫽ 0.36, p⬍0.05). All measurements were well tolerated by patients and the techniques were quickly learned by investigators. Conclusions: Durometer readings of skin hardness in scleroderma offer a precise, continuous outcome measure that correlates with MRSS. Similarly, spectrophotometric measurements appear to correlate with severity of skin disease in scleroderma and may also offer a non-invasive method to measure biological activity in the skin. Compared to MRSS, durometer and spectrophotometric assessments may also offer complementary and different information regarding disease process and activity and provide a greater dynamic range of measurements. Continuation of this work will refine these devices’ precision, sensitivity to change, and utility for adoption in clinical trials in scleroderma. Disclosure: Supported by a research grant from the Scleroderma Foundation and by the Boston University General Clinical Research Center (NIH).

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TREATMENT WITH L-CARNITINE IN PATIENTS WITH SYSTEMIC SCLEROSIS (SSc). A ONE YEAR OPEN CONTROLLED LONGITUDINAL STUDY. Flavio Fantini, Silvana Zeni, Nicoletta Del Papa, Amedeo Soldi, Roberta Cossutta, Wanda Maglione, Denise P Comina Milan, Italy

DIGITAL IONTOPHORESIS OF VASOACTIVE CHEMICALS AS MEASURED BY LASER DOPPLER IMAGING - A NEW NONINVASIVE TECHNIQUE BY WHICH TO STUDY RAYNAUD’S PHENOMENON. Marina E Anderson, Tonia L Moore, Mark Lunt, Ariane L Herrick Salford and Manchester, United Kingdom

Aims: to evaluate the effects of treatment with L-carnitine on the skin involvement, the Raynaud’s phenomenon (Rp), the ulcers, the disability and the quality of life in patients affected by Systemic Sclerosis (SSc). Methods: we studied 50 patients affected by SSc (17 dSSc, 29 lSSc, 4 intermediate group). The patients were randomised to receive L-Carnitine (2g/day) (23 patients) or not (27 patients) according to the date of their birth. All the patients were on standard treatment with vasoactive and immunosuppressant drugs according to the severity of disease. The skin involvement was evaluated by the modified Rodnan skin score. The ulcers were scored according to their number and severity. The Rp was scored taking into account discomfort, duration and elicitability.The disability was evaluated by the disability index of HAQ. The quality of life was evaluated by the SF-36 questionnaire. Results: the table shows the mean values at baseline (To) and after one year (T1) in both groups and the statistical significativity of the comparison between the differences (D T1-T0) of the two groups.


Controls Intra 0.76 0.54

L-Carnitine

Skin Score

Raynaud’s P

Ulcers

HAQ

*dP(SF-36)

Mean Values T0 Mean values T1 Difference% ⌬T1-T0 MEAN (⫾ ⌺⌬) Controls Mean values T0 Mean values T1 Difference% ⌬T1-T0 MEAN (⫾ ⌺⌬) Comparison

7,96 6,06 -23% -1,9(⫾5,3) Skin Score 12,33 12,36 0,24% 0,03(⫾ 9,2) p⫽0,377

4,01 2,98 -24% -1,03(⫾2,57) Raynaud’s P 4,05 5,37 27,9% 1,32(⫾ 2,74) p⫽0,0031

1,74 1 -43% -0,74(⫾2,4) Ulcers 1,23 2,18 77,2% 0,95(⫾2,09) p⫽0,0105

0,6359 0,7734 21,6% 0,1375(⫾0,7337) HAQ 0,8843 1,03 16,9% 0,14(⫾ 0,85) p⫽0,958

56,64 64 12,9% 7,36(⫾31,3) *dP(SF-36) 56,56 62,85 11,1% 6,29(⫾ 35,26) p⫽0,91

*dP(SF-36)⫽PA (physical activity) ⫹ RP (role of physical activity) ⫹ BP (bodily pain)/3 A statistically significant improvement was found in the L-Carnitine group compared with the control group in respect to the Raynaud’s phenomenon (p ⫽ 0,0031) and the ulcers ( p ⫽ 0,0105). The skin involvement and the physical dimension of quality of life showed a not statistically significant trend to improvement in the L-Carnitine group. Conclusion: L-Carnitine seems to be effective in improving the vascular component of SSc. Further double blind studies are needed to confirm this effect. Disclosure: The drug was supplied for free by Sigma Tau

Purpose To compare digital cutaneous microvascular responses noninvasively in patients with primary Raynaud’s phenomenon (PRP), limited cutaneous systemic sclerosis (LCSSc) and healthy control subjects by measuring blood flow responses to iontophoresis of the endothelial-dependent vasodilator acetylcholine (ACh) and the endothelialindependent vasodilator nitroprusside (NP) with laser Doppler imaging (LDI), as opposed to single point laser Doppler which has previously given conflicting results. Patients and Methods We studied 10 patients with PRP, 10 patients with LCSSc and 11 healthy control subjects at 23°C. Two chambers (1cm diameter circular aperture, platinum wire electrode) were attached, one to each of two adjacent fingers of the left hand (middle phalanx). One chamber was filled with 1% ACh and the other with 1% NP solution. Baseline skin microvascular blood flow within the area of the apertures was measured using scanning 633nm LDI prior to iontophoresis. At commencement of iontophoresis at 30 ␮amps for 120 sec (2 chambers simultaneously), the LDI initiated recording of a set of 25 serial scans (each scan at 20 sec intervals) measuring skin blood flow at the areas of iontophoresis. The procedure was repeated on 2 adjacent fingers of the right hand. Results Median normalised area under the curve for blood flow in perfusion units (AUC) was significantly lower in the LCSSc group for both ACh (p⫽0.014) and NP (p⫽0.006). [Repeated measures ANOVA]

Controls PRP LCSSc

ACh - mean AUC (95% CI)

NP - mean AUC (95% CI)

6 213 (4 319, 8 108) 7 698 (5 323, 10 073) 3 008 (913, 5 102)

7 766 (5 745, 9 786) 7 690 (5 571, 9 809) 2 937 (568, 5 305)

Conclusions Endothelial-independent and possibly also endothelial-dependent cutaneous vasodilation are impaired in LCSSc in keeping with permanent structural microvascular damage, whereas vasodilatory responses to iontophoresis are unimpaired in PRP as would be expected in a completely reversible disorder without permanent tissue damage. This study demonstrates the ability of LDI, which measures blood flow over an area rather than a single site, to quantify local vasodilatory effects in patients with Raynaud’s phenomenon. Disclosure: LDI purchased with a joint equipment grant funded by Moor Instruments (Axminster, UK), MRC & Scleroderma Society

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HIGH DOSE CYCLOPHOSPHAMIDE FOLLOWED BY AUTOLOGOUS BLOOD STEM CELL TRANSPLANTATION FOR THE TREATMENT OF SYSTEMIC SCLEROSIS. R Verheesen, J M van Laar, A VMB Schattenberg, W Fibbe, F C Breedveld, T de Witte, L BA van de Putte, F HJ van den Hoogen Nijmegen and Leiden, Netherlands

NONINVASIVE TESTS FOR ATHEROSCLEROSIS IN SLE. M Petri Baltimore, MD Angina and myocardial infarction occur in about 8% of SLE patients in cohorts worldwide. The prevalence of myocardial infarction and stroke is greatly increased over controls. In this study we asked whether coronary and carotid atherosclerosis are concordant in SLE. Methods: 53 SLE patients were studied. Duplex carotid sonography was performed using commercially available high resolution linear transducers (5-10 MHz, Acuson 128XP, Hewlett Packard Image Point or ATL HDI 3000). Images were acquired of the distal common carotid arteries (CCA), carotid bulb and proximal internal carotid arteries (ICA) in the sagital plane. If atherosclerotic plaque was identified, transverse images and measurements of the plaque were also obtained. Helical CT was used to measure coronary calcification score. Results: 12 of 30 patients who did not have carotid artery plaque had coronary calcification on helical CT. These 12 patients did not differ from the other 18 in age, sex, race, cholesterol, weight, diabetes, hypertension, RVVT, or aCL status, and would not have been suspected as being “high risk”.

Background: Although beneficial effects of methotrexate and cyclophosphamide (CY) on the course of systemic sclerosis (SSc) have been reported, some patients experience a rapid progression of skin and internal organ involvement leading to severe disability and often to death. Remission of autoimmune diseases after bone marrow transplantation and data from animal models of autoimmune diseases indicate that stem cell transplantation (SCT) could be a potential treatment for patients with autoimmune diseases. We report the preliminary results of SCT in 10 patients with SSc. Patients and methods: Ten patients (seven of female gender), with progressive diffuse SSc were selected for this therapy. Two patients had skin involvement only, five patients had pulmonary, two pulmonary and heart and one pulmonary and kidney involvement. The age of the patients varied from 27 to 58 [mean 42] years. Patients were treated with SCT according to the EBMT/EULAR SSc Protocol. Stem cell mobilization occurred with CY 4 gr/m2 followed by G-CSF 10 microgr/kg/day sc for 9 to 12 days. Leukapheresis was performed on day 12 to 15. The graft was enriched for CD34 positive cells (Clinimacs) and between 0.4 and 3.6 x 104 T cells/kg were left in the graft. Conditioning consisted of 200 mg/kg CY alone. Results: The procedure appeared feasible in all patients: no major complications occurred. The aplastic period lasted less than 3 weeks in all patients. Toxicity consisted of alopecia (n⫽10), nausea and vomiting (n⫽10) and fever due to infection of an indwelling catheter (n⫽2), necessitating temporary treatment with antibiotics. One patient with known pericardial effusion developed a pericardial tamponade immediately after stem cell mobilisation, one patient got a myocardial infarction during conditioning. Two patients experienced a moderate myositis 3 months after SCT, that responded well on a short course of low-dose corticosteroids. Preliminary efficacy data , with follow-up ranging from 6-40 months, showed 70% [range 50-90%] improvement in skin score. In 6 patients DLco worsened 15% when measured 6 months after SCT, but returned to pretransplant levels 6 months thereafter. In one patient DLco worsened 2 years after SCT. No change in creatinine clearance was observed. Conclusion: These preliminary results underline the feasibility and potential efficacy of this treatment.

Conclusion: Coronary calcification was detected in 40% of patients with no carotid plaque. This suggests that atherosclerosis occurs more frequently in the coronary than in the carotid distribution in SLE. Patients without traditional cardiovascular risk factors had abnormal coronary calcification. Measurement of coronary calcification may be the preferred method of ascertainment of atherosclerosis in noninvasive studies because of its greater sensitivity.

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AN ECOLOGICAL ANALYSIS OF THE EFFECTS OF SOCIOECONOMIC STATUS (SES) AND HISPANIC ORIGIN ON MORTALITY FROM SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). Stephen J Walsh Farmington, CT

DOES THE USE OF THE WEIGHTED CRITERIA IMPROVE OUR ABILITY TO CAPTURE A LARGER NUMBER OF LUPUS PATIENTS INTO OBSERVATIONAL AND INTERVENTIONAL STUDIES THAN THE ACR CRITERIA? Martha L Sanchez, Rhondrea M Copeland, Gerald McGwin, Jr, Barri J Fessler, Ellen Sowell, Robert P Kimberly, Graciela S Alarcon Birmingham, AL

Objective: To determine the relative contributions of SES and Hispanic origin to geographical variation in SLE mortality rates in the United States. Methods: Univariate and multivariate Poisson regressions were performed using SLE mortality counts in 3111 U.S. counties for 1984-93 as the dependent variable. For each county, 25 measures of SES from the 1990 census and the percentage of residents of Hispanic origin constituted the independent variables. An offset variable adjusted for age, sex, and race. Race, defined as black, white, or other, constituted a different measure than Hispanic origin. SES measures were represented as principal component factors in the multivariate regressions due to high pairwise correlations. Geographical cluster analysis was performed to identify regions of high and low SLE mortality. The impact of SES and Hispanic origin on clustering was evaluated using analysis of deviance. Results: In univariate analyses, lower SES was significantly predictive of higher SLE mortality for 23 SES measures. Univariate results for Hispanic origin indicated that the SLE mortality rate would be 1.93 times greater [95% C.I. (1.68, 2.22)] in a county with all residents of Hispanic origin than in a comparable county with no residents of Hispanic origin. In multivariate analysis, the three most influential variables were a SES factor related to urban poverty, another factor related to median income, and Hispanic origin. The adjusted estimate for the effect of Hispanic origin was 1.90 (1.57, 2.29). Cluster analysis identified 8 multi-county regions with significantly high or low SLE mortality. Analysis of deviance revealed that, within clusters, 34.4% of the deviance between observed and expected mortality was explained by SES factors alone, 8.6% was explained by Hispanic origin alone, and 42.1% was explained by SES factors and Hispanic origin together. Conclusions: SES may be associated with over a third of the geographic variation in SLE mortality rates across the U.S. SES does not appear to confound the relationship between Hispanic origin and SLE mortality. Instead, Hispanic origin seems to affect SLE mortality independently or, perhaps, through interaction with SES.

Coronary Calcification Carotid Plaque

Absent 18 5

Absent Present

Present 12 17

Purpose/Background: The ACR criteria (ACR, and updates) for the classification of SLE have been used since 1972, and patients included or excluded in clinical and interventional studies based on them. Clinicians, however, are often times frustrated by the exclusion of patients with “clinical” SLE by these criteria (and by the possible inclusion of non-SLE patients). The aim of the study was to determine if the weighted criteria (WeC) [Clough, et al, Arch Intern Med, 144:281-85, 1984], as recently modified by Costenbader, et al, (Lupus 10:S117, 2001), (weights given according with seriousness and objectivity of manifestations) have better overall metric properties than the ACR. Methods: A computerized list of patients with the billing diagnosis of SLE (ICD9 710.0) in a large University-based Rheumatology practice was obtained. Physicians (MD, n⫽7) were asked to validate their patients’ diagnosis. 363 patients were considered by their MDs to have definite or probable SLE (other diagnoses excluded). ACR and WeC were applied after careful review of primary data in patients’ medical records. Sensitivity, specificity and overall predictive values were calculated using MD diagnosis as the gold standard. Results: 90 % of the patients were women, 51% Caucasians, 47% African-American. Their mean age was 44 years ⫾ 14.4. Data are otherwise shown below. Conclusions: The WeC have an increased sensitivity, which may allow the inclusion of patients with bona fide SLE into lupus studies. However, this gain is tempered by the loss of specificity and thus the inclusion of patients that are not classified as SLE by their physicians. MD⶿ACR

Yes

No

Metric properties

Yes No Total

231 36 267

27 69 96

86.5%⫽Sensitivity⫽90.3% 71.9⫽Specificity⫽60.4% 82.6%⫽Pred Val⫽82.4%

MD⶿WeC

Yes

No

Yes No Total

241 26 267

38 58 98

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BALES: THE BALTIMORE LUPUS ENVIRONMENTAL STUDY. M Petri, E Thompson, R Abusuwwa, J Huang, E Garrett Baltimore, MD

ETHNIC DIFFERENCES IN THE INCIDENCE OF KAWASAKI DISEASE, HENOCH-SCHONLEIN PURPURA AND OTHER VASCULITIS IN 586 CHILDREN. Janet M Gardner-Medwin, Pavla Dolezalova, Taunton R Southwood Birmingham, United Kingdom

Environmental triggers of SLE, such as ultraviolet light and sulfonamide antibiotics, likely play a role as large as genetic predisposition in the causation of human SLE. We investigated environmental triggers in the 5-years prior to diagnosis in SLE patients and in the same time frame in relatives and friends. Methods: SLE patients (n⫽545, for the 5 year period before diagnosis), relatives (n⫽339) and friends (n⫽372) completed a detailed questionnaire. Data were analyzed using both matched and unvatched methods. In the matched analysis, patients were matched with relatives and friends separately and also combining the two control groups. Maximum likelihood estimates of odds ratios (with p-values) for risk of lupus versus exposure were estimated using conditional logistic regression. The unmatched analysis was performed analogously using ordinary logistic regression. Results: Smoking, echinacea, and pets were not significant risk factors.

The aim of this study was to determine the incidence and ethnic distribution of Henoch-Schonlein Purpura (HSP), Kawasaki Disease (KD), and rare vasculitides in children resident in a region of the UK with a richly diverse ethnic mix, including a large Asian population originating from the Indian subcontinent. Methods: Prospective monthly questionnaires were sent to 321 paediatricians, rheumatologists and other relevant consultants, with a mean return rate of 71.1% over 3 years (1996-99). Case ascertainment was verified by review of a further 406 case notes not identified by the questionnaire but with diagnostic codes for vasculitis, and a single questionnaire to 2860 primary care doctors (GPs) (return rate 59.7%) at the end of the study. Results: 586 new cases of vasculitis fulfilling established diagnostic criteria were collected prospectively from the 1.2 million children resident in the region. All Asian children originated from the Indian subcontinent, none were of oriental origin. Asian children had a higher incidence of KD and rarer primary systemic vasculitis(PSV) than white children. Asian and black children had a higher incidence of systemic lupus erythematosus (SLE), juvenile dermatomyositis (JDMS). Table: Incidence (95% CI)/100,000 children/year. *p⬍0.01 between ethnic groups.

Progest. Pill OR 0.39 0.48

(Relatives) Matched Unmatched

Oral Contraceptives

p-value 0.05 0.06 Measles

0.58 0.64

OR 1.58 1.38

p-value 0.07 0.13

Sunburns OR 1.53 1.31

p-value 0.01 0.07

German Measles 0.06 0.04

2.12 2.37

7.77 5.58

Eggs OR 1.34 1.15

p-value 0.01 0.10

Mono 2.12 2.37

Alfalfa OR 1.39 1.41

p-value 0.009 0.002

All cases Asian Black White

10% 3% 86%

HSP (2 year data) n⫽463

KD n⫽73

SLE n⫽28

JDMS n⫽14

rarer PSV n⫽8

20.4 (18.6-22.4) 24.0 (18.2-31.2) 6.2*(1.7-16.0) 17.8 (16.0-19.8)

2.1 (1.7-2.7) 4.9*(2.8-7.8) 2.1 (0.3-7.5) 1.9 (1.4-2.4)

0.8 (0.5-1.2) 5.6 (3.0-9.5) 3.1 (0.6-9.1)) 0.4*(0.2-0.7)

0.4 (0.2-0.7) 0.6 (0.1-2.1) 2.1*(0.3-7.5) 0.3 (0.2-0.6)

0.24(0.1-0.5) 1.7* (0.5-4.4) 0.14 (0-0.4)

Parvovirus 0.21 0.09

0.01 0.01

0.21 0.09

Conclusion: Parvovirus and mono increase the risk of lupus, whereas measles and german measles are protective. Oral contraceptives increase the risk, but the progesterone pill is protective. Alfalfa sprouts (as in monkeys) increase the risk, as, surprisingly, do eggs. In contrast, no risk was associated with smoking, in comparison to previous case-control studies. This study strongly incriminates some environmental exposures in the causation of SLE. Disclosure:

% paediatric population

The annual incidence of HSP was higher than previously described, rising to 22.1/100,000 under 14 years (95% CI 19.2-25.0), and 70.3*/100,000 between the ages of 4-6 years (95% CI 54.2-86.4). The annual incidence of KD rose to 5.5/100,000 in children under 5 (95% CI 3.1-7.8), and 14.6* /100,000 (95% CI 1.3-27) in Asian children under 5 years. In girls over 11 years the annual incidence of SLE was 12.3* /100,000 (95% CI 7.3-17.3). Further analysis revealed clustering (at p⫽0.05 level) of KD (time: 180 days, space 1500 metres) and HSP (time: 120 days, space 1500 metres). Conclusion: Our study has recognised a high incidence of vasculitis in non-oriental Asian and black children, and a higher incidence of HSP than previously reported. Genetic and environmental influences may be involved. Disclosure: The enormous contribution of all consultants, general practitioners and medical record staff in the West Midlands supporting this study is acknowledged. J Gardner-Medwin is an Arthritis Research Campaign clinical lecturer.

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(Combined) Matched Unmatched

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HENOCH-SCHONLEIN PURPURA IN CHILDREN FROM NORTHWEST SPAIN: A 20-YEAR EPIDEMIOLOGIC AND CLINICAL STUDY. Miguel A Gonzalez-Gay, Carlos Garcia-Porru ´ a, Maria C Calvino, Jose L Fernandez-Iglesias, Pilar Rodriguez-Ledo, Javier Llorca Lugo, Lugo and Santander, Cantabria, Spain

CANCER RISK IN A POPULATION BASED COHORT OF PATIENTS WITH WEGENER’S GRANULOMATOSIS. Ann M Knight, Anders Ekbom, Johan Askling Uppsala and Stockholm, Sweden

OBJECTIVE: To examine incidence, clinical spectrum, and renal outcome in children with Henoch-Schonlein purpura (HSP). METHODS: Retrospective study of unselected children (age 14 and younger) diagnosed with HSP at the single hospital for a defined population in NW Spain between 1980 and 1999. Patients were classified according to the criteria proposed by Michel et al in 1992. To assess renal outcome only those patients with a follow-up of at least 1 year were reviewed. RESULTS: Seventy-eight children (median age 5.5 years) were classified with HSP. Girls slightly outnumbered boys. Cases occurred more commonly in fall and winter. A previous history of upper respiratory infection was frequent. The overall average annual incidence rate was 10.45/100,000. Abdominal pain and or joint manifestations preceded the onset of purpura in 31% of the cases. All children developed nonthrombocytopenic palpable purpura during the course of the disease. Arthritis was seen in 65%, gastrointestinal bleeding in 31% and renal manifestations in 54% within the first 3 months after the onset of symptoms. All but one of the patients with renal manifestations had hematuria, associated with proteinuria in 19 of 41 cases. Sixty-nine cases were followed for at least 1 year. After a median follow-up of 7 years, 8 (11.6%) patients had persistent hematuria with proteinuria. However, none of them had developed renal insufficiency. Hematuria at the onset of the disease or renal manifestations within the first 3 months after the onset of the symptoms and relapses were significantly more common in the group of patients with renal sequelae. The presence of nephrotic syndrome during the course of the disease was generally associated with renal sequelae. In contrast, the age at the onset of the disease was not associated with a different disease severity and outcome. Using discriminant analysis the best predictor for renal sequelae was the presence of nephrotic syndrome during the first 3 months after the onset of symptoms. Using this model 65 of 69 patients with a follow-up of at least 1 year were classified correctly for the risk to develop renal sequelae. CONCLUSIONS: In NW Spain, HSP is a common, generally benign and self-limited, vasculitis. Some children, however, in particular those who develop nephrotic syndrome, suffer permanent renal involvement.

Wegener’s granulomatosis is a necrotizising granulomatous vasculitis of unknown origin, which untreated has a high mortality within the first year of onset.The introduction of cyclophosphamide in the treatment has considerably improved survival rates, but may, according to past studies, involve an increased risk for cancer of the urinary bladder. The aim of this study was to assess the overall cancer incidens in patients with Wegener⬘s granulomatosis and to specifically study the risk of bladder cancer.We followed a population-based cohort of patients with Wegener’s granulomatosis, derived from Swedish registry data.We identified 1,065 patients and followed them for up to 26 years.Standardized incidence ratios (SIR) between observed and expected numbers of cancers were used as a measure of risk. There was a two-fold increase in cancer incidence in these patients.The risk was most pronounced for bladder cancer( SIR⫽4,8;95%CI 2.6-8.1),non-melanoma skin-cancer(SIR⫽7.3;95%CI4.4-11.6), leukemias(SIR⫽5.7; 95%CI 2.3-12) and for malignant lymphomas (SIR⫽4.2; 95%CI 1.8-8.3). These results confirm indications of an increased risk of cancer of the urinary bladder but also point to an increased risk of cancers at other sites,including those affected by the granulomatosis.The study also highlights the necessity of further research to establish the causes of cancer in this group and especially to distinguish the effects of treatment from the effects of the disease itself. Disclosure:

Disclosure:

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PREVALENCE AND INCIDENCE OF POLYARTERITIS NODOSA (PAN), MICROSCOPIC POLYANGIITIS (MPA), WEGENER’S GRANULOMATOSIS (WG) and CHURG - STRAUSS SYNDROME (CSS) IN A FRENCH SUBURBAN POPULATION IN 2000: A CAPURE - RECAPTURE ANALYSIS. Alfred Mahr, Segolene Ayme, Loic Guillevin Bobigny and Villejuif, France

NO INCREASED FREQUENCY OF MALIGNANT NEOPLASMS IN POLYMYALGIA AND TEMPORAL ARTERITIS. A PROSPECTIVE LONGITUDINAL STUDY OF 398 CASES AND MATCHED POPULATION CONTROLS. Geirmund Myklebust, Tom Wilsgaard, Bjarne Koster Jacobsen, Jan Tore Gran Tromso, Norway

Background: Because PAN, MPA, WG and CSS are rare diseases, only a few population-based studies have been conducted to estimate their precise frequencies, particularly in urban populations. Objective: To estimate the prevalences and the incidences of these 4 vasculitides in a Parisian suburban population during the year 2000 using capture - recapture analysis. Study design: Cases were collected in Seine - Saint-Denis County, a northern suburb of Paris, which has 1,382,928 inhabitants, 1,093,515 of whom are ⱖ15 years old. The study period encompassed the entire calendar-year 2000. The cases were identified by 2 separate survey sources: 1) mailing with follow-up letter to all the general practitioners (n⫽1119); 2) mailing with telephone follow-up to the departments of internal medicine, rheumatology, nephrology, pneumology and hemodialysis of all the public hospitals and 2 big private clinics (n⫽20). The medical charts of all such identified cases were reviewed and diagnoses confirmed according to the Chapel Hill Consensus Conference nomenclature. The point prevalences and incidences were estimated by 2-source capture - recapture analysis using Chapman and Seber’s formula; the 95% confidence intervals (95% CI) were calculated using the Poisson distribution. Results: The response rates were: 52% for general practitioners and 100% for hospital departments. Of the 59 cases identified (PAN, n⫽20; MPA, n⫽11; WG, n ⫽21; CSS, n⫽7), 17 (29%) cases were retrieved from both sources (matches). The mean age was 59.1 yr (range: 19 - 84 yr) and the male/female ratio was 1.27. Ten diagnoses were made during 2000 (PAN, n⫽1; MPA, n⫽4; WG, n⫽4; CSS, n⫽1); 0 source matches. The table gives the estimated prevalences and year 2000 incidences (95% CI) in the ⱖ15-year-old population.

Conclusion: Compared to previous estimates based mostly on rural populations, our results suggest, in particular, a slightly lower frequency of WG that more probably reflects methodological and/or diagnostic differences rather than specific environmental etiological factors.

Objective: To ascertain the frequencies and types of malignant neoplasms in polymyalgia rheumatica (PMR) and temporal arteritis (TA) and compare them to a population of matched controls. Methods: In a population based study 1987 - 1997, 338 patients were diagnosed with PMR or TA. In addition, 60 cases were diagnosed in the same period, but admitted to hospital for reasons other than PMR and TA. The 398 patients were each randomly assigned four age and sex matched controls from the same county, totally 1592 controls. All patients and controls enrolled in the study were crosschecked with the data files at the Cancer Registry of Norway, and followed to the end of 1998. Results: Among patients, 66 cases (16.6 %) with cancer were found compared to 306 controls (19.2 %). Prior to inclusion, 32 patients (8.0 %) and 153 controls (9.6 %) with cancer were found. When previous cases of cancer were excluded, malignant neoplasms were discovered in 34 patients (9.3 %) compared to 143 controls (10.8 %) after inclusion. There was no difference between patients and controls regarding frequency, types and sites of cancers discovered. The interval between the inclusion and the time of diagnosis of malignant neoplasm did not differ between patients and controls. Conclusion: No differences in frequencies and types of malignant neoplasms between PMR or TA and their controls were found. Neither PMR nor TA as defined by present diagnostic criteria appears associated with cancer and routine screening of malignancy in such patients should be avoided.

Disclosure:

Disclosure:

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EPIDEMIOLOGY OF POLYMYALGIA RHEUMATICA IN OLMSTED COUNTY, MINNESOTA 19701999. Michele F Doran, Gregory R Pond, Cynthia S Crowson, W Michael O’Fallon, Gene G Hunder, Sherine E Gabriel Rochester, MN

SMALL-FIBER NEUROPATHY IN SLE - A CONTROLLED STUDY. Roald Omdal, Svein Ivar Mellgren, Lasse Goransson, Sigurd Lindal, Wenche Koldingsnes, Gunnar Husby Stavanger, Tromso and Oslo, Norway

Disease


PAN MPA WG SCS All 4

Prevalence/1,000,000

Incidence/1,000,000

22.3 (13.8 - 33.3) 11.7 (6.2 - 21.0) 25.7 (17.0 - 38.1) 7.3 (2.8 - 14.4) 70.2 (54.6 - 88.4)

0.9 (0.03 - 5.6) 3.7 (1.1 - 10.2) 3.7 (1.1 - 10.2) 0.9 (0.03 - 5.6) 17.4 (9.9 - 27.2)

Objective. To determine time trends in the incidence of polymyalgia rheumatica (PMR) over a 30-year period in Olmsted County, Minnesota, and to compare survival in patients with PMR with that in the general population. Methods. Using the unified medical record linkage system of the Rochester Epidemiology Project, we identified all incident cases of PMR among residents of Olmsted County, MN between January 1, 1970 and December 31, 1999. For inclusion, cases were required either to fulfil all 3 of the following criteria, or to fulfil 2 of the 3, and have documentation of a prompt response to corticosteroid therapy: age ⱖ50 years, bilateral aching and stiffness in specified areas, ESR elevated to ⱖ40mm/hour. Incidence rates were estimated and age- and sex-adjusted to the 1990 US white population. A Poisson regression model was used to evaluate predictors of PMR incidence. Survival rates among individuals with PMR were compared with expected rates in the population. Results. There were 378 incident cases of PMR during the 30-year study period. Of these, 66.6% were female and the mean age at incidence was 72.8 years. The overall age- and sex-adjusted annual incidence of PMR per 100,000 population aged ⱖ50 years was 58.7(95% confidence interval (CI) 52.8, 64.7). Incidence rates increased with age in both sexes, but in women, unlike in men, incidence fell after age 80. The incidence rates varied over the period of observation, but no significant trends over time were found. In the multivariable analysis, gender(p⫽0.023), age(p⬍0.001), and age2(p⬍0.001), but not calendar year(p⫽0.24) were significant predictors of incidence. The standardized mortality ratio for PMR was 0.88 (95% CI 0.75-1.01). Conclusion. Although there is variability in the incidence of PMR in this population over the past 30 years, no distinct time trends are present.

Objective. Patients with SLE report far more neuropathic symptoms than age- and sex-matched healthy controls, and many SLE patients with no other evidence of peripheral neuropathy (PN) display high scores for neuropathic symptoms. There is also an impairment of the ability to recognize warmth and heat pain. These factors indicate that there may exist a small-fiber neuropathy in SLE. Since immunostaining with antibodies against the panaxonal marker “protein gene product 9.5” (PGP 9.5) allows visualization of these small nerve fibers, we wished to find out if the numbers of epidermal nerve fibers (ENF) were reduced in patients with SLE. Methods. Fifteen consecutive and non-selected SLE patients were included in the study. Ages ranged from 25 to 65 years with a mean of 47.3⫾10.2 years, and disease duration 2-28 years with a mean of 14.8⫾8.6 years. No patient had diabetes, uremia or other known causes of PN. The criteria for selecting healthy controls were sex and age ⫾2 years of the individual SLE patients. Briefly, skin punch biopsies were immediately fixed, rinsed and placed in cryoprotectant overnight. Thereafter 50 mm sections were produced and immunostained with 0.1% rabbit polyclonal antibodies to human PGP 9.5 (Chemicon International). After rinsing and incubation with secondary antibody, blocking for endogenous peroxidase, rinsing, and coloring with peroxidase substrate, the number of ENF was counted. The total length of epidermis was measured with the NIH Image 1.61 morphometry program. The number of nerve fibers per mm was then reported as the mean of counts in six slides, three from each of the two biopsies. Results. The median number of ENF in patients with SLE was 8.6 (range 5.0-9.9) per mm, while the matched controls had 11.5 (range 6.8-18.6) ENF per mm. P⫽0.002 (Wilcoxon Signed Rank Test). Conclusion. This study proves the existence of a small-fiber neuropathy in SLE, and gives a pathophysiological explanation for the prevalent neuropathic symptoms (burning pain, tingling, aching) and impaired warm sense in such patients, by demonstrating a reduced number of A-␦ and C fibers. Medication for SLE, age, disease duration, or routine hematological-, biochemical-, or immunological variables, did not influence the number of ENF in the SLE patients


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PREDICTORS OF OSTEONECROSIS (AVASCULAR NECROSIS) IN SYSTEMIC LUPUS ERYTHEMATOSUS. M Petri Baltimore, MD

ATOPIC AND NON-ATOPIC ALLERGIES IN SYSTEMIC LUPUS ERYTHEMATOSUS. Martin Rudwaleit, Bianka Andermann, Helmut Soerensen, Rieke Alten, Joachim Listing, Joachim Sieper, Juergen Braun Berlin, Germany

Osteonecrosis (ON) is highly associated with higher doses of prednisone. It remains controversial whether ON is associated with other risk factors, such as antiphospholipid antibodies, Raynaud’s, and vasculitis. Methods: 109 (11.5%), in a prospectively-followed cohort of 949, developed ON by CT/MRI. There were 875 women, 42% African-American (55% Caucasian). Demographic, clinical, and laboratory variables were ascertained at cohort entry and updated at least yearly thereafter. Results: Only 2 pts developed ON without steroids. The highest dose of prednisone was highly associated with ON (mean 68.9 vs 41.3 mg/day, p⫽0.0001). African-Americans had a higher risk (17.5% vs 8.4%, p ⬍ 0.0001). Univariate and multivariate results (adj.) are shown. Demographic

with

w/o

P-value

adj

Yrs-education Priv. Insur. Smoker-curr. Clinical Vasculitis Raynauds Laboratory LA (RVVT) Anticardiolipin Elevated LFT High ESR

12.8 9% 18.9% with 23.7% 12.9% with 16.5% 10.2% 16.1% 13.2%

13.7 18.5% 9.4% w/o 9% 9.6% w/o 10.2% 12.3% 8.1% 4.2%

0.003 ⬍0.0001 0.0005 P-value ⬍0.0001 NS P-value 0.036 NS 0.0002 0.0003

NS NS 0.01 adj ⬍0.0001 NS adj 0.07 0.08 0.04 0.03

Introduction: The T helper (Th) cell cytokine pattern of patients with systemic lupus erythematosus (SLE) is considered to be deviated towards Th2 with increased production of IL-10 and decreased production of IFNg. The few studies investigating the relation between SLE and non-atopic allergies and atopic disorders (Th2) revealed contradictory results. Methods: A questionnaire to assess the prevalence of asthma, hay fever, and atopic eczema / neurodermatitis was sent to 170 SLE patients and 900 healthy controls (hospital workers). The questionnaire incorporated validated questions from the European Community Respiratory Health Survey (ECRHS) and the International Study of Asthma and Allergies in Childhood (ISAAC). Non-atopic allergies were also assessed. Results: 104 SLE patients (61%) and 536 controls (60%) responded to the questionnaire. The median age was 42 yrs (SLE) and 48 yrs (controls). Surprisingly, atopic disorders such as asthma, hay fever, or atopic eczema were less frequently reported by SLE patients compared to controls (10.6% vs 20.7%, OR 0.45, 95% CI 0.23-0.88, p⫽0.02). On the contrary, allergies to medication, particularly penicillin, chemicals, cosmetics, metals, and food compounds were reported more often by SLE patients compared to controls (27.8% vs 14.9%, p⫽0.002). Conclusions: In this cohort, atopic disorders (Th2) are decreased but non-atopic allergies are increased in SLE patients compared to healthy controls. This stresses the different pathophysiology underlying various forms of allergies. Disclosure:

Anti-b2 GPI and anti-prothrombin were not associated with ON. ON was highly associated with lupus involvement that might require high dose steroids, including alopecia, nephrotic syndrome, encephalopathy, hemolytic anemia, thrombocytopenia, and myocarditis (p-values 0.003 to 0.0001). It was not associated with elevated triglycerides. ASA or NSAID use was not protective. Conclusion: Prednisone, race, smoking, vasculitis, ESR and elevated LFT remain predictors of ON in multiple variable models. The strong association with smoking should certainly renew physician and patient efforts to eliminate this risk factor, but may also lead physicians to use steroid-sparing drugs earlier in this subset. Disclosure:

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DIGITAL INFARCTION IN TWO PATIENTS TREATED WITH A SELECTIVE CYCLOOXYGENASE-2 INHIBITOR. Devra B Baryshnik, Rachel Shupak Toronto, Ontario, Canada

PREVALENCE AND CORRELATES OF FIBROMYALGIA IN A PROSPECTIVE, MULTIETHNIC LUPUS COHORT. M B Tew, A W Friedman, S-H Kang, H M Bastian, G McGwin Jr, B J Fessler, B A Baethge, J D Reveille, G S Alarcon Houston and Galveston, TX; Birmingham, AL

Cyclooxygenase (COX)-2 inhibitors are being widely prescribed in place of standard NSAIDs as their anti-inflammatory, anti-pyretic and analgesic efficacy is similar, but their risk of serious GI complications is significantly less [1]. However, unlike NSAIDs, COX-2 inhibitors do not inhibit COX-1 activity. Thus platelet aggregation and vasoconstriction are promoted via thromboxane A2 (TXA2), potentially shifting the hemostatic balance toward thrombosis. This was a theoretical concern until the recent publication of 4 case reports describing patients with connective tissue diseases (CTD) and thrombotic risk factors, who developed thromboses while taking celecoxib, a COX-2 inhibitor [2]. We present 2 additional cases, including the first case of a woman without a CTD, who developed digital ischemia in the context of celecoxib use. In both cases, the patients had risk factors for thrombosis. The first patient, a 29 year old woman with no CTD diagnosis, had Raynaud’s phenomenon, used an oral contraceptive and was a smoker. The second patient, a 60 year old woman with SLE, had a concurrent diagnosis of metastatic colon carcinoma. In both cases, the ischemic insult resolved subsequent to discontinuation of celecoxib. COX-2 inhibitors may not be as safe an alternative to NSAIDs as initially thought. When used by individuals with thrombotic risk factors, COX-2 inhibitors may shift the balance between TXA2 and prostacyclin, leading to thrombotic events. Thus individuals with such risk factors should be prescribed COX-2 inhibitors with caution and close monitoring. [1]Bombardier M, Laine L, Reicin A, et al. Comparison of upper gastrointestinal toxicity of Rofecoxib and Naproxen in patients with rheumatoid arthritis. N Engl J Med 2000;343:1520-8. [2]Crofford LJ, Oates JC, McCune WJ, et al. Thrombosis in patients with connective tissue diseases treated with specific cyclooxygenase 2 inhibitors: A report of four cases. Arthritis Rheum 2000:43:1891-6 Disclosure:

Purpose: Fibromyalgia (FM) reportedly occurs in 8-61% of patients with systemic lupus erythematosus (SLE) and its presence is associated with poorer indices of function (e.g. employment status, levels of disability, social support) than those with SLE alone. The wide variation in reported prevalence reflects differing modes of case ascertainment and possibly differences in FM-associated factors among populations studied. We sought to determine the prevalence and correlates of FM in a prospective, multiethnic SLE cohort. Methods: 266 SLE patients (as defined by ACR criteria) with disease duration of ⱕ5 years at study entry were evaluated systematically for the presence of FM (per ACR criteria). All patients were part of a longitudinal SLE cohort study in which subjects were evaluated at entry and annually thereafter for sociodemographic and clinical features (SLE-related and unrelated, including self-reported history of anxiety or affective disorder) as well as for multiple measures of functioning. These measures included the IBQ, SF-36 for physical (PCS) and mental function, social support (ISEL), Helplessness Index, and fatigue (FSS). Disease activity and damage were measured with the SLAM and SLICC damage index, respectively. The prevalence of FM was then calculated as was the prevalence of FM-like widespread pain (FMWP) in the absence of 11/18 tender points. Variables were evaluated for association with FM or FM⫹FMWP in separate univariate analyses, and factors with p⬍0.2 were included in separate multivariate regression analyses. Results: FM was present in 23 patients (8.6%; 12/92 Caucasians [C], 8/109 African-Americans [AA], 3/65 Hispanics[H]) and FM⫹FMWP in 48 (18%; 21 C, 15 AA, 12 H) at a mean disease duration of 55 months. Significant findings are shown in table. Group

Variable

p value

FM FM FM⫹FMWP FM⫹FMWP

PCS of SF-36 Anxiety/affective disorder FSS Anxiety/affective disorder

0.0017 0.0137 0.0007 0.0028

OR (95% CI) 0.92 3.52 1.03 3.57

(0.88-0.97) (1.29-9.58) (1.01-1.04) (1.55-8.20)

Conclusions: FM and FM-like symptoms correlated best with the presence of concomitant anxiety or affective disorder and to a lesser extent with poorer self-reported physical functioning. Neither measures of disease activity nor damage correlated with FM. Disclosure:

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ACUTE ABDOMINAL PAIN IN SYSTEMIC LUPUS ERYTHEMATOSUS: FOCUSED ON LUPUS ENTERITIS. Chang K Lee, Man S Ahn, Eun Y Lee, Jung H Shin, Yoo S Cho, Hyun K Ha, Bin Yoo, Hee B Moon Seoul, Republic of Korea

THROMBOTIC THROMBOCYTOPENIC PURPURA IN SYSTEMIC LUPUS ERYTHEMATOSUS: DISEASE ACTIVITY AND THE ROLE OF CYTOTOXIC DRUGS. Sheila Vasoo, Julian Thumboo, Kam-Hon Yoon, Kok-Yong Fong Singapore

Objective: 1) To determine the causes of acute abdominal pain in SLE. 2) To evaluate the relationship between the causes of acute abdominal pain and SLE disease activity index (SLEDAI). 3) To describe the characteristic symptoms, laboratory findings, and abdominal computed tomography (CT) findings of lupus enteritis. Methods: A retrospective review was carried out for all medical admission of patients with SLE from 1993 to March 2001. All patients fulfilled the American College of Rheumatology criteria for the classification of SLE. SLEDAI and laboratory data were collected at diagnosis of SLE and at the time of acute abdominal pain. Lupus enteritis was diagnosed if at least three of the following signs were seen on CT scan: bowel wall thickening, target sign, dilatation of intestinal segments, engorgement of mesenteric vessels, and increased attenuation of mesenteric fat. Results: Chart review identified 175 patients (20 male, 155 female) with diagnosis of SLE. Of these patients, 38 patients (21.7%) presented with acute abdominal pain. The causes of acute abdominal pain were composed of the following: 17 lupus enteritis (44.7%), 6 urinary tract infection (15.8%), 5 acute gastroenteritis (13.6%), 2 pancreatits (5.3%), and so on. Patients were divided in three groups: Group 1: lupus enteritis (n⫽17), Group 2: acute abdominal pain without lupus enteritis (n⫽21), and Group 3: SLE without acute abdominal pain (n⫽137). Age and sex were not different among three groups. Antiphospholipid antibody, anti-RNP antibody, anti-Sm antibody, anti-Ro antibody, and anti-La antibody were not different among three groups. There was no difference in SLEDAI at diagnosis (13.7⫾ 5.98 vs. 17.0⫾ 8.6, p⫽0.19) and at the time of acute abdominal pain (10.3⫾ 5.98 vs. 12.5⫾ 8.09, p⫽0.75) between group 1 and 2. C3 (56.2 ⫾20.1 vs. 55.1 ⫾35.2, p⫽0.75), C4 (13.51⫾6.51 vs. 17.4 ⫾10.9, p⫽0.09), ESR (47.6 ⫾23.7 vs. 59.0 ⫾45.2, p⫽0.39), CRP (2.34⫾2.78 vs. 3.24⫾ 4.78, p⫽0.53), and anti-ds DNA (234.5 ⫾379.8 vs. 107.9 ⫾208.5, p⫽0.10) calculated at the time of acute abdominal pain were not different between group 1 and 2. In lupus enteritis, the jejunum (80%) and ileum (85%) were the most common site of involvement. Rectal involvement (14%) was rare. Even though 4 patients relapsed, all patients with lupus enteritis including relapsed episodes responded well to corticosteroid. Conclusion: Lupus enteritis is the most common cause of acute abdomen in SLE. All patients with lupus enteritis responded well to high dose of corticosteroid without surgical intervention. SLEDAI and laboratory data do not correlate with the occurrence of lupus enteritis. Key words: Systemic lupus erythematosus, Enteritis, Abdominal Pain

Introduction and Objectives Thrombotic thrombocytopenic purpura (TTP) is a rare and at times fatal hematologic disorder that can coexist with autoimmune diseases including systemic lupus erythematosus (SLE). The relationship between TTP and SLE activity in patients with active disease as well as the role of cytotoxics in such patients has however not been well-defined. We therefore studied these issues. Methods Medical records of all patients with SLE and TTP admitted to a tertiary referral hospital from 1998 to 2001 were identified through a computerised database and reviewed by a single abstractor using a pre-tested, standardized data collection form. SLE was defined using ACR criteria and TTP as Coomb’s negative microangiopathic hemolytic anaemia and thrombocytopenia in the absence of other causes (Murphy. Blood Coagul Fibrinolysis 1992; 3:655). Disease activity was assessed using the SLE Disease Activity Index (SLEDAI) at TTP diagnosis and fortnightly thereafter. A complete response (CR) of TTP was defined as the resolution of clinical symptoms and normalisation of platelet counts and lactate dehydrogenase (LDH) levels. Results All patients had active SLE (SLEDAI ⬎ 4) at diagnosis of TTP and received high dose steroids, pulse methylprednisolone and plasmapheresis (see Table). One patient had synchronous onset of SLE and TTP, 3 had pre-existing SLE and all had prior renal involvement. SLE activity paralleled TTP activity in all patients, with SLEDAI scores improving as platelet and LDH levels normalised. 3 patients had TTP refractory to plasmapheresis but went into complete remission with the addition of cytotoxics. However one of these patients perished from CMV encephalitis and pneumonia. Conclusions SLE and TTP activity run a parallel course in patients with active SLE. Cytotoxics may be of value in treating TTP in such a setting.

Disclosure:

Age (years)/ Sex Duration of SLE before TTP diagnosis (years) SLEDAI at TTP onset Plasmapheresis- No. of cycles to CR Plasmapheresis - Total plasma volume (L) IV Cyclophosphamide (gm) IV Vincristine (mg) IV Gammaglobulin (gm) Time to CR after start of cytotoxics (days) Outcome Disclosure:

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Patient 1

Patient 2

Patient 3

28/F 0.0 23 23 61 1.3 5 180 24 Dead

36/F 3.7 18 3 7 120 Alive

24/F 1.5 10 19 42 0.4 20 14 Alive

Patient 4 47/M 7.7 11 14 22 0.4 15 Alive


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ETIOLOGY AND OUTCOME OF ACUTE PANCREATITIS (AP) IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). PascualRamos Virginia, Duarte-Rojo Andres, Robles-Diaz Guillermo, Villa R Antonio, Alarcon-Segovia Donato, Alcocer-Varela Jorge Mexico, D.F., Mexico

OSTEOPOROSIS IN SLE II: PREVALENCE AND ASSOCIATED CLINICAL FACTORS IN WOMEN. Christian A Pineau, Murray B Urowitz, Paul R Fortin, Dominique Ibanez, Dafna D Gladman Toronto, Ontario, Canada

Knowledge regarding possible causation and clinical course of AP in SLE is scarce and controversial. In order to contribute to the understanding of these two diseases, a systematic analysis of our cases was performed. Forty episodes of AP (typical clinical symptoms, ⬎ 3-fold increase in amylase or lipase, and/or evidence on CT scan) were detected in 29 SLE patients (ACR criteria). Cases diagnosed at necropsy were not included. A single event was present in 22 patients, two events occurred in 5, and four in 2 cases. Mean age was 27 ⫾ 10 years. Twenty seven patients were females (93%). Clinical data regarding AP were available in 37 episodes (29 patients). Mean follow-up was 30 months (range: 1 day to 111 months). Episodes were grouped according to AP etiology. Group I (GI), mechanical : Biliary (n⫽8), obstructive (n⫽3), post-ERCP (n⫽1); Group II (GII), toxic-metabolic : Hypertriglyceridemia (n⫽3), chronic renal insufficiency (n⫽3), alcoholic (n⫽2); and Group III (GIII), idiopathic (n⫽17). SLE activity according to Mex-SLEDAI in GIII (mean: 10 ⫾ 5) tended to be higher compared to all other etiologies (p⫽0.09) and to GI (mean: 6 ⫾ 6) (p⫽0.05). In 11 episodes, at least one drug with strong evidence to induce AP (azathioprine, furosemide, sulindac, isoniazid) was used at onset (GI⫽2, GII⫽3, GIII⫽6); while in 18, corticosteroids alone were in use (GI⫽8, GII⫽3, GIII⫽7). No new AP events occurred when half of the patients treated with the former drugs, and most of the patients with corticosteroids, were re-exposed. There were no differences in the usage of AP-inducing drugs among groups. Twenty patients (69%) developed severe AP (Atlanta Criteria) in their first episode, without differences among groups (GI⫽5, GII⫽4, GIII⫽14). None of the subsequent episodes was severe. Five severe cases died (25%) (GI⫽2, GII⫽1, GIII⫽2), being AP the direct cause in 3. Severity and mortality were not related to Mex-SLEDAI. In conclusion, a recognized cause of AP was identified in half of the episodes. Increased SLE activity was found during idiopathic AP, suggesting an etiopathogenic role on AP development. A direct link between drugs and AP was not supported, however, its contribution as an etiologic co-factor can not be ruled out. Severity and mortality of AP in SLE were significantly higher when compared to a historic cohort (Am J Gastroenterol 1998;93:1324). This outcome may be influenced by co-morbid factors associated with SLE.

AIM: To determine the prevalence of, and factors associated with osteoporosis in women with SLE. METHOD: All women with SLE who had a DEXA done between January 1st 1995 and December 31st 2000 were identified from a large lupus cohort. All DEXA studies were done at the discretion of the treating physician. For each patient, the first DEXA done within that time period was used. Clinical data collected from cohort entry to the last visit prior to the initial DEXA was analyzed. First, various clinical factors were compared between normal and osteoporotic patients. We then studied the association between these factors and the T scores of all 205 patients to evaluate the effect of the spectrum of normal-osteopenia-osteoporosis. The adjusted mean SLEDAI (AMS) was used to estimate lupus disease activity over time. RESULTS: Of the 516 women followed over that period, 205 (39.7%) had a DEXA performed. Of these, 68 (33.2%) had a normal bone density, 100 (48.8%) had osteopenia and 37 (18%) had osteoporosis.

Age at DEXA (years) Post-menopausal patients Patients with a fracture (peripheral) SLE disease duration (years) AMS (SLEDAI units) Steoid dose at DEXA (mg/d) Mean steroid dose (mg/d) Cumulative steroid dose (g) Last SLICC (excluding osteroporosis) Patients with coronary disease (ever)

Normal (n⫽68)

Osteoporosis (n⫽37)

P value

42.8 ⫾ 11.3 21 (30.9%) 5 (7.4%) 11.8 ⫾ 9.5 6.98 ⫾ 5.85 13.6 ⫾ 12.2 12.3 ⫾ 11.4 38.8 ⫾ 40.6 1.46 ⫾ 1.42 3 (4.4%)

50.6 ⫾ 13.2 24 (64.9%) 11 (29.7%) 16.7 ⫾ 11.3 6.01 ⫾ 4.16 14.0 ⫾ 13.0 10.9 ⫾ 8.6 41.0 ⫾ 36.8 2.85 ⫾ 2.51 7 (18.9%)

0.0018 0.001 0.002 0.021 0.33 0.57 0.52 0.79 0.0059 0.001

Disclosure: For all 205 patients, the T scores and clinical variables showed similar associations. A linear regression model revealed that age (p⫽0.0019) and SLICC score (p⫽0.02) were the main variables associated with lower T scores. CONCLUSION: In women with SLE, osteoporosis and lower T scores are associated with increased age and increased damage. Lupus disease activity and steroids are not associated with lower bone densities. Disclosure:

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JOINT LAXITY IN SYSTEMIC LUPUS ERYTHEMATOSUS. Monica Guma`, Alex Olive, Jordi Forcada, J Carles Duro, Josep Roca, Susana Holgado, Enric Casado, Xavier Tena Badalona, Barcelona, Spain

OSTEOPOROSIS IN SLE III: CONTRIBUTORS OF INCREASED DAMAGE IN OSTEOPOROTIC PATIENTS. Christian A Pineau, Murray B Urowitz, Paul R Fortin, Dominique Ibanez, Dafna D Gladman Toronto, Ontario, Canada

Aim: To investigate joint laxity in patients in Systemic Lupus Erythematosus (SLE). Methods: Setting: University Hospital; 80 patients with SLE (1999 ACR criteria), 90% females and 10% males, mean age 44 ⫾ 16, who regularly attended a specialist SLE clinic were examined. Control group was composed of 280 patients attending a GP office, 70% females and 30% males, mean age 46 ⫾ 19. Joint laxity was measured according to Carter and Beighton criteria (total score 4 or more). A regression analysis was performed (SAS statistical package). Results: Forty-eight% or patients with SLE and 12% of the control group were hypermobile. Regression analysis showed that after adjustment for the age and sex. SLE patients who were younger (⬍ 49 years ) were more hypermobiles than control group (odds ratio: 2.3). Older SLE patients (⬎49 years old) have 18 times more probability to be hypermobiles than control population. Neither the clinical and analytical profile nor the use of corticosteroids was related to joint laxity. Conclusion: Patients with SLE are more hypermobile that control group. No clinical or analytical profile is related. Steroids are not related to the presence of joint laxity in patients with SLE. Arthralgias in SLE patients might be related to hipermobility.

AIM: To determine whether osteoporosis is associated with increased damage in SLE, and to identify the contributors of such damage. METHOD: All women with SLE who had a DEXA done between January 1st 1995 and December 31st 2000 were identified from a large registry. DEXA studies were done at the discretion of the treating physician. For each patient, the first DEXA done within that time period was used. Patients with either osteoporosis (t ⬍ -2.5 ) or normal bone densities (t ⬎ -1) were included in the analysis. The last SLICC scores (excluding the osteoporosis with fracture item) obtained prior to the DEXA were compared between those two groups. The presence of 23 of 24 SLICC items (excluding the osteoporosis with fracture item) was then compared by t test. Finally, a stepwise multiple logistic regression analysis using age, disease duration and the 23 SLICC items was done. Only patients who had a SLICC score recorded before their first DEXA scan were included. RESULTS: Of the 516 women followed over that period, 205 (39.7%) had a DEXA performed. Of those, 68 (33.2%) had a normal bone density, 100 (48.8%) had osteopenia and 37 (18%) had osteoporosis. Amongst normals and osteoporotics, 52 and 33 patients respectively had a SLICC done prior to their DEXA.

Age at DEXA (years) SLE disease duration at DEXA (years) SLICC score (excluding osteoporosis) Muscle atrophy or weakness Deforming or erosive arthritis

Disclosure:

Normal (n⫽52)

Osteoporosis (n⫽23)

P value

42.8 ⫾ 11.3 11.8 ⫾ 9.5 1.46 ⫾ 1.42 1/52 (1.9%) 5/52 (9.6%)

50.6 ⫾ 13.2 16.7 ⫾ 11.3 2.85 ⫾ 2.51 10/33 (30.3%) 12/33 (36.4%)

0.002 0.021 0.006 0.0002 0.005

The two groups were not different with regard to any other SLICC item. In addition, there was no difference in the mean or cumulative steroid dose, antimalarials or immunosuppressants use. In the multiple logistic regression analysis, only age (p⫽0.0083) and muscle atrophy or weakness (p⫽0.0082) were significantly different between normals and osteoporotics. CONCLUSION: SLE patients with osteoporosis have increased damage which is contributed to mainly by age and muscle atrophy or weakness.


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CLINICAL FEATURES AND OUTCOME OF THE SHRINKING LUNG SYNDROME IN SYSTEMIC LUPUS ERYTHEMATOSUS. L Miranda, Y Karim, C Tench, P Gordon, D P D’Cruz, M A Khamashta, G RV Hughes London, UK; Lisbon, Portugal

PREVALENCE AND PREDICTORS OF OSTEOPOROTIC FRACTURES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS. Caroline Gordon, Nicola Crabtree, Janet Skan, Simon Bowman, Deva Situnayake Birmingham, United Kingdom

Introduction: Pleuropulmonary involvement in systemic lupus erythematosus (SLE) occurs in 60-80% of patients, and is commoner in SLE than in any other collagen vascular disease. Shrinking lung syndrome (SLS) is a relatively rare complication - from 1965 to 1997, only 49 cases of SLS were clearly documented in the literature. Case reports: We describe 7 cases of SLS differing in terms of the clinical features of the underlying SLE, age at onset of SLS, time of onset of SLS after diagnosis of SLE, auto-antibody profile, and treatments received. They did however display similar diagnostic features. Presenting symptoms were progressive breathlessness and chest pain. Investigations typically showed reduced lung volumes on pulmonary function testing and a normal or low transfer factor corrected for lung volume. Characteristically thoracic CT scans showed no evidence of parenchymal lung disease and ventilation-perfusion scanning showed no evidence of recurrent pulmonary embolism. In terms of treatment, these patients received a variety of medications, mostly immunosuppressant drugs, including prednisolone, azathioprine, methotrexate, and cyclophosphamide with good overall results. Following treatment, 4 out of 7 patients stabilised both symptomatically and on measurement of lung function and 3 of these showed further improvement. One patient deteriorated despite aggressive immunosuppression. Two patients have only recently been diagnosed. Conclusions: SLS is a rare disease manifestation limited to a small subset of SLE patients. SLS should be considered as a possible diagnosis in SLE patients with dyspnoea. The diagnosis is to some extent one of exclusion. It is important to establish a correct diagnosis of SLS in patients with SLE and in particular to exclude fibrosing lung disease, so that informed decisions regarding treatment and prognosis can be made.

Introduction. Using the WHO criteria, reduced bone mineral density (BMD) in the osteopenic range has been reported in up to 57% and osteoporosis in up to 23% of patients with systemic lupus erythematosus (SLE). However, there has been very little work on the risk of fractures in these patients and the relationship of fractures to BMD and other risk factors for osteoporotic fractures. Aims. We wanted to establish the prevalence of reduced BMD and of fractures, and risk factors for fractures in a cross-sectional study of a large cohort of SLE patients followed prospectively since 1989. Methods. All SLE patients willing to take part in this study had bone densitometry performed in 1999/2000 and completed a validated questionnaire on risk factors for osteoporosis. Medication use and disease activity using the BILAG index is assessed at each visit. Accumulated damage is assessed every 6 months using the SLICC/ACR damage index (DI). Only fractures since the onset of lupus and unrelated to trauma were included and the DI score was modified to exclude osteoporotic fractures (mDI) for this analysis. Statistical analysis was by Chi-square, ANOVA, linear and logistic regression. Results. We studied 242 patients: median age 39.9 (range 18-80) years with median disease duration 7.0 (range 0-42) years. There were 63% Caucasians, 17% Afro-caribbeans, 15% Asians from the Indian subcontinent and 5% other/mixed racial origin. Of the 231 females, 126 (55%) were pre-menopausal, 39 (17%) had a premature menopause and 65 (28%) had a normal menopause. Drug use at the time of bone densitometry included: 89 (37%) on calcium/Vitamin D3 and 14 (6%) on a bisphosphonate; with 9 (4%) previously on bisphosphonates. Steroids had never been taken by 57 (24%), moderate doses up to 29mg daily prednisolone by 124 (51%) and higher doses by 61 (25%) patients. Normal BMD was found in 119 (49%), osteopenic BMD in 98 (41%), and osteoporotic BMD in 22 (10%) patients. Osteoporotic fractures occurred in 22 (9.1%) patients, all women (9.6%). Of those with a history of fractures 2 (9%) had normal BMD, 13 (59.1%) had osteopenia and 7 (31.8%) had osteoporosis on BMD. Ethnic group, steroid use and disordered menstrual history were associated with reduced BMD but not with fractures. Impaired mobility was strongly associated with low BMD and fractures on univariate analysis but multiple logistic regression showed that age was the best predictor of fractures (p⬍0.001) with modified DI score and osteoporotic BMD exerting less influence (p⫽0.03). Impaired mobility and menopausal status were not independent predictors of fractures. Conclusion. Age and not steroid exposure is the biggest risk factor for fractures.

Disclosure:

Disclosure: This study was funded in part by an unconditional grant from Merke, Sharpe and Dohme and by grants from LUPUS UK.

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FLOW CYTOMETRY ANALYSIS OF ANTI-ENDOTHELIAL CELL ANTIBODY REACTIVITY: A NEW PARAMETER FOR ASSESSEMENT OF LUPUS DISEASE ACTIVITY. Lais LF Mendonca, Solange Carrasco, Bruno Della-Maggiore, Claudia Goldenstein-Schainberg, Renato C Monteiro Filho Sao Paulo, SP, Brazil and Paris, France

DYSREGULATION OF INSULIN RECEPTOR BY INSULIN LIKE GROWTH FACTOR-1 (IGF-I) AND INSULIN GROWTH FACTOR BINDING PROTEIN (IGFBP-3) LEADING TO HYPOGLYCEMIA IN SLE. Ranju Singh, Raquel S Cuchacovich, Wenqun Huang, Cindi E Jones, John Amos, Luis R Espinoza New Orleans, LA

Circulating immunoglobulins exert a key role in vascular homeostasis, modulating the endothelial response to antigenic stimulus. Conversely, lupus sera may have antibodies that cause activation and damage of the endothelial cell surface of most organs and systems. We have investigated, using a new approach, the ability of lupus antibodies to bind to endothelial cell surface antigens by flow cytometry (FACS), using a pure population of endothelial cells in suspension. Objectives: To detect anti-endothelial cell antibodies (AECA) using FACS and investigate the possible relationship between AECA and SLE Disease Activity Index (SLEDAI). Patients and Methods: Twenty-five SLE patients were enrolled for this study. Clinical assessment involved the determination of SLEDAI and collection of sera. AECA were determined by FACS using HUVEC as antigen. Fifteen sera from blood bank donors were used as control. Briefly, EC were harvest with trypsin and kept in TC-199/10% FCS at room temperature, for 2 hours on a platform shaker. Subsequently, 106 cells were placed in each tube and washed twice with PBS/2% FCS. Sera diluted 1:20 were incubated for 30 minutes, at 0° C. Tubes were washed 3 times and 10 ␮g of anti-IgG conjugated to FITC (Dako, USA) was added and incubated for an additional 30 minutes, at 0° C. The cells were washed 3 times and fixed with 2% paraformaldehyde and analyzed by flow cytometry. Peak channel values (a parameter that measures the fluorescence intensity obtained at binding of antibodies to cells) above the mean of the control population plus three standard deviations were considered as positive for IgG-AECA. Results: Fourteen patients presented a high IgG antibodies binding to endothelial cells as determined by FACS. There was a significant association between positive AECA and SLEDAI ⱖ 4 (OR⫽6; p⫽0.0001), since 10/14 (71%) patients with SLEDAI ⱖ 4 presented higher peak channel values than the control population. Moreover, there was a significant association between patients IgG binding to endothelial cells and the presence of altered urinary sediment, cutaneous ulceration and increased ESR (OR⫽ 5; 2,7 and 3,5, respectively; p⬍0.03). Conclusion: FACS is a widely used method to measure fluorescence intensity produced by labeled antibodies or ligands that bind to specific cell-associated molecules. By this technique, our findings demonstrated an increased binding of lupus antibodies to endothelial surface, which is most marked in patients with active disease. This phenomenon might be implicated in the initiation or perpetuation of the widespread activation of the vascular endothelium, an important characteristic of the lupus disease.

Introduction: Hypoglycemia as a presenting feature of SLE is a rare event. Most of these cases have been associated with insulin receptor antibodies leading to either insulin resistant diabetes or hypoglycemia due to unclear mechanism. We report a patient with severe hypoglycemia as initial manifestation of SLE in which treatment with corticosteroids resulted in complete resolution of symptoms. IGF-1/somatomedin C and IGFBP-3 were measured by a specific ELISA (R&D systems). IGF-1 mainly mediates actions of growth harmone, induces protein synthesis and glucose utilization enhancing insulin sensitivity. Also, it is an acute phase reactant, being produced by hepatocytes. IGFBP-3 is major transporter of IGF limiting its bioavailability. It prevents IGF induced hypoglycemia. The complex IGF-1/IGFBP-3 downregulates proinflammatory cytokines. Case report: A 23 year old black female with no past medical history presented with altered mental status and right sided weakness. Patient found to have initial blood glucose of 4mg/dl and repeat of 12mg/dl. While in the hospital , she continued to have severe hypoglycemia (4-60mg/dl) despite continues dextrose 10% infusion. Clinical and laboratory evaluation revealed right sided hemiperesis and other features suggestive of SLE including anemia, thrombocytopenia, serositis, positive antinuclear /anti-ds DNA / Sm antibodies, low complements and massive proteinuria (⬎10gm/24 hrs). Both serum C-peptide and fasting insulin levels were elevated. Antiphospholipid antibodies, urine drug screen, cortisol stimulation test, thyroid function tests and antibodies to human/pork/beef insulin were normal/negative. Patient was treated with I/V methylprednisolone (60 mg twice/day) with dramatic improvement of hypoglycemia within 24 hours. No further episodes of hypoglycemia were noticed after 6 months of follow up. IGF-1 and IGFBP-3 levels were measured at time of hypoglycemia and after 1 month of treatment. The ratio IGF-1/IGFBP-3 decreased from (2850/5700ng/ml⫽0.5) to (330/1253ng/ml⫽0.2), indicating that the synthesis of this acute phase reactant protein (IGF-1) decreased with corticosteroid treatment in parallel to SLE activity leading to improvement of hypoglycemia. Conclusion: Current case emphasizes the potential clinical importance of IGF-1/IGFBP-3 induced hypoglycemia in SLE since early institution of corticosteroid therapy can be life saving in this situation. Disclosure:

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POTENTIAL ROLE OF ANTI-C1Q ANTIBODY IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS. Pei-Chih Chen, Chrong-Reen Wang, Ming-Fei Liu Tainan, Taiwan

SERUM LEVELS OF IGE, IL-4, AND IL-13 IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS. Jun-Ki Min, Mi-La Cho, So-Youn Min, Myung-Su Lee, Chul-Soo Cho, Ho-Youn Kim Bu-Chen and Seoul, Republic of Korea

Objectives: The presence of antibodies against C1q (anti-C1q) in patients with systemic lupus erythematosus (SLE) has been demonstrated to be associated with renal involvement. In present study, we investigated the serum titers of anti-C1q in SLE with and without nephritis and correlated with renal pathology. Methods: Serum anti-C1q was measured in 26 healthy controls (Group1) and 47 SLE patients who were divided into groups of non-nephritis (Group 2) and nephritis (Group 3). Anti-C1q levels were assessed by ELISA method. We analyzed the relationship between the anti-C1q titers and SLE, renal C1q staining and WHO classification of lupus nephritis. Results: The anti-C1q levels in SLE patients were significantly higher than those of normal controls (53.187⫾73.616 U/ml v.s. 15.750⫾2.530 U/ml, p⬍0.0005). Compared with normal controls, only patients with nephritis reached a statistical significance (61.540⫾87.720 U/ml v.s. 15.750⫾2.530 U/ml, p⫽0.00492). The anti-C1q levels were significantly correlated with the presence of C1q deposition in the kidney tissue (66.038⫾91.141 U/ml v.s. 16.652⫾3.097 U/ml, p⬍0.01). Further analysis showed that the anti-C1q levels in serum appeared to bear a negative relationship with the staining intensity of C1q in renal tissues. However, the correlation coefficient did not demonstrate a significant difference (r⫽-0.209, p⬎0.1). No difference was noted in the anti-C1q levels between the WHO class IV and V lupus nephritis. Conclusions: The study showed that anti-C1q level was significantly elevated in lupus patients with nephritis, which suggests its potential pathogenesis in the development of nephritis. In addition, the correlation between serum anti-C1q level and the presence of C1q staining in kidney indicates that anti-C1q may play a role in C1q deposition in kidney, possibly through the immune-complexes formation. The negative correlation between anti-C1q levels and the renal C1q staining intensity may be explained by consumption of anti-C1q. In conclusion, our study suggests that the presence of high anti-C1q level may predict renal involvement in SLE patients and serial measurements of anti-C1q levels may have clinical implications in the management of lupus nephritis.

Objective: High serum levels of IgE and their association with disease activity have been reported in patients with systemic lupus erythematosus(SLE). IL-4 and IL-13 are major cytokines which promote the synthesis of IgE. IL-4 gene polymorphism is suggested to regulate IgE production. This study was designed to investigate whether the serum levels of IgE are associated with the circulating levels of IL-4, IL-13, and IL-4 repeat polymorphism in SLE or not. Methods: 107 patients with SLE and 66 healthy controls were enrolled in this study. Serum levels of IL-4 and IL-13 were measured by the ELISA method. In all SLE patients, evaluations for serum levels of IgE, C3, C4, anti-ds DNA antibodies were performed. Genomic DNA were examined by polymerase chain reaction for the analysis of GT nucleotide repeat in intron 2 of IL-4 and variable number of tandem repeat polymorphism located in the third intron of IL-4 genes. Results: The serum levels of IgE were higher in patients with SLE than in healthy controls (314.7 ⫾ 59.5 IU/ml vs. 56.8 ⫾ 17.9 Iu/ml, P⬍0.01, mean ⫾ SEM). Patients with SLE showed higher circulating IL-4 and IL-13 levels than those in controls (81.4 ⫾ 6.7, 414.6 ⫾ 120.9 pg/ml, P⬍0.01 vs. 25.4 ⫾ 1.1, 41.2 ⫾ 1.8 pg/ml P⬍0.01, respectively). Serum IgE levels were not correlated with the levels of IL-4 and IL-13 and were not associated with IL-4 repeat polymorphism. There were no correlations between the levels of serum IgE, IL-4, IL-13 and serum levels of C3, C4, anti-ds DNA antibodies. Conclusion: Serum IgE was significantly increased in patients with SLE without association with disease activity markers, serum levels of IL-4, IL-13, and IL-4 repeat polymorphism. Disclosure: This work is supported by a grant from Catholic Research Institute of Medical Science.

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ACQUIRED C1 ESTERASE INHIBITOR DEFICIENCY PRESENTING AS A LUPUS-LIKE SYNDROME WITH ANTICARDIOLIPIN ANTIBODIES. Maria Louise Barilla-LaBarca, C Yung Yu, Daniela Gioffre, Andrea Zanichelli, Marco Cicardi, John P Atkinson St. Louis, MO; Columbus, OH and Milan, Italy

CAPILLARY MICROCIRCULATION STUDY IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). CORRELATION WITH DISEASE PARAMETERS & ANTI CARDIOLIPIN ANTIBODIES (ACA). E Anwar, A El-Ashmawy, T Heshmat, N Zoheir, K Gado Egypt

Clinical and laboratory features may substantially overlap between SLE and inherited (HAE) or acquired (AAE) C1 esterase inhibitor (C1-Inh) deficiency. Angioedema is not an uncommon manifestation of SLE and AAE may present with minimal or no angioedema. We have studied two patients with C1-Inh deficiency secondary to an autoantibody who were diagnostic dilemmas because of clinical syndromes and laboratory profiles suggesting SLE, the presence of anticardiolipin antibodies and lack of typical angioedema. The first patient, with cognitive dysfunction, neutropenia and severe (Hct of 12%) autoimmune hemolytic anemia, was referred to us with a diagnosis of ANA negative lupus in the setting of a putative complete C4 deficiency. However, recognition of the distinct complement profile typical for AAE (low C1q, very low but detectable C4, low C2, normal C3, and low antigenic and functional C1-Inh) led to the search for and identification of an autoantibody to the C1-Inh. A non-Hodgkin’s B-cell lymphoma (immunocytoma) subsequently evolved. Splenectomy followed by Rituxan (rituximab) led to a remission, although C4 levels have remained low. In a second individual, AAE was identified in a patient with an ANA of 1:640. Both patients also have antiphospholipid antibodies and well documented venous thrombotic events. During a two-year follow up, neither patient developed angioedema despite ongoing complement activation. We hypothesize that the presence of the antiphospholipid antibodies prevented the angioedema by blocking kinin generation. These two cases also highlight the diagnostic difficulties that can arise in separating AAE from SLE. Guidelines to the workup of a low or absent C4 in such rheumatic disease patients will be presented.

We studied capillary microcirculation in 40 SLE patients (36 females&4 males) and 20 age & sex matched healthy controls. Study entailed: 1) segmental plethysmography (SP). 2) digital plethysmography (DP) before & after cooling. 3) capillary morphologic grading (CMG) according to Fagrell classification (1973). 4) red cell blood velocity (RCBV) & recovery time (RT) after CO2 application. SP in SLE patients was not different from control subjects suggesting no larger vessel affection. 38 SLE patients showed abnormal DP (19 before&19 after cooling) compared to none of the control group. All control subjects had normal CMG (grades 0&1) while 95% of SLE patients showed capillary morphologic alterations (CMA) from grades 2-6. RCBV was significantly lower (p⬍0.001) & RT was prolonged (p⬍0.001) in SLE patients than controls. There was no significant correlation between telagiectasia, livedo reticularis, musculoskeletal & cardiopulmonary affection and all microcirculation studies. Patients with Raynauds phenomenon (RP) showed higher CMA (p⬍0.001) & prolonged RT (p⬍0.05) than those with -ve RP. Also, patients with thrombotic events showed significantly prolonged RT (p⬍0.05) than those with no such events. Patients with ⫹ve ACA had significantly more frequent digital affection (p⬍0.05), higher CMA (p⬍0.001), lower RCBV (p⬍0.01) & more prolonged RT (p⬍0.01) than those with -ve ACA. Patients with renal hypertension had a significantly lower CMA (p⬍0.01) & less frequent digital affection (p⬍0.01) while a significant negative correlation was noted between RCBV & 24h.urinary proteins. We conclude that study of capillary microcirculation is a useful tool in clinical evaluation of patients with SLE.

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ANTI-52kD Ro, ANTI-60kD Ro AND ANTI-La ANTIBODY PROFILES IN NEONATAL LUPUS. P Gordon, M A Khamashta, E Rosenthal, J Simpson, G Sharland, A Brucato, R Franceschini, K De Bosshere, L Meheus, P L Meroni, G RV Hughes, J Buyon London, United Kingdom; Milan and Brescia, Italy; Ghent, Belgium; and New York

PREDICTORS OF QUALITY OF LIFE IN POSTMENOPAUSAL WOMEN AFTER LOW IMPACT DISTAL FOREARM FRACTURE. Maria-Teresa MP Cuddihy, L Joseph Melton III, Jeff S Sloan, Cynthia S Crowson, Sherine E Gabriel Rochester, MN

Aim: To assess the sensitivity of anti-52kD Ro, anti-60kD Ro and anti-La in mothers whose children have Neonatal Lupus utilizing several different methods of detection. Methods: 112 mothers whose children have neonatal lupus erythematosus(NNLMs) were studied. A serum sample from each mother was tested for anti-52kD Ro, anti-60kD Ro and anti-La antibodies by a line immunoassay (INNO-LIA® ANA Update)(LIA). The LIA uses recombinant 52kD Ro and La protein and purified human 60kD Ro antigens. In addition some samples were tested by counterimmuno-electrophoresis (CIE)(bovine antigen), ELISA(recombinant protein(rELISA) or bovine protein(bELISA)) or immunoblot(denatured human protein). Statistical analysis was by chi-square and McNemar’s test. Results: 10 NNLMs had children with rash, 93 had children with congenital heart block and 9 had both. By LIA, sensitivity to detect the NNLMS by anti-60kD Ro was 94.6%, compared to 85.7% for anti-52kD (p⬍0.05) and 76.8% for anti-La(p⬍0.001vs anti-60kD Ro). The sensitivity increased to 95.5% for any combination of the three antibodies. The sensitivity for skin rash alone or heart block alone was not significantly different for the three antibodies. By immunoblot of 65 NNLMs there were fewer positive results than by LIA for anti-60kD Ro(p⬍0.001) and anti-52kD Ro(p⬍0.05). By rELISA of 28 NNLMs there also were fewer positive results than by LIA for anti-60kD Ro(p⬍0.01). By immunoblot, sensitivity to detect the NNLMS by anti-La was 84.6%, compared to 81.5% for anti-52kD Ro(p⫽NS) and 53.8% for anti-60kD Ro(p⬍0.01vs anti-La). By rELISA the sensitivity to detect the NNLMS by anti-52kD Ro was 75%, compared to 69.2% for anti-La(13 samples, p⫽NS) and 50% for anti-60kD Ro(p⫽NS). The bELISA(67 NNLM) and CIE(29 NNLM) use bovine antigen which essentially only contains 60kD Ro. There were no statistical differences between LIA and bELISA results for anti-60kD Ro or La. CIE was significantly less sensitive than LIA for anti-La antibodies(p⬍0.01). Conclusion: By INNO-LIA® ANA Update the anti-60kD Ro antibody was the most sensitive antibody for neonatal lupus (94.6%). This contrasts with immunoblot where anti-52kD Ro was more sensitive for NNL(81.5%) than anti-60kD Ro(53.8%). The specificity needs to be evaluated in a relevant control group.

Purpose: Distal forearm fracture (DFF) is one of the earliest signs of osteoporosis occurring well before hip or spine fractures. Hip and spine fractures affect QOL, yet little is known about the impact of DFF on quality of life (QOL). We assessed QOL among a population-based cohort of 343 postmenopausal women of Olmsted County, MN who had sustained a DFF between 1993-1997. Methods: The DFF cohort was assembled using the resources of the Rochester Epidemiology Project, and survivors were sent a mailed survey in 1999 to assess their quality of life and osteoporosis preventive behaviors. The Osteoporosis QOL Questionnaire (OPToQOL) provided three domains: physical, fear and adaptations. We also asked a single question on perception of overall health. Preventive behaviors included exercise frequency and intensity, calcium intake (adequate ⫽⬎ 1200 mg/day versus low ⫽⬍1200 mg/day) and pharmacologic interventions for osteoporosis. We examined age at time of fracture, fracture history, past osteoporosis diagnosis, and Charlson co-morbidity index. Results: Among the 285 survivors, 168 (59%) responded (mean age 66 years). The median QOL scores (scale 0-100) were 91 for physical, 78 for fear and 62 for adaptations; QOL scores were lower on these 3 domains (p⬍.01) for women over 65 and those with prior osteoporotic fracture, by as much as 18%. Median overall health perception was only 2(scale 1-5)and was lower (p⬍.01) for women ⬍ 65 years and those who had ⬎ high school education. Daily calcium intake was adequate in 114 (68%) and low in 52 (31%). Only 20 (12%) exercised vigorously at least once per week, while 98 (60%) exercised moderately and 46 (28%) little or none. Women who exercised vigorously had higher QOL scores (p⬍.01)compared to women who exercised at lower intensity, by as much as 22%. QOL scores were not associated with use of calcium or with osteoporosis treatment(past or current). Conclusion. This study demonstrates that low impact DFF appears to have a substantial negative impact on quality of life. Aspects of QOL including physical, fear, and adaptations were lower in women over 65 years, those who are less physically active and those who have sustained a prior low impact trauma fracture. Yet, overall health perception was reduced in women under 65 and those who were more educated. Thus distal forearm fracture appears to have a major negative impact on quality of life among postmenopausal women who have had prior fragility fractures.

Disclosure: Innogenetics supported this project. Dr Gordon is supported by the British Heart Foundation

Disclosure: This work was funded through an Eli Lilly and company junior faculty development award in postmenopausal womens health

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1702

FACTORS ASSOCIATED WITH FEMORAL NECK BONE MINERAL DENSITY IN OLDER CAUCASIAN MEN: THE BALTIMORE MALE OSTEOPOROSIS STUDY (MOST). Marc C Hochberg, J Kathleen Tracy, Raymond H Flores Baltimore, MD

CONTINUOUS ORAL GLUCOCORTICOID THERAPY IS ASSOCIATED WITH A GREATER RATE OF BONE LOSS IN OLDER WOMEN: THE STUDY OF OSTEOPOROTIC FRACTURES (SOF). M C Hochberg, J K Tracy, M Vogt, K Stone, K Ensrud Baltimore, MD; Pittsburgh, PA; San Francisco, CA and Minneapolis, MN


MOST is a longitudinal cohort study designed to identify factors associated with low bone mass and bone loss in elderly men. Participants are volunteers recruited through mailings to holders of Maryland drivers licenses. All men completed a detailed questionnaire and underwent a standardized examination. Bone mineral density (BMD) was measured at the femoral neck (FN), lumbar spine (LS), and total body (TB) using a Hologic QDR-2000; quantitative ultrasound (QUS) was performed at the calcaneus using a Sahara. Presented herein are baseline data on 500 Caucasian men aged 64 to 93 years (mean [SD] 75.1 [5.8]) recruited from July 2000 thru May 2001. Mean [SD] FNBMD, LSBMD, TBBMD and QUS index (QUI) were 0.77 [0.15], 1.08 [0.20], 1.18 [0.13] g/cm2, and 93.8 [20.0], respectively. Only 38 (7.6%) men had osteoporosis defined by World Health Organization criteria. Significant (P ⬍ .01) correlations were noted between FNBMD and LSBMD (R ⫽ 0.28), FNBMD and TBBMD (R ⫽ 0.54), and LSBMD and TBBMD (R ⫽ 0.67); QUI was not significantly correlated with BMD at any site. Demographic, anthropometric, family and history, and clinical variables were assessed for their association with FNBMD initially in bivariate analyses and then in age-, and weight-adjusted multiple variable models using SPSS. Current weight was a significant predictor of FNBMD (adjusted R2 ⫽ 0.16). Men with prostate cancer (N⫽81) had significantly higher adjusted mean FNBMD than those without prostate cancer: 0.798 vs 0.765 g/cm2, P ⫽.044, These data demonstrate that older Caucasian men who have a higher weight have greater bone mass. Furthermore, higher FNBMD may be a risk marker for prostate cancer. Future analyses from MOST will compare baseline and longitudinal data between Caucasian and African-American men.

Oral glucocorticoid (GC) therapy is the most common cause of secondary osteoporosis and is associated with an increased risk of fracture. Dual xray absorptiometry scans obtained at visits 2 and 4 (mean 3.5 years apart) were analyzed from 4,258 white women in SOF (mean [SD] age 77.1 [5.1] years) who had complete data on use of GCs at visits 1, 2 and 4. Percent change in total hip and femoral neck bone mineral density, adjusted for age, weight, physical activity, smoking and self-reported health status at visit 4, and weight change between visits 2 and 4, were compared between continuous users at all three visits (N⫽125), former users (users at either visit 1 or 2 but not at visit 4, N⫽202), and never users (N⫽3931). %/yr Total hip Femoral neck

Continuous Users

Former Users

Never Users

1.30 (0.13)* 0.89 (0.17)*

0.42 (0.10) 0.25 (0.13)

0.55 (0.02) 0.39 (0.03)

Adjusted rates of bone loss were significantly higher in continuous users than in both the former users and the never users (*P ⬍ .01 for all comparisons). None of the differences between former users and never users were significant. These data demonstrate that continuous users of oral GCs lose bone at a greater rate than those not on such therapy. Elderly women on oral GC therapy should receive treatment to prevent bone loss and reduce their increased risk of fractures.

Disclosure: Supported by grants from the Department of Veterans Affairs and Arthritis Foundation, Maryland Chapter.

Disclosure:

1700

1703

SELF-REPORT OF PHYSICAL ACTIVITY VS.WEIGHT-BEARING MEASURE: RELATION TO BONE MASS. Kathleen M Coyle, Robert McLean, Douglas P Kiel, Marian T Hannan Boston, MA and Philadelphia, PA

A PROSPECTIVE STUDY OF RISK FACTORS FOR FALLS IN ELDERLY INSTITUTIONALISED INDIVIDUALS. Lyn M March, Tang C Lau, Alan B Brnabic, Jane M Zochling, Yih Y Sitoh, Stephen J Lord, Jennifer M Schwarz, Ian D Cameron, Robert G Cumming, Philip N Sambrook St Leonards, Ryde, and Randwick, Sydney, NSW, Australia

Physical activity has a large impact upon bone health, yet no standardized method exists to measure it in large studies. Our goal was to compare self-reported physical activity to weight-bearing activity measured with an ambulatory monitor as they relate to bone mass. We examined these relations in a cross-sectional study of adults. Subjects were recruited from both the Framingham Offspring Study (n ⫽ 79) and Harvard Cooperative Program on Aging research subject registry (n⫽28). We obtained 2 ultrasound measures of calcaneal bone mass using the Sahara bone sonometer (Hologic): broadband ultrasound attenuation (BUA) and speed of sound (SOS). Higher values indicate greater bone mass. Self-reported physical activity was assessed by using the Physical Activity Scale for Elderly (PASE) questionnaire and weight-bearing activity was measured as number of steps over 3 days via the Ambulatory Monitoring System (AMS), an accelerometer worn on top of a shoe to count steps. PASE is validated for adults ages 50⫹ and produces a score based on activities over the previous 7 days. Correlation analyses examined relation between bone mass and PASE as well as AMS. Linear regression tested relation of bone mass for PASE and AMS controlling for age, sex, weight and height. Separate models were also considered for each sex. Mean age was 62 ⫾11 years (range 36-86) for the 107 subjects (47 women, 60 men); mean PASE 139 ⫾86; mean AMS 7990 ⫾3783; mean ultrasound BUA 76.4 dB/MHz ⫾19.1 and mean SOS 1556 m/second ⫾33.0. Ultrasound measures were weakly correlated to PASE (r⫽0.11, p⫽0.26) but more strongly correlated to AMS count (r⫽0.21, p⫽0.03). Regression models continued to show a strong relation between bone mass and PASE (p⫽0.66). AMS contributed an additional 6% to R2 in regression models, whereas PASE did not add any information to explain variance in models. Results were similar for sex-specific analyses. In sum, AMS activity provides a more direct count/measure of weight-bearing activity than PASE score, and is also a better indicator of bone mass. Indeed, in our study, PASE was not related to bone mass measures. Collecting physical activity data in large studies is a daunting task, and our study indicates that compared to PASE, the AMS may be a better tool for measuring weight-bearing activity. Disclosure:

Osteoporosis is a major public health issue and will become increasingly common as our population ages. Falls are an important risk factor for osteoporotic fractures. To study potential predictors for falls in elderly insitutionalised individuals, 896 residents of nursing homes and hostels in North Sydney were interviewed after giving either self or proxy informed consent. Demographic data, comorbid health conditions, measurements including cognitive function assessed by SMMSE, Melbourne Edge Visual Test, lower limb proprioception, quadriceps muscle strength (QMS), body sway, reaction time, and serum vitamin D levels were collected. Fall data were recorded prospectively. There were 416 nursing home (mean age 85.3, M:F ⫽ 92:324) and 480 hostel residents (mean age 84.4, M:F ⫽ 147:333 ). A total of 1103 falls (number of fallers ⫽ 348, incidence of fall per person year ⫽ 1.388) and 111 deaths were recorded after a median follow up time of 336 days. Older age, lower SMMSE scores, nursing home residents, use of walking aids and low quadriceps muscle strength values were independent predictors of time to first fall using univariate analysis. Vitamin D levels were low in this population however no association with falling was found. A sex stratified Cox model with no-interaction assumption was used to evaluate the time to first fall with deaths censored. After controlling for sex and age, people with lower mean QMS [hazard ratio (HR) 0.98, confidence interval (CI) 0.952-0.998, p⫽0.039], nursing home residence (HR 2.78, CI 1.801-4.275, p⬍0.001) and people using walking aids (HR 1.85, CI 1.294-2.648, p⫽0.001) were more likely to fall. Lower quadriceps muscle strength, nursing home residential status and use of walking aids are significant predictors of falls in insitutionalised elderly people. Strength and mobility programs could be useful in preventing falls in this population. Disclosure:

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1707

TRENDS AND ETHNIC DIFFERENCES IN HIP FRACTURE MORTALITY IN TEXAS, 1990-1998. Carlos H Orces, Benjamin S Bradshaw

TAPASIN POLYMORPHISM IS ASSOCIATED WITH SYSTEMIC ONSET JUVENILE RHEUMATOID ARTHRITIS (SoJRA). Hulya Bukulmez, Susan D Thompson, Paul Horn, Edward H Giannini, David N Glass, Robert A Colbert Cincinnati

Objective: To determine the age, sex, and ethnic group adjusted-death rates due to hip fracture and to describe the trends in hip fracture death rates in Texas during 1990-1998. Methods: Death certificates in which hip fracture (ICD 820) was mentioned as the cause of death (underlying or multiple cause) were obtained from Texas Department of Health for the years 1990-1998. Population estimates for Texas by age, sex, and ethnic groups from 1990 through 1998 were obtained for each year from the Bureau of Census. Age-specific (5-year group) and age-adjusted death rates for ages 50 years and older were calculated by the direct method using the U.S 1990 population as the standard. Age-adjusted percent changes in rates were calculated for the differences between years 1990-98. Proportional mortality rates (PMR) using death rates for hip fracture and total deaths in Texas for each year by age, sex, and ethnic group as denominator were calculated. Results: Table 1 demostrates % changes in age-adjusted death rates, sex mortality ratio and PMR from 1990-98. Conclusion: Hip fracture-associated mortality rates observed for white men are becoming a major public health problem that will assume even greater significance as the population ages. Further research is needed to explore cause of death patterns associated with hip fracture using multiple cause models. Sex

1990

men women

21.6 18.5

men women

7.4 4.6

men women

1998 Whites 25.7 20.7 Blacks 10.1 6.6 Hispanics 6.8 7.1

11.3 7.9

% change

Ratio(M/F)

PMR

19 11.9

1.3

0.6 0.9

36.5 43.5

1.5

0.2 0.2

-39.8 -10.1

1.2

Objective: Numerous human leukocyte antigen (HLA) alleles encoded in the major histocompatibility complex (MCH) have been associated with susceptibility to pauci- and polyarticular onset JRA, and some with SoJRA. To further investigate candidate susceptibility genes we examined the tapasin polymorphism. Tapasin, a protein involved in HLA-class I assembly, is encoded in the MHC, centromeric to HLA-DP and has 2 known alleles (01 and 02) defined by a single amino acid difference. Methods: Tapasin genotypes were determined for 291 children with JRA, 203 of their parents, and 113 healthy individuals by differential digestion of a PCR amplified DNA fragment with restriction enzymes Bfa I and Sfc I. Chi-square statistic was used to test for association, and transmission disequilibrium testing (TDT) was used to assess linkage. Results: The tapasin genotype distribution was different in SoJRA group when compared to the pauci/polyarticular onset group or healthy controls, with 0101 being more frequent (Table). This difference was statistically significant when the SoJRA group was compared to the pauci/polyarticular group (␹2⫽7.13, p⫽0.028, 2df), but not when compared with healthy controls (␹2⫽5.0, p⫽0.08, 2df). A higher proportion of patients with SoJRA were homozygous for the 01 allele as compared to the pauci/polyarticular JRA group (␹2⫽4.02, p⫽0.04). Controls had a lower frequency of 01 homozygosity than did SoJRA patients, but this was not statistically significant (␹2⫽1.93, p⫽0.16). Tapasin genotypes 0101 0102 0202

0.3 0.5

Pauci-and Poly JRA n ⫽ 199 57 93 49

28.6% 46.7% 24.6%

SoJRA n ⫽ 92 38 42 12

41.3% 46.2% 12.5%

Control n ⫽ 113 35 56 22

31.0% 49.5% 19.5%

We next determined whether there was evidence for linkage in SoJRA families. In 20 simplex families, 24 parents were informative (i.e. heterozygous for the tapasin alleles). The 01 allele was transmitted seventeen times to the affected offspring (TDT ⫽ 4.166, p ⫽ 0.039). Conclusions: These results suggest that the tapasin 01 allele may be associated and linked to susceptibility to SoJRA. Further studies investigating whether tapasin polymorphisms are functionally significant may enhance our understanding of the etiopathogenesis of SoJRA. Disclosure: Hulya Bukulmez, M.D., Third year fellow in Pediatric Rheumatology Department in Childrens Hospital Medical Center Cincinnati, Cincinnato, Ohio

Disclosure:

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1708

LOWER EXPRESSION OF GRANZYME B IN CD8⫹ T CELLS OF PATIENTS WITH SYSTEMIC ONSET JUVENILE RHEUMATOID ARTHRITIS. Neil J Normand, Thomas JA Lehman, Michael Henrickson, Keith B Elkon, Karen B Onel New York, NY and Madera, California

MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) IN PATIENTS WITH JUVENILE IDIOPATHIC ARTHRITIS. Cristina Meazza, Patrizia Pignatti, Silvia Magni-Manzoni, Federica Rossi, Alberto Martini, Fabrizio De Benedetti Pavia, Italy

The acute hemophagocytic syndrome (HPS) is a severe and potentially fatal complication of Systemic Onset Juvenile Rheumatoid Arthritis (SOJRA) that is manifested by profound cytopenias and disseminated intravascular coagulation (DIC). This syndrome has been ascribed to intrinsic macrophage hyperactivity or defective T cell function, and is frequently triggered by viruses or medications. Recently, the molecular defects underlying two related forms of hemophagocytosis, Familial Hemophagocytic Lymphohistiocytosis (FHL) and X-Linked Lymphoproliferative Disease (XLP) have been identified. Both syndromes share abnormalities in T cell cytotoxic function. Because hemophagocytosis is common to all three syndromes we evaluated cytotoxic T cells in SOJRA. We previously observed low levels of perforin, a pore-forming protein essential for T cell cytotoxicity, in some patients with SOJRA, especially those with long-standing disease. Granzyme B, is a serine protease that also functions in the perforin-granzyme pathway of cell death. In this study we asked whether granzyme B expression is reduced in SOJRA patients with normal levels of perforin. To assess granzyme B levels, PBMC’s were isolated from patients with SOJRA, Pauciarticular JRA (PAJRA), Rheumatoid arthritis (RA), Systemic Lupus Erythematosus (SLE) and normal healthy individuals. Cells were stimulated with PHA (2ug/ml) and IL-2 (20 units/ml) for 6 days with appropriate monitoring for activation status and CD4/CD8 ratio. Whole cell lysates were separated by SDS-PAGE and levels of granzyme B evaluated by western blot analysis. Granzyme B expression was defined as low if the result was less than half of that of the normal control. Results

MIF shows a broad range of immunostimulatory and proinflammatory activities. It induces T cell proliferation and IL-2 production and macrophage production of the proinflammatory cytokines IL-6, TNF-a, IL-1, and IL-8. In addition, MIF has the unique ability to override the inhibitory effects of glucocorticoids on immune cell activation and cytokine production. We evaluated MIF production in patients with juvenile idiopathic arthritis (JIA). Serum MIF levels, measured by ELISA, were significantly higher (p⬍0.001) in patients with active JIA (48.4⫾52.4 ng/ml) than in controls (4.1⫾4.0) and in JIA patients in remission (6.1⫾5.2). Higher serum MIF levels were found in systemic JIA (69.7⫾70.1) compared to patients with polyarticular (20.6⫾22.8) or oligoarticular JIA (42.0⫾36.3). In addition, in unstimulated conditions peripheral blood mononuclear cells of patients with systemic JIA released higher amounts of MIF (2.4⫾0.9 ng/ml) compared to patients with oligoarticular JIA (1.7⫾0.7) or to healthy controls (1.5⫾0.4). Synovial fluid MIF levels of patients with systemic JIA (75.5⫾78.0 ng/ml) were significantly higher than those of patients with oligo-JIA (16.1⫾17.5). In individual joints, SF MIF levels were inversely correlated with the duration of the clinical remission induced by intraarticular administration of triamcinolone hexacetonide in both systemic (R⫽-0.714, p⫽0.05) and oligoarticular JIA (R⫽-0.508, p⫽0.003). These data show that MIF levels are increased in patients with JIA and particularly in patients with systemic JIA and the presence of a significant correlation with the clinical response to intraarticular steroid injection, suggesting the involvement of MIF in the pathogenesis of JIA.

Diagnosis and No. No. with HPS Low Granzyme

SOJRA (n⫽10)

RA (n⫽4)

SLE (n⫽4)

PAJRA (n⫽1)

Normal (n⫽9)

5 7

0 1

0 0

1 1

0 2

Disclosure:

These results suggest a role for the low expression of granzyme B as well as perforin in susceptibility or pathogenesis of hemophagocytosis associated with systemic inflammatory disease in childhood. Disclosure:

1709

A NOVEL 5⬘FLANKING REGION POLYMORPHISM OF MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) IS ASSOCIATED WITH UK JUVENILE IDIOPATHIC ARTHRITIS. Rachelle Donn, Emma Shelley, William Ollier, Wendy Thomson, THE BPRG Study Group Manchester, England United Kingdom

STUDY OF THE INSULIN-LIKE GROWTH FACTOR-I (IGF-I) SYSTEM IN INTERLEUKIN-6 (IL-6) TRANSGENIC MICE. Cristina Meazza, Patrizia Pignatti, Marina Vivarelli, Simona Garrone, Antonina Barreca, Alberto Martini, Fabrizio De Benedetti Pavia and Genova, Italy

Background: MIF is a ubiquitously expressed protein that has pro-inflammatory, hormonal, and enzymatic activities. In the presence of MIF macrophage intracellular killing and phagocytic function is up-regulated, as is macrophage secretion of TNF-alpha. Uniquely, MIF release from immune cells can be biphasically regulated by glucocorticoids. Meazza et al. recently described significant increased levels of MIF within the serum and in the synovial fluids of children with JIA. This increase was most marked for children with systemic-onset juvenile idiopathic arthritis (SYS-JIA)(1). No previously reported single nucleotide polymorphisms (SNPs) have been described for the MIF gene. We used dHPLC technology (Wave, Transgenomic, Crewe, UK) to screen the MIF gene. This identified a G to C polymorphisms at position -173, in the 5⬘ flanking region of the gene (ref: Genbank Accession no: L19686). This nucleotide change creates an activator protein 4 (AP-4) transcription factor binding site. Methods: A large panel of UK caucasian JIA patients (n⫽526) and unrelated healthy controls (n⫽259) were genotyped for the MIF-173 G/C polymorphism using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP). The presence of a C at -173 of MIF introduces an AluI restriction enzyme site. Primers: MIF-173 forward - 5⬘ACT AAG AAA GAC CCG AGG C 3⬘; MIF-173 reverse - 5⬘ GGG GCA CGT TGG TGT TTA C 3⬘. Amplified PCR product (3ul) was digested in a 10ul final reaction volume using 1ul 10x Reaction Buffer 2 and 4 units Alu I restriction enzyme (New England Biolabs), at 37°C overnight. Results: The allele and genotype frequencies were significantly different between the JIA patients and the controls. Specifically, possesion of a MIF-173*C allele gives an approximately two fold increased risk of JIA susceptibility (34.8% vs 21.6%; OR 1.9; 95% CI 1.4-2.7). Conclusions : Polymorphisms within MIF may predict both an individual’s predisposition to chronic inflammation and their sensitivity in terms of response to exogenously administered steroid treatment. The -173 G/C polymorphism we describe is the first report of a SNP in the MIF gene. This polymorphism is associated with UK JIA. Luciferase reporter gene assays are currently being carried out to determine the functional significance of the MIF-173 polymorphism. References: 1. Meazza C, Pignatti P, Magni-Manzoni et al. (2000) Arth & Rheum. 43 (9); Abs. 1739.

Low levels of IGF-I and IGF binding protein-3 (IGFBP-3) are present in chronic inflammatory diseases associated with stunted growth. IL-6 transgenic NSE/hIL-6 mice, expressing elevated levels of IL-6 since birth, present growth impairment associated with low levels of IGF-I and IGFBP-3. Here we demonstrate in NSE/hIL-6 mice proteolytic degradation of IGFBP-3 by a serum protease activity that appears, by zymogram, to be secondary to metalloproteinase-2 (MMP-2). Therefore, decreased circulating levels of IGFBP-3 are, at least in part, due to increased degradation. Gel chromatography show that low levels of IGFBP-3 cause impaired formation of the ternary complex, comprising IGF-I, IGFBP-3 and ALS. Pharmacokinetic studies following an i.v. bolus of IGF-I show a 2-fold increase in total plasma clearance in transgenic (0.029ml/min/g) compared to wild-type mice (0.015), associated with a decreased T1/2(beta) . Therefore, impaired ternary complex formation causes an increased clearance of IGF-I. Administration to NSE/hIL-6 mice of IGF-I (1mg/gr s.c. twice daily) or of IGF-I complexed with IGFBP-3 (9 mg/gr s.c. twice daily) led to increased mortality (p⬍0.05) in transgenic mice with no deaths in wild-type littermates. Defective ternary complex formation due to low IGFBP-3 levels and increased proteolytic degradation of IGFBP-3 appears to explain the increased mortality to administration of IGF-I alone and of IGF-I complexed with IGFBP-3 respectively, exposing the animals to the acute toxic insulin-like effects of IGF-I. This study provides information relevant to the development of therapeutic approaches aimed at correcting abnormalities of the IGF-I system and subsequently of stunted growth in chronic inflammatory diseases. Disclosure:

Disclosure:

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1710

1713

REGULATORY ROLE OF PEPTIDES FROM HUMAN HOMOLOGUES OF E.COLI HSP DNAJ IN THE AUTOIMMUNE INFLAMMATION OF OLIGOARTICULAR JIA (O-JIA). Margherita Massa, Maristella Passalia, Fabrizio De Benedetti, Silvia Magni Manzoni, Stefania Viola, Angela Pistorio, Berent Prakken, Alberto Martini, Salvatore Albani Pavia, Italy; Netherlands

EVIDENCE OF LINKAGE TO HLA-A AND HLA-DRB1 IN UK CAUCASIAN JUVENILE IDIOPATHIC ARTHRITIS. Eleftheria Zeggini, Rachelle Donn, William Ollier, Wendy Thomson, The BPRG Study Group Manchester, United Kingdom

Immune responses to heat shock proteins (hsp) appear to play a relevant role in the induction and/or regulation of chronic autoimmune inflammation. We showed that synovial fluid mononuclear cells (SFMC) from patients with o-JIA had higher proliferative responses and IFN␥ production to rec. E.coli hsp dnaJ (rdnaJ) than peripheral blood MC. To evaluate correlation with the course of the disease, patients were divided in persistent (n⫽16) and in extended o-JIA (n⫽15). The proliferative responses to rdnaJ of SFMC from patients with extended o-JIA were lower (p⫽0.014) than those of SFMC from patients with persistent o-JIA; IFN␥, TNF␣, and IL-10 production were comparable in the two groups. Studies with 8 synthetic peptides from E.coli dnaJ showed that in patients with persistent o-JIA responses were mainly directed to 4 of the 8 peptides (B22-36, B61-75, B174-188 and B268-282). In patients with extended o-JIA lower proliferative responses were found with peptide B174-188 (p⬍0.03). No IL-10 was detected in the two groups. Stimulation of SFMC with 3 peptides, derived from regions of three human hsp dnaJ (HDJ1, HDJ2, and HSJ1) with sequence homology with the bacterial peptide B174-188, induced detectable IL-10 (pept H164-178:10.2⫾13.8 pg/ml; pept H167-181:7.5⫾9.6; pept H176-190: 10.6⫾13.1). On the contrary, IL-10 production in response to 9 peptides derived from human dnaJs and non homologous to E.coli peptides was present in higher frequency in patients with persistent o-JIA than in those with extended o-JIA. These results suggest that T lymphocyte reactivity to unique human peptides may be part of T cell regulatory mechanisms affecting the course of joint inflammation. Disclosure:

Background. Previously we have reported multiple associations between HLA alleles and oligoarticular disease in an ILAR-defined UK Caucasian JIA cohort1. However, true susceptibility genes can only be identified by linkage. Linkage to HLA loci in ILAR-defined JIA in UK Caucasians has not previously been examined. Objective. To establish linkage to HLA-A and HLA-DRB1 in oligoarticular (persistent and extended) JIA in UK Caucasians. Methods. A total of 117 families consisting of affected offspring and their parents were typed for HLA-DRB1 and HLA-A using a commercially available PCR-SSOP typing system (DynalRELI SSO, Dynal, UK). Of these, 76 patients had persistent, while 41 had extended oligoarticular JIA. Analysis was performed using the extended transmission disequilibrium test (ETDT)2 that detects linkage in the presence of association. Significance was assessed using the likelihood ratio test and p values were corrected by using Monte Carlo simulations. Results. Linkage to HLA-DRB1 was established for all oligoarticular patients (p⫽0.003, 115 informative transmissions). Excess transmission of alleles DRB1*08 and *11 was demonstrated, whilst alleles *04 and *07 were transmitted significantly less frequently than expected. Linkage to HLA-DRB1 was also established for persistent oligoarticular JIA (p⫽0.002, 72 informative transmissions), whereas the p value for extended oligoarticular disease became nonsignificant after correction (p⫽0.070, 43 informative transmissions). Oligoarticular JIA was linked to HLA-A (p⫽0.003, 84 informative transmissions), with significant deviation from random segregation for alleles A*02 (excess transmission) and *03 (reduced transmission). Linkage to HLA-A was established for persistent oligoarticular JIA (p⫽0.045, 52 informative transmissions), with A*01 significantly transmitted in excess and *02 exhibiting a trend for excess transmission. Extended oligoarticular JIA was also found to be linked to HLA-A, A*02 being the contributory allele transmitted in excess. Conclusions. This is the first study to establish linkage to HLA-A and HLA-DRB1 in oligoarticular JIA in UK Caucasians. Further studies are being undertaken to determine whether it is these loci or other genes in linkage disequilibrium with them that are the major contributors to JIA susceptibility. References. 1. Thomson W, Southwood T, Woo P, Pepper L, Barrett J, Donn R, Ollier WER On behalf of the BPRG (1999) Annals of the Rheumatic Diseases 58; suppl. 333. 2. Sham PC and Curtis D (1995) Annals of Human Genetics 59; 323-336. Disclosure:

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CHEMOKINE RECEPTORS CCR1, CCR3, CCR5 AND CXCR3 EXPRESSION IN SYNOVIAL T CELLS OF PATIENTS WITH JIA. Margherita Massa, Maristella Passalia, Paola Travaglino, Patrizia Pignatti, Cristina Meazza, Alberto Martini, Fabrizio De Benedetti Pavia, Italy

ANTI-CYCLIC CITRULLINATED PEPTIDE ANTIBODIES IN JUVENILE RHEUMATOID ARTHRITIS PATIENTS. Terry L Moore, Alexander K Chung, Daniel A Kietz, Peri H Pepmueller St. Louis, MO

Chronic arthritis is associated with the accumulation of Th1 cells into inflamed joints. We evaluated, by two color flow cytometry, the expression of the chemokine receptors CCR5 and CXCR3, characteristic of Th1 cells, and CCR1 and CCR3, characteristic of Th2 cells, on synovial fluid (SF) and peripheral blood (PB) T cells of patients with polyarticular (po-JIA), or oligoarticular JIA (o-JIA), and from PB of healthy controls. We measured SF levels of chemokines IP-10 and the functionally related MIG, that share CXCR3, and MIP-1␤ that binds CCR5. No difference was found in the expression of chemokine receptors between PB T cells of JIA patients or controls. In JIA patients expression of CCR1 and CCR3 (Th2 related) was comparable in PB and SF, while a significantly (p⬍0.01) higher percentage of T cells expressing CCR5 or CXCR3 cells (Th1 related) was found in SF of patients with po-JIA or o-JIA. The fold increase of the percentage of CXCR3⫹ T cells in SF relative to PB was significantly higher (p⬍0.03) in patients with extended o-JIA than of persistent o-JIA. SF levels of the chemokines related to CXCR3 (IP-10 and MIG) or to CCR5 (MIP-1␤) were detectable and higher in patients with po-JIA or extended o-JIA compared to persistent o-JIA (Table).

IP-10 MIG MIP-1b

persistent o-JIA

extended o-JIA and polyarticular JIA

P value

9552 ⫾ 18065 3578 ⫾ 3320 67.4 ⫾ 46.1

14652 ⫾ 26236 14055 ⫾ 14063 272 ⫾ 360.6

0.09 0.024 0.012

Introduction - Previous studies in patients with rheumatoid arthritis (RA) have revealed that anti-cyclic citrullinated peptide antibody (anti-CCP) presence has specificity of 95% and are found in 60-75% of these patients. The object of this study was to evaluate the predictive value of anti-CCP antibodies in patients with juvenile RA (JRA). Methods - The presence of anti-CCP antibody was determined using a single citrullinated peptide variant optimized for detecting JRA-specific autoantibodies in serum by an ELISA-based test. We evaluated 66 JRA patients (16 seropositive polyarticular, 18 seronegative polyarticular, 19 pauciarticular, and 13 systemic-onset). We also tested RA, systemic lupus erythematosus, and healthy patients as controls. Results - Significant levels of anti-CCP antibodies were detected in 51 of 66 (77%) JRA patients including 12/16 (75%) seropositive polyarticular, 15/18 (83%) seronegative polyarticular, 16/19 (84%) pauciarticular, and 8/13 (62%) of systemic-onset. Healthy controls showed no antibody expression. The presence of anti-CCP antibodies correlated with disease activity and with disease response to therapy with falling levels, but did not statistically correlate with rheumatoid factor presence. Conclusions - Our data indicate anti-CCP antibody is highly specific for JRA patients and is present in the early stages of disease. It may be a disease marker for JRA and indicates a possible role of citrulline-containing epitopes in the pathogenesis of JRA. Disclosure:

These data show expression of the Th1 related chemokine receptors CCR5 and CXCR3 and of the Th1 related chemokines IP-10, MIG and MIP-1␤ in JIA patients and suggest that increased expression of both the chemokines and the chemokine receptors is associated with a polyarticular course.


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EPITOPE MAPPING OF DEK, A MAJOR AUTOANTIGEN IN JUVENILE RHEUMATIOD ARTHRITIS. Ilona S Szer, Weiguo Zhu, Eng M Tan, Wlodzimierz Szer San Diego and La Jolla, CA

DETECTION OF BACTERIAL DNA IN SYNOVIAL FLUID AND PERIPHERAL BLOOD FROM CHILDREN WITH JUVENILE IDIOPATHIC ARTHRITIS. Fazeelat Bibi, Clive A J Ryder, Janet M Gardner-Medwin, Elsadeg Sharif, Nigel L Brown, Gabrielle Kingsley, Taunton R Southwood Birmingham and London, United Kingdom

Epitope mapping often provides insights into the role of an autoantigen in normal metabolism and helps to define its functional domains, e.g. active and/or binding sites of the protein. We have previously shown that anti-DEK antibodies are frequently detected in the sera of children with Pauciarticular JRA, particularly when iritis is present. Methods: The Glutathion S-Transferase Fusion System was used to generate recombinants containing from 20 to 175 amino acids of DEK (the protein is 376 aa long). The antigenicity of the individual fragments was assayed by Western Blotting (WB). Results: WB of three partially overlapping recombinants (150-175 aa in length each) covering the entire length of DEK demonstrated that the antigenicity is located primarily in the C-terminal domain (position 220-376). Focusing on this domain, we have further narrowed the active region to a 60 aa stretch (position 315-376). This region was analyzed using recombinants of about 20 to 40 aa long. The major epitope appears to be located in the middle portion of the region (position 335-355). However, the C-terminal 21 aa fragment (position 355-376), while totally inactive by itself, is most essential since only those larger fragments that include it, are strongly reactive. It appears that the upstream 20 aa fragment (position 315-335) plays a similar role: inactive by itself, its presence enhances, or confers reactivity on larger recombinants. Conclusions: A complex antigenic site near the C-terminus of DEK appears to contain both linear and conformational recognition elements within a stretch of 60 aa. The central region of some 20 aa (position 335-355) reacts with only the strongest sera but reactivity is enhanced by the presence of one or, better, of both of the flanking regions. This system of different recognition elements may be responsible for discrimination between weakly and strongly reactive sera and, for the fragments reported here, for multiple and variable specificities of autoimmune sera. The results also indicate that the use of short peptides(e.g. 15-mers) is not sufficient for the determination of complex epitopes.

An infectious aetiology has been implicated in juvenile idiopathic arthritis (JIA), although JIA synovial fluid (SF) culture is, by definition, sterile. We have previously reported indirect evidence that antigens may drive intra-articular inflammation by demonstrating that JIA SF T cells recognise a variety of infectious agents, particularly gram -ve enteric organisms. The aim of the current project was to use a eubacteria-specific, culture-independent approach (bacterial 16S ribosomal DNA nested polymerase chain reaction: rDNA PCR) to detect bacterial DNA in SF and peripheral blood (PB) from patients with JIA. Broad range primers specific for conserved regions on the 16S ribosomal gene were used to detect bacterial DNA and the associated hypervariable regions enabled identification of the bacteria. Positive PCR products were isolated, sequenced and identified using the BLASTn tool to search for homologous sequences in both the GenBank and EMBL databases. 110 paired SF and PB samples were obtained from 46 JIA patients (oligoarthritis 23, extended oligo 7, RF- polyarthritis 6, systemic arthritis 6, enthesitis related arthritis 2, unknown 2) undergoing therapeutic arthrocentesis, and control PB samples were collected from 12 healthy adult volunteers. Bacterial DNA was detected in SF and/or PB in 13/46 JIA patients (28.3%) and 0/12 controls. 26% of oligoarthritis or extended oligo patients were positive, compared with 12.5% of other articular patterns. In 4/13 patients, bacterial products were detected in SF alone; the remainder had bacterial products in either PB alone (4) or both SF and PB (5). Bacterial species previously associated with arthritis that were detected in JIA SF included Bacillus, Micrococcus, Nocardia and Staphylococcus. Bacterial species detected that had not previously been recognised as pathogenic in humans included Acidovorax, Bradyrhizobium and Clavibacter. Several previously unrecognised bacterial sequences were also isolated. No bacterial species specific for any subgroup of JIA were found. In conclusion, over 25% of this JIA population had detectable bacterial DNA, detected by rDNA PCR, in SF, PB or both compartments. Microbial invasion of these compartments may contribute to the chronic inflammatory process of JIA. Disclosure: This project is supported by a grant from the Arthritis Research Campaign UK

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IDENTIFICATION OF JOINT INFLAMMATION SPECIFIC T-CELL RESPONSES IN JUVENILE IDIOPATHIC ARTHRITIS. Sylvia Kamphuis, Marca Wauben, Jolanda van Bilsen, Margje Haverkamp, Grace Gordon, Ger Rijkers, Wietse Kuis, Berent Prakken Utrecht, Netherlands

IMMUNOLOGIC EFFECTS OF ACUTE EXERCISE ON INFLAMMATORY MEDIATORS IN CHILDREN WITH JUVENILE IDIOPATHIC ARTHRITIS. Kathleen M O’Neil, Nadine Fisher, Jaya Venkatraman Buffalo, NY

Objective: Juvenile Idiopathic Arthritis (JIA) is an auto-immune disease in which autoreactive T-cells play a central role in joint inflammation and destruction. The immune responses are thought to be directed against self-antigens present in the (inflamed) joint. The goal of our present study was to identify T cell responses in JIA against joint inflammation specific antigens. Methods: The putative self-epitopes were selected by a computer search strategy designed for the rat model of Adjuvant Arthritis (AA). In this model the arthritogenic T cell clone A2b was identified, recognizing the mycobacterial HSP65 178-186 peptide and cross-reacting with cartilageassociated components suggesting molecular mimicry between a mycobacterial and a cartilagederived epitope. A selection of human analogues of the recognized peptides in AA was made, and tested for T cell recognition in JIA patients by measuring proliferative activity of peripheral blood mononuclear cells. Results: Four out of 11 of the selected self-peptides were recognized by at least 20-40 % of JIA patients. Among these are peptides from the matrix metalloproteinase (MMP) family and peptides from the aggrecan/versican proteoglycan family. MMP’s are known to be involved in rheumatoid arthritis joint destruction, whereas the aggrecan protein plays a role in repair processes of cartilage. This suggests that we have found autoreactive T-cells, recognizing joint inflammation specific self-antigens based on molecular mimicry with mycobacterial antigens. Conclusion: With the identification of these self-epitopes, inducing proliferative responses in JIA patients, we have found tools for a better understanding of the role of autoreactive T cells in JIA. In addition the identified self-epitopes might become targets for immunotherapy in future. Further analysis is currently being performed to find out whether the found reactivity has to do with disease induction/maintenance or disease regulation/suppression.

Exercise is used to treat inflammatory arthritis, but data regarding its effect on inflammation is sparse. The immunologic consequences of acute exercise in children with stable juvenile idiopathic arthritis (JA, n⫽19) and healthy control children (C, n⫽23) were studied using a graded bicycle stress test (GXT) to determine if acute exercise promotes or inhibits inflammatory mediator release. TNF-alpha, sTNF-RI, sTNF-RII, IL1-beta, IL1RA, IL-6 and IL-10 were measured in plasma and cell culture supernatants of peripheral blood mononuclear cells (PBMC) from JA and C subjects immediately prior to and following GXT. Children with JA differed from controls in their responses to acute exercise in LPS-induced TNF production (p⫽0.047), with a rise in TNF releasability after exercise (from 1267⫾245 to 1402⫾256 pg/ml), compared to a decrease in controls (791⫾71 to 656⫾83 pg/ml). Plasma TNF concentrations were generally undetectable. Unstimulated PBMC TNF production did not differ in response to GXT between subjects and controls. IL-6, IL-10 and IL-1-beta responses to GXT also did not differ between groups. IL1RA in plasma was similar in JA and C, but LPS-induced production from PBMC decreased more dramatically after GXT in C (from 4838⫾1171 to 4639⫾1086 pg/ml) than JA subjects (from 1933⫾574 to 1404⫾325 pg/ml; p⫽0.029). Unstimulated PBMC IL1RA production after GXT was more suppressed by GXT in JA than controls (p⫽0.054). Both TNF-RI and RII were affected similarly by GXT in JA and C subjects, although LPS-induced sTNF-RI was increased slightly in JA (from 28.5⫾4.3 to 34.5⫾6.3 pg/ml) and decreased more substantially in C (from 85.7⫾25.4 to 18.4⫾1.6 pg/ml; p⫽0.078). LPS-stimulated PBMC from JA subjects produced significantly more IL-10 than those of controls before exercise (1927⫾536 vs. 527⫾56 pg/ml; p⫽0.042), more IL-1 after exercise (2535⫾245 vs. 1716⫾53pg/ml; p⫽0.040) and more IL1RA after exercise (4639⫾1085 vs. 1404⫾325 pg/ml). Thus, children with JA responded to acute exercise with a substantial increase in LPS-induced TNF releasability of PBMC versus a minimal decline in controls, and though not significant, a trend toward less IL1RA produced by unstimulated PBMC after exercise in JA than in controls. This suggests the possibility that acute aerobic exercise could increase inflammatory activity in children with JA. In other studies, we have shown resistance training may have antiinflammatory effects. Disclosure: Supported by NIDRR #H133G70156.

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MANNOSE BINDING LECTIN (MBL) POLYMORPHISMS INFLUENCE SUSCEPTIBILITY TO SYSTEMIC ONSET JUENILE IDIOPATHIC ARTHRITIS (SYS-JIA) IN AN AGE DEPENDENT MANNER. Wendy Thomson, Daniel Crosdale, Kay Poulton, Rachelle Donn, William Ollier, The BPRG Study Group Manchester, United Kingdom

OSTEOPROTEGERIN SERUM LEVELS ARE ELEVATED IN CHILDREN WITH JUVENILE IDIOPATHIC ARTHRITIS. Fernanda Falcini, Rolando Cimaz, Gabriele Simonini, Giuseppe Bindi, Sandra Trapani, Susanna Benvenuti, Elisa Piscitelli, Laura Masi Florence and Milan, Italy

Background: SYS-JIA can occur at any age but disease onset is often under the age of 5 years. Children with SYS-JIA are acutely unwell and often present with a high fever and evervescent rash, features that are highly suggestive of an infectious trigger. MBL is a serum protein important in innate immune defence. MBL binds to sugar units found on many microbial surfaces leading to activation of the complement system, thereby promoting phagocytosis and complement mediated killing of microorganisms. Serum levels of MBL are influenced by three mutations in exon 1 of the gene and are modulated by a number of promoter polymorphisms. The exon 1 polymorphisms (codons 52, 54 and 57) result in secondary structural abnormalities known to effect the biological function of MBL. A study of MBL in children with immunodeficiencies demonstrated an overrepresentation of JRA in children with MBL deficiency. Aim: To determine whether any association exists between the structurally encoding mutations of MBL and SYS-JIA. Methods: 121 UK caucasian children with SYS-JIA and 156 UK Caucasian controls were typed for the two structural polymorphisms found in Caucasians (codon 52 and 54) using PCR-SSP based techniques. Results: Within the SYS-JIA patients as a whole there were no significant differences in allele frequencies for codon 52 (p⫽0.8) or 54 (p⫽0.5) compared with controls. However, age specific effects were observed. Children with an age at disease onset of ⬍2 years of age had a higher frequency of codon 54 mutant alleles than those with an age at disease onset ⱖ2 (20.8% vs 9.3%, p⫽0.03). Whereas, children with an age at disease onset of ⱖ 5 years had a higher frequency of codon 52 mutant alleles than those with an age at disease onset ⬍ 5 years(11.2% vs 3.9%, p⫽0.03). Conclusions: MBL is thought to play an important role in host defence during the “window of vulnerability”, which is the period of time during which maternal antibody titre is declining and the infants adaptive immune system is still immature. MBL deficiency due to codon 54 mutation causes an increased susceptibility to SYS-JIA in children with an age of disease onset before the age of 2 years. In contrast, in children with a disease onset over the age of 5 years an increased frequency of codon 52 mutant alleles is detected. These findings imply that, dependent on the age of onset of disease, different infectious triggers are important in susceptibility to SYS-JIA.

Aim Skeletal complications of Juvenile Idiopathic Arthritis (JIA) include focal bone erosions and iuxtarticular osteopenia at sites of active inflammation, as well as systemic osteopenia. Osteoprotegerin (OPG), the soluble decoy receptor of receptor activator of nuclear factor ␬B ligand (RANK-L, or OPGL), has been recently identified as a novel cytokine that inhibits differentiation and activation of osteoclasts: OPG could protect from cartilage destruction, a critical and irreversible step in the pathogenesis of arthritis. The aim of our study was to evaluate serum levels of OPG in patients with JIA and healthy controls. Subjects and Methods Eighty-four patients (66 girls and 18 boys) with JIA (30 pauciarticular and 54 polyarticular RF negative) and 20 sex and age-matched controls were enrolled in our study. Patients were further divided in four groups: 21 without therapy, 12 on corticosteroid therapy, 26 on metrotrexate, and 25 on the association of corticosteroid and metrotrexate. Serum OPG was measured using an enzyme-linked immunosorbent assay. OPG levels were correlated with bone mineral density values. Statistical analyses were performed using Mann-Whitney U test, Tukey’s test, and Ancova analysis, as indicated. Results We observed that JIA patients had significantly higher serum levels of OPG than controls (mean 0.866 ⫾0.31 ng/ml vs 0.652 ⫾0.14; P⫽0.001). Moreover, we observed a statistically significant difference in the amount of serum OPG between patients with and without therapy (ANCOVA, P⫽0.002). In particular, patients on steroid therapy had higher serum OPG levels than patients without any therapy (1.10 ⫾0.57 vs. 0.670 ⫾0.22 ng/ml; P⫽0.01). Finally, no significant differences in the amount of OPG were observed between patients with and without bone erosions. Conclusions This preliminary study shows that JIA patients have elevated serum levels of OPG. This might reflect a compensatory response to degeneration of bone and cartilage. Steroid treatment is associated with higher OPG levels: steroid therapy could increase OPG levels through a reduction of osteoclast activity and cartilage destruction. Further prospective studies are needed in order to better understand the significance of these findings. Disclosure:

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IMPAIRED SYNOVIAL EXPRESSION OF MATRIX METALLOPROTEINASES’ TISSUE INHIBITOR-1 (TIMP-1) IN JUVENILE IDIOPATHIC ARTHRITIS. Marco Gattorno, Valeria Gerloni, Paolo Picco, Adriana Morando, Antonella Buoncompagni, Flavio Fantini, Claudio Gambini

HLA-ALLELE FREQUENCIES IN JUVENILE IDIOPATHIC ARTHRITIS (JIA) IN RELATION TO OUTCOME. Kirsten Minden, Martina Niewerth, Joachim Listing, Constanze Schoenemann, Marion Nagy, Angela Zink Berlin, Germany

Disclosure:

Objective: To investigate the HLA phenotype frequencies in a followup cohort of JIA patients; to determine if specific alleles are predictors of remission. Methods: HLA B27, DRB1, DPB1, DQA1 and DQB1 alleles were analysed by using PCR-based methods (SSP, SSOP) in a group of 193 out of 215 JIA patients, who comprised 83% of a 17-year-followup JIA cohort. Positive or negative associations were assumed for allele frequencies of controls being outside the subgroup-related confidence intervals. positive association All JIA patients (n⫽193)

B27;DR8;DQA4,5

- Systemic arthritis (n⫽28) - Oligoarthritis (n⫽77)

B27;DR8;DP2;DQA4,5

- Seronegative polyarthritis (n⫽25) - Enthesitis-related arthritis (n⫽27)

DP3;DQA4;DQB4; B27

negative association DR4,7;DP1,4;DQA1,2,3; DQB2,3,5,6 DP4;DQB3 DR4,7;DQB3,6;DP4; DQA1,2,3;DQB2,3,5,6 DQB3 DQA1;DQB3

Results: In addition to known positive associations with certain JIA subtypes, negative associations were seen with various DP, DQA and DQB alleles. In oligoarthritis, for DR4 and DQA3 not only negative associations were found, but patients with one of these alleles also had a higher probability of remission (significant only for DQA3 with p⫽0.036; Odds Ratio 2.9). Furthermore, there was a tendency of DR3, DP3 and B27 being associated with a lower probability of remission in oligoarthritis. For the whole JIA group DQA3 was associated with a higher, while DP3 and B27 were associated with a lower probability of remission. Conclusion: For JRA well documented HLA alleles were also found in a German JIA cohort, of which DQA3 might be a predictor of a higher rate of remission. Disclosure: The study has been funded by the German Federal Minister of Research (01VM9707/1)

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Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes involved in the remodelling of extracellular matrix in many physiological (embryonic development, organ morphogenesis?.) and pathological (chronic inflammatory diseases, tumours..) conditions. Four different natural inhibitors of MMPs are known (TIMP-1 to -4), which interact with activated MMPs forming a 1:1 stoichiometric complex). Aim of the study was to investigate the synovial membrane (SM) expression of MMP-1, MMP-3, MMP-13, as well as of TIMP-1 in juvenile idiopathic arthritis (JIA). Patients and Methods. SM obtained at synoviectomy or arthroplasty from 9 JIA patients diagnosed according Durban’s criteria were studied. SM from an adult RF positive RA patient and from a 13-year old girl who underwent post-traumatic meniscectomy were studied as positive and negative controls, respectively. Eleven SM specimens sized from 5 to 12 mm of maximum diameter (mean 8mm) were studied. Immunohistochemical study was performed according to standard technique. The following monoclonal antibodies were used: anti CD68, clone kp1 (Dako, Denmark); anti MMP-1, clone 41-1E5; anti MMP-3, clone 55-2A4; anti MMP-13, clone 181-15A12; anti TIMP-1, clone 147-6D11(Chemicon International, Canada). Slides were evaluated by 2 expert pathologists unaware of diagnosis. A semi-quantitative evaluation was performed at the level of lining and sublining layer, according to the following criteria: number of positive cells/high power field (hpf/40x) and considered to be i) absent (-, ⬍ 3 positive cells/hpf), ii) weakly positive (⫹, 3 to 10/ hpf), iii) moderately positive (⫹⫹, 10 to 30/hpf), iv) strongly positive (⫹⫹⫹, 30-50/hpf) or v) intensively positive (⫹⫹⫹⫹, ⬎ 50/hpf). Results In JIA patients and RA control, MMP-1 and MMP-3 displayed a prevalent localization at the level of the lining and sublining layers. A clear, even weaker expression was noted in post-traumatic SM too. In JIA patients, a high correlation (Spearman’s rank test) with macrophagic infiltration (CD68 expression: r ⫽ 0.95, p ⬍ 0.001; r ⫽ 0.83, p ⫽ 0.003, respectively) was noted. MMP-13 was expressed in RA control, in 8/9 JIA patients, but not in post-traumatic SM. In JIA, a lower intensity of staining in respect to MMP-1 and -3 was noted, with localization almost restricted to the sublining areas. TIMP-1 tissue distribution was similar to that observed for MMP-1 and -3, both in JIA patients and controls, even if with a clear lower intensity of staining. Conclusion the present study shows that overexpression MMP-1 and MMP-3 is clearly related with the degree of synovial inflammation in JIA patients. Inadequate expression of their tissue inhibitors may represent a crucial event for the development and perpetuation of tissue damage.

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SEROPREVALENCE OF HUMAN PARVOVIRUS B19 IN CHILDREN AFFECTED BY JUVENILE IDIOPATHIC ARTHRITIS. Yvonne J Suess, Benedikt Weissbrich, Hermann J Girschick Wurzburg, Germany

PREGNANCY OUTCOME AND anti-Ro/SSA IN AUTOIMMUNE DISEASES: A PROSPECTIVE STUDY. Antonio Brucato, Micol Frassi, Gabriella Castellino, Andrea Doria, Pierluigi Meroni, Maria Pia Pisoni, David Faden, Andrea Lojacono, Laura Solerte, Marina Muscara, Rolando Cimaz, Franco Franceschini, Ilaria Cavazzana, Chiara Rebaioli Biasini, Pier Franca Gambari, Anna Ghirardello, Francesca Vescovi, Bianca Canesi, Angela Tincani Milano, Brescia and Padova, Italy

Recently the role of human parvovirus B19 in the etiology and pathogenesis of adult rheumatoid arthritis has been discussed controversely. Studies analyzing a potential pathogenic role of parvovirus B19 in juvenile idiopathic (rheumatoid) arthritis (JIA) are limited in all ethnic groups. We analyzed the prevalence of anti-parvovirus B19 IgG antibodies in the serum of 382 children who were referred to the section of pediatric rheumatology at the University of Wurzburg, Germany. In addition 146 age-matched healthy controls were analyzed. The studies were performed according to policies established by the institutional ethics review board at the University of Wurzburg. Patients and controls uniformly were of white caucasian descent. The subgroups were oligoarthritis n⫽ 80, polyarthritis (RF ⫹ and -) n⫽19, systemic arthritis n⫽12, psoriatic arthritis n⫽ 11, enthesitis related arthritis n⫽66, reactive arthritis n⫽ 38, Lyme arthritis n⫽37, “other” arthritis n⫽28, arthralgias n⫽85, systemic lupus erythematosus n⫽4, iridocyclitis n⫽6. The frequency of antiparvovirus B19 IgG antibodies were 35% (oligoarthritis, ), 58% (polyarthritis), 62.5% (systemic arthritis), 63.6% (psoriatic arthritis), 72.2% (enthesitis related arthritis), 39.5% (reactive arthritis), 67% (Lyme arthritis), 57% (other) 62%, 62.5% (arthralgias), 100% (SLE) and 33% (iridocyclitis), respectively. The seroprevalence in the reactive arthritis group was significantly less than expected from the control group (p⬍0.05). With inclusion of additional 5 patients with erythema infectiosum and subsequent arthritis (all seropositive) into “reactive arthritis” the difference did not reach statistical significance. All seroprevalence in the different groups did not reach statistical significant difference from the age matched controls which were adjusted for the mean of age and the standard deviation of the age distribution. Analysis of the seroprevalence of anti-parvovirus B19 IgG antibodies in european caucasian children affected with arthritis did not support the hypothesis that human parvovirus B19 is involved in the pathogenesis of JIA.

Introduction. Anti-Ro/SSA antibodies are associated with neonatal lupus but also considered a possible cause for unexplained pregnancy loss and adverse pregnancy outcome. Materials and methods. We have prospectively followed 100 anti-Ro/SSA positive women with an autoimmune disease during their pregnancies (53 SLE, 25 primary Sjogren syndrome, 19 UCTD, 1 SSP, 1 primary APS and 1 MCTD). Anti-Ro/SSA antibodies were tested by Counterimmunoelectrophoresis and Immunoblot. Results. A total of 118 pregnancies in 100 women ended with the delivery of 112 living newborns (4 twins pregnancies). Pregnancy losses were 8%(10/118): 6% (7/118) occurred within 10 weeks of gestation and 2.5% (3/118) after 10 weeks of gestation. There were also 4 therapeutic abortions. Three out of 10 pregnancy losses occurred in mothers with positive Lupus anticoagulant and/or a positive anticardiolipin antibody (aCL) test. There was 1 extrauterine pregnancy. Prematurity, defined as delivery before the completion of 37 weeks of gestation, occurred in 19% of pregnancies (23/118). Mean gestational age at delivery was 37 weeks (range 34-41). Sixty-five % of pregnancies ended with spontaneous labors and 35% with cesarean sections. Ten cases of premature fetal membranes rupture and 2 gestosis were observed. One hundred twelve newborns were delivered: 13 newborns (11,6%) were small for gestational age, 3 (2.6%) developed intra-uterine growth retardation, 2 (2%) had a complete atrio-ventricular heart block. Conclusions. Based on the hypothesis that adverse pregnancy outcome may represent intrauterine expression of neonatal lupus, we have tried to elucidate the possible contribution of the anti-Ro/SSA antibody system to reproductive failure of autoimmune patients. Our study confirm previous data which had already demonstrated how the presence of anti-Ro/SSA antibody is responsible for neonatal lupus syndrome but does not seem to affect pregnancy outcome. Comparison of our anti-Ro/SSA positive group with an anti-Ro/SSA negative control group and a normal population will be useful to fortify this hypothesis. Disclosure:

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MATERNAL MICROCHIMERISM IN THE HUMAN THYMUS. Anne M Stevens, Heidi Hermes, Tracy Tylee, J Lee Nelson Seattle, Washington

Fc␥R ALLELIC POLYMORPHISMS: A CANDIDATE FETAL FACTOR IN THE DEVELOPMENT OF CONGENITAL HEART BLOCK (CHB). M Eugenia Miranda-Carus, Robert P Kimberly, Jeffrey C Edberg, Jill P Buyon New York, NY and Birmingham, AL

PURPOSE: Maternal cells that pass into the fetal circulation can be detected in the blood of her immunocompetent offspring well into adult life. The presence of maternal cells in the immature immune system may have profound effects on development of self vs. non-self recognition and subsequent autoimmune disease. We have studied maternal microchimerism in the major organ for T lymphocyte development, the thymus. METHODS: Fluorescence in situ hybridization (FISH) for X- and Y-chromosome-specific sequences was used to assay for maternal cells in 15 thymuses from male children five days to 15 months old. Subsets of thymocytes were isolated by flow cytometry to characterize the types of maternal cells present. To increase the sensitivity of the assay and to enable detection of maternal DNA in both male and female thymuses, maternal DNA was detected by Taqman real-time quantitative PCR amplification of noninherited maternal genes: the glutathione-S-transferase M1 insertion allele and HLA-B44. RESULTS: Using the FISH method on total unfractionated thymocytes, maternal nuclei were found in two of seven thymuses assayed, at a level of 2/1000 infant cells. In four additional thymuses the maternal/child inheritance pattern was informative for the assay by the Q-PCR method. Three of these thymuses,aged six days to 15 months, were found to carry 2.8 to 48 maternal cells per million thymocytes. In the immature CD4-/CD8- subset, 0.5 to 5/1000 maternal cells were detected by FISH in three of eight thymuses, aged two to eight months. Maternal cells were detected in the mature CD4⫹/CD8- subset of two of six thymuses and at the rate of 2 to 4/1000. In the CD4-/CD8⫹ subset 1 to 1.5/1000 maternal cells were detected in two of 7 thymuses examined. No maternal cells were found in the intermediate CD4⫹/CD8⫹ subset, which makes up 85% of the thymocytes. The oldest child in which maternal cells were detected was 15 months. CONCLUSIONS: Maternal cells are present in normal human infant thymus where they persist at least throughout the first year of life. The presence of maternal cells at different stages of thymic development suggests that maternal hematopoietic stem cells transfer into the fetal thymus and undergo differentiation alongside the fetal cells. The presence of maternal antigens during thymic development could affect the infant’s T cell repertoire, and lead to tolerance of maternal antigens and persistence of maternal microchimerism. Disclosure:

Anti-SSA/Ro and anti-SSB/La antibodies are nearly universal in mothers of children with neonatal lupus, but the risk of having children with CHB is low. A fetal factor may contribute to CHB, and Fc␥R genotype modulating antibody-mediated inflammatory effector cell activity represents one such factor. Families enrolled in the Research Registry for Neonatal Lupus provided DNA from 13 anti-SSA/ Ro⫹ mothers who had at least one child with neonatal lupus and from 48 children (19 with CHB, 8 with rash, 21 unaffected). Genotyping for Fc␥RIIA, Fc␥RIIIA and Fc␥RIIIB was performed by allele specific PCR. Children with CHB showed an increased frequency of the low binding homozygous genotype RR131 of Fc␥RIIA (42%) compared with the general population (23%), children with rash (25%), unaffected siblings (29%), and mothers (31%) (CHB vs general population, p⫽0.09). HH131 homozygosity was 21% in children with CHB, 13% in those with rash, 29% in normal sibs, 8% in mothers, and 29% in the general population. There was a corresponding enrichment of R131 alleles in children with CHB compared to the general population (0.61 vs 0.50). Although for Fc␥RIIIA there were no striking differences in the genotype distribution or allelic frequencies, a trend toward an increase in homozygous NA2 genotype of neutrophil-specific Fc␥RIIIB in CHB (NA2/2⫽44%, NA1/1⫽12%) was apparent compared to unaffected sibs (NA2/2⫽30%, NA1/1⫽15%). These results suggest that Fc␥R allelic polymorphisms may contribute to fetal susceptibility; perhaps low binding Fc␥IIa triggers an aberrant macrophage signalling pathway skewed toward the release of proinflammatory mediators capable of inducing fibrosis of the atrioventricular node. This is of interest since IgG2, a subclass bound only by Fc␥RIIa, is present in one-third of anti-48 kD SSB/La and anti-52 kD SSA/Ro responses. In contrast, the decrease in low binding IIIb in unaffected siblings may be a protective factor.


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EXTENDED SPECTRUM OF CONDUCTION ABNORMALITIES IN INFANTS BORN TO MOTHERS WITH ANTI-Ro/La ANTIBODIES. AD Askanase, DM Friedman, P Katholi, JP Buyon New York

HEPATOBILIARY DISEASE IN NEONATAL LUPUS: PREVALENCE AND CLINICAL CHARACTERISTICS IN CASES ENROLLED IN A NATIONAL REGISTRY. Lela A Lee, Ronald J Sokol, Jill P Buyon Denver, CO and New York, NY

The classic cardiac manifestation of neonatal lupus is 3rd° heart block (HB), attributed to Ab-mediated inflammation and subsequent fibrosis of the atrioventricular (AV) node. It is likely that injury progresses through stages. Identification of 1st° block would support this pathologic model and serve as a potential marker for treatment if the conduction defect could be shown to progress. Moreover, experimental evidence suggests that maternal anti-Ro/La Ab inhibit calcium fluxes, which should result in dysfunction of the sinoatrial (SA) as well as the AV node. To ascertain the spectrum of arrhythmia associated with maternal anti-Ro/La Ab, records of all children enrolled in the Research Registry for Neonatal Lupus were reviewed. Of 185 children with any conduction abnormality, 6 had a prolonged PR interval on EKG at birth: 1 progressed to 2nd° requiring a pacemaker by age 18 mo; 1 progressed to 3rd° after age 3 yr; 1 child treated with dexamethasone for 2nd° block diagnosed in utero was born with 1st° block and progressed to 2nd° by age 1 yr; 1 child progessed to 2nd° after age 1 wk; 1 child remains in 1st° at age 6 mo; 1 died of dilated cardiomyopathy at age 5 mo. A child whose younger sibling had 3rd° block was diagnosed with 1st° block at age 10 yr at the time of surgery for a broken wrist. Two children diagnosed in utero with 2nd° block were treated with dexamethasone and reversed to normal sinus rhythm at birth, but ultimately progressed to 3rd° block. Four children had 2nd° block at birth: 1 progressed to 3rd° block by 1 mo and required a pacemaker by age 2 yr; 1 progressed to 3rd° by 2 mo and required a pacemaker by age 5 yr; 2 remain in 2nd° block at ages 1 yr and 4 yr, respectively. Sinus bradycardia (⬍100 bpm) was present in 2 (3%) of 75 fetuses where atrial rates were recorded by echocardiogram. The mean atrial rate was 139⫾16 bpm (range 68-160). Of 39 neonates where EKGs were available, the mean atrial rate was 136⫾22 bpm (range 75-200). The one slow rate of 75 bpm was obtained during sleep and increased to 140 bpm when awake. In summary, these data have important research and clinical implications. Perhaps many fetuses sustain mild inflammation, but resolution is variable, as suggested by the presence of incomplete HB. However, subsequent progression can occur. In contrast to the AV node, permanent SA nodal involvement is not clinically apparent. These findings emphasize the importance of obtaining an EKG on all infants born to mothers with anti-Ro/La Ab.

Hepatobiliary disease may occur as a manifestation of neonatal lupus (NLE), but only a handful of cases have been reported previously, and consequently there is little information about its clinical features and prognosis. The present study was performed by chart review of the NLE National Research Registry database to determine the frequency, clinical characteristics, and outcome of NLE hepatobiliary disease. Nineteen of 219 cases (9%) of NLE enrolled in a national registry had probable or possible NLE hepatobiliary disease. In sixteen cases, hepatobiliary disease occurred in addition to cardiac or cutaneous NLE. In three cases, hepatobiliary disease occurred as the sole clinical manifestation of NLE. Three clinical variants of hepatobiliary disease were observed: 1) severe liver failure present during gestation or in the neonatal period, often with the phenotype of neonatal iron storage disease (6 cases); 2) conjugated hyperbilirubinemia with mild or no elevations of aminotransferases, occurring in the first few weeks of life (5 cases); and 3) mild elevations of aminotransferases occurring at approximately 2 to 3 months of life (6 cases). (In two cases, there was insufficient information to determine whether the disease course fit better into category 2 or category 3.) The prognosis for the children in the latter two categories is excellent. Hepatobiliary disease is a relatively common finding in NLE and can be the sole clinical manifestation of NLE. A broad range of hepatobiliary disease may occur in children with NLE, varying from liver failure to mild enzyme abnormalities. Disclosure:

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SYNOVIAL OVEREXPRESSION OF VASOACTIVE INTESTINAL PEPTIDE IN POLYMYALGIA RHEUMATICA. R Meliconi, P Dolzani, T Silvestri, L Pulsatelli, L Boiardi, P Macchioni, C Salvarani, R De Giorgio, L Frizziero, A Facchini Bologna, Italy

MANNOSE-BINDING LECTIN VARIANT ALLELES AND HLA-DRB1*04 ALLELES ARE INDEPENDENTLY ASSOCIATED WITH GIANT CELL ARTERITIS. Soren Jacobsen, Bo Baslund, Hans O Madsen, Niels Tvede, Arne Svejgaard, Peter Garred Copenhagen, Denmark

Objective. In polymyalgia rheumatica (PMR) the histological and laboratory findings do not fully justify the very intense pain in the neck, shoulder and pelvic girdle. Since there is increasing evidence regarding the interplay between the immune and neuroendocrine system, we evaluated the expression of three neuropeptides (VIP, substance P and somatostatin) involved in modulation of pain trasmission and immune functions in synoviall tissue from patients with PMR. Methods. All patients were diagnosed according to the criteria of Healey. Synovial biopsies were obtained from 15 PMR patients (9 untreated and 6 corticosteroid treated patients), 5 patients with rheumatoid arthritis (RA) and 7 patients with osteoarthritis (OA). The neuropeptide expression in biopsies was analyzed by immunohistochemistry and results were evaluated by image analysis. Results. While substance P and somatostatin staining were absent in PMR, an elevated expression of VIP was found in PMR and RA synovial tissue. In OA only two out of seven patients showed high number of VIP positive cells. Furthermore, while in PMR we observed a prevailing immunostaining in the lining layer and in the nearby sublining area, in RA, VIP synovial expression was localized in deep sublining area and in perivascular infiltrate. Corticosteroid treatment of PMR patients did not significantly modify VIP lining layer expression but was associated with disappearence of VIP positivecells in the sublining. Conclusion. Local VIP production in PMR synovial tissue could contribute to cause the typical muscoloskeletal discomfort of this disease and it may have a role in the immunomodulation of synovial inflammation.

Objective: To determine whether variant alleles of the mannose-binding lectin (MBL) gene causing low serum concentrations of MBL and/or polymorphisms of HLA-DRB1 are associated with increased susceptibility to polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) or particular clinical phenotypes of GCA/PMR. Methods: MBL and HLA-DRB1 alleles were determined by polymerase chain reaction in 102 Danish patients with PMR (n⫽37) or GCA (n⫽65). Two hundred and fifty and 193 healthy persons served as controls for MBL and HLA genotyping, respectively. Results: MBL variant alleles were present in 46% of all patients and in 53% of patients with GCA. The latter differed significantly from the 37% found in controls (P⫽0.01). HLA DRB1*04 was found in 52% of all patients and 54% of patients with CCA, which differed significantly from the 35% found in controls (P⫽0.01). There was no interaction between MBL variant alleles and HLA DRB1*04 alleles. HLA DRB1*04 alleles were not associated with any clinical phenotypes of GCA/PMR, whereas MBL variant alleles were associated with early disease onset in men, cranial affection, high erythrocyte sedimentation rate, and low B-hemoglobin. Conclusion: We found MBL variant alleles and HLA-DRB1*04 alleles to be independent weak susceptibility markers for GCA and that in patients with PMR/GCA MBL variant alleles predicted increased inflammatory activity and clinical signs of arteritic manifestations. These findings indicate that MBL and DRB1*04 contribute to the pathophysiology of GCA at different levels in the disease process.

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VASCULAR ENDOTHELIAL GROWTH FACTOR POLYMORPHISMS IN POLYMYALGIA RHEUMATICA. Luigi Boiardi, Bruno Casali, Pierluigi Macchioni, Maria Grazia Catanoso, Lia Pulsatelli, Fulvia Rossi, Gianluigi Bajocchi, Davide Nicoli, Enrico Farnetti, Fabrizio Cantini, Ignazio Olivieri, Carlo Salvarani Reggio Emilia, Bologna, Prato and Potenza, Italy

ANTI-BETA2-GPI (a␤2-GPI), ANTICARDIOLIPIN (aCL) AND ANTI-HUMAN PARAINFLUENZA TYPE 1 (HPIV-1) ANTIBODIES IN GIANT CELL ARTERITIS (GCA) AND POLYMYALGIA RHEUMATICA (PMR): A MULTICENTER CASECONTROL STUDY. Pierre Duhaut, Marie-Laure Marie-Laure, Sylvie Bosshard, Elwire Devaux, Dominique Seydoux, Helene Pellet, Jean-Charles Piette, Micheline Berruyer Paris, Lyon, Saint-Etienne and Bron, France

Objective. Circulating serum levels and in vitro production of vascular endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMC) ares higher in untreated polymyalgia rheumatica (PMR)patients than in controls. VEGF genes may play an important role in PMR pathogenesis. A 18-bp insertion/deletion (I/D) polymorphism in the VEGF promoter region and a ⫹405 G 3 C polymorphism have been described. The aim of this study was to evaluate potential associations of these two polymorphisms with PMR susceptibility. In vitro production of VEGF by lipopolysaccharide (LPS) stimulated PBMC was also evaluated. Methods. We enrolled 122 consecutive PMR patients without histological or clinical evidence of giant cell arteritis. 100 healthy blood donors from the same geographic area were selected as controls. All the patients and controls were genotyped by polymerase chain reaction technique for VEGF I/D polymorphism, while ⫹405 polymorphism was determined in 77 PMR patients. VEGF concentrations in PBMC culture supernatants after LPS stimulation were evaluated using a commercial ELISA kit in 32 controls. Results. The frequency of allele I was significantly higher in PMR patients (44.7% vs 35.0%, p ⫽ 0.03, OR ⫽1.5) compared to healthy controls, while the distribution of allele frequencies of ⫹405 polymorphism did not differ significantly between PMR patients and healthy controls. LPSstimulated PBMC VEGF levels from controls with II genotype (n ⫽ 15) were significantly higher than those from controls with DD genotype (n ⫽ 17) (51.0 ⫹/- 89.5 pg/ml versus 13.8 ⫹/- 24.5 pg/ml, p ⫽ 0.01). No correlation between ⫹405 genotype and LPS-stimulated production was observed. Conclusions. Our data show that VEGF I/D polymorphism is associated with PMR susceptibility.The higher levels of VEGF producted by allele I polymorphism may predispose to PMR

On behalf of the GRACG study (Groupe de Recherche sur l’Arterite a Cellules Geantes). Background: The prevalence of aCL amounts to 32% in patients with biopsy-positive GCA, but aCL are not associated with thrombosis in this setting. HPIV-1 re-infection has been reported to be associated with GCA onset. Objectives: To assess the prevalence of a␤2-GPI and aCL, and their potential association with anti-HPIV-1 antibodies in patients with GCA or PMR. Methods: 242 patients (176 females aged 74.5⫾8.4 yrs, and 67 males aged 73⫾7.7 yrs) with either positive biopsy GCA (n ⫽ 124 cases), negative biopsy GCA (n ⫽ 50), negative biopsy PMR (n ⫽ 42) or GCA/PMR without biopsy (n ⫽ 26) and 181 randomly selected population based, age- and sex-matched controls (123 females aged 75⫾7.5 yrs, and 58 males aged 73⫾7.5 yrs) have been prospectively included. Results: IgG a␤2-GPI were present in 2.9% of cases vs 6.1% of controls (p ⫽ 0.11), and IgM a␤2-GPI in 3.7 % of cases vs 4.9 % of controls (p ⫽ 0.74). IgG or IgM a␤2-GPI were found in GCA, but not in PMR (p ⫽ 0.19). Within patients who underwent temporal artery biopsy, a␤2-GPI were exclusively associated with the presence of giant cells (p ⫽ 0.009). Clinical manifestations of occlusion in external carotid territory were not clearly associated with a␤2-GPI, but were strongly associated in multivariate analysis with a positive temporal artery biopsy (p ⫽ 0.002). aCL of both isotypes were present in 31.2% of biopsy-proven GCA, 16.7% of biopsy-negative GCA, 2% of biopsy-negative PMR vs 2.9% of controls (p ⫽ 2.8 10-13, 6.3 10-4, and 1.0, respectively, versus controls). IgM aCL were strongly associated with IgM anti-HPIV-1 in patients (p ⫽ 0.006), whereas aCL IgG were not (p ⫽ 0.15). Neither IgG nor IgM aCL were associated with IgM anti-HPIV-1 in controls (p ⫽ 0.53 for both). Conclusion: The prevalence of a␤2-GPI was low and similar in cases and controls. The presence of IgM aCL was associated with IgM anti-HPIV-1 in patients. Further studies are needed to determine whether GCA-related aCL are virus-induced. Disclosure:

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INCREASED SERUM LEVELS OF ANTI-HUMAN HSP ANTIBODIES IN POLYMYALGIA RHEUMATICA AND GIANT CELL ARTERITIS AND DECLINE AFTER CLINICAL REMISSION. Marcos Lopez-Hoyos, Victor M Martinez-Taboada, Maria J Bartolome, Ricardo Blanco, Vicente Rodriguez-Valverde Santander, Cantabria, Spain

TISSUE EXPRESSION OF PRO-INFLAMMATORY CYTOKINES (IL-1␤, IL-6 AND TNF␣ ) IN GIANT CELL ARTERITIS (GCA) PATIENTS. CORRELATION WITH THE INTENSITY OF THE SYSTEMIC INFLAMMATORY RESPONSE. Maria C Cid, Jose Hernandez-Rodriguez, Montse Sanchez, Maria J Esteban, Ana Garcia-Martinez, Carme Queralt, Josep M Grau

OBJECTIVE: The expression of a number of heat shock proteins (hsp) is induced after infection. Such hsp are well-conserved interspecies and their overexpression after an infectious episode can generate autoimmune reactions against self-hsp. Thus, hsp have been involved in the development of several autoimmune diseases. However, there are no reported data about their role in polymyalgia rheumatica (PMR) and giant cell arteritis (GCA), two related diseases in which an infectious trigger seems to participate.Our aims were to analyze whether patients with PMR and GCA show increased levels of anti-human hsp60 and -hsp70 antibodies and to study if those levels change with therapy and remission of symptoms. METHODS: The serum levels of anti-human hsp60 and -hsp70 IgG were measured in patients newly diagnosed of PMR (n⫽64) and GCA (n⫽21) by a household ELISA and calculated as absorbance units at 405nm (AU). As control population 24 aged healthy blood donors were included into the study. We further measured the serum levels of those autoantibodies in 18 PMR and 11 GCA patients after clinical remission with corticosteroids. RESULTS: Levels of both antibodies were higher in PMR and GCA as compared to control subjects (p⬍.01 and p⬍.05, respectively). After clinical remission, anti-hsp60 and -hsp70 IgG levels decreased significantly in both groups of patients (p⬍.01, see table). There were not significant differences between PMR and GCA. Altogether serum anti-hsp60 and-hsp70 IgG kept a good correlation (r⫽.710; p⬍.001). Such correlation was even better when considered only PMR (r⫽.805; p⬍.001) or GCA (r⫽.835; p⬍.001). CONCLUSIONS: The increased levels of anti-human hsp antibodies in PMR and GCA suggest a previous exposure to infectious agents that would induce the expression of hsp. Those antibodies might be produced by a mechanism of molecular mimicry. The decrease after remission further strengths such hypothesis that must be confirmed with viral and bacterial analyses.

Background: The systemic inflammatory response is mediated by pro-inflammatory cytokines, mainly IL-1␤, IL-6 and TNF␣ which are synthesized mostly by activated macrophages. TNF␣, IL-1␤ and IL-6 mRNAs have been detected in temporal arteries from patients with GCA, a disease characterized by a remarkable acute phase reaction. Objective: To assess the relationship between tissue expression of pro-inflammatory cytokines (IL-1␤, IL-6 and TNF␣) in temporal artery biopsies from GCA patients and the intensity of the systemic inflammatory response. Patients and Methods: Temporal artery sections from 50 GCA patients with a similar degree of histologic involvement were immunostained with antibodies against IL-1␤, IL-6 and TNF␣. Ten normal temporal arteries from patients in whom a surrogate diagnosis was obtained were also studied. Four inflammatory parameters were considered to evaluate the intensity of the systemic inflammatory response (fever, weight loss, ESR ⱖ 85 mm, and Hb ⬍ 11 gm/dL). Immunostaining was blindly quantitated using a predefined score considering the percentage of cell staining at the intima/media junction (1:⬍25%, 2:26-50%, 3:51-75% and 4:76-100%). Results: Tissue expression of IL-1␤, IL-6 and TNF␣ was intense and occurred mainly within the granulomatous reaction at the intima/media junction. Cytokine expression was variable among patients even displaying a comparable degree of inflammatory changes. No cytokine expression was observed in control samples. Patients with a strong systemic inflammatory response (4 parameters) exhibited significantly higher scores for IL-6 (5 vs 8 patients, p⫽0.034) and for TNF␣ (7 vs 12 patients, p⫽0.025) than patients with a weak systemic inflammatory reaction (0 parameters). No relationship between IL-1b expression and the intensity of the inflammatory response was found. Conclusions: Tissue expression of pro-inflammatory cytokines IL-1␤, IL-6 and TNF␣ is prominent in full-blown giant-cell arteritis lesions. IL-6 and TNF␣ expression correlates with the intensity of the systemic inflammatory response.

PMR GCA Aged healthy control

hsp60 Abs at diagnosis

hsp60 Abs after remission

hsp70 Abs at diagnosis

hsp70 Abs after remission

250 ⫾ 156 326 ⫾ 237 137 ⫾ 76

169 ⫾ 102 201 ⫾ 162

631 ⫾ 248 774 ⫾ 456 540 ⫾ 199

493 ⫾ 230 501 ⫾ 270

Disclosure: FIS 98/0443, FIS 00/0689, Fundacio Pedro Pons, Hospital Clıńic Research Award, DAKO (M Sanchez)

Disclosure: Fundacion Valdecilla 1999/00; FIS 98-0846

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TUMOR NECROSIS FACTOR ALPHA IS EXPRESSED IN GRANULOMATOUS INFILTRATES IN GIANT CELL ARTERITIS - POSSIBLY SUPPORTED BY TOLL-LIKE RECEPTOR 4. Annette D Wagner, Ulrike Wittkop, Angelika Prahst, Wolfgang A Schmidt, Erika Gromnica-Ihle, Klaus Vorpahl, Alan P Hudson, Henning Zeidler Hannover and Berlin, Germany and Wayne State University School of Medicine, MI

CIRCULATING ANTI-INFLAMMATORY CYTOKINES (IL-10, IL-13 and TGF-b ) IN GIANT CELL ARTERITIS (GCA). Jose Hernandez-Rodriguez, Marta Segarra, Carme Vilardell, Eva Badia, Josep M Grau, Maria C Cid Barcelona, Spain

Background: Giant cell arteritis (GCA) is a vasculitis which predominantly affects medium- and large-sized arteries. Recent data show the co-localisation of dentritic cells (DCs) and Chlamydia pneumoniae (Cpn). The process of leukocyte migration to sites of infection is stimulated by tumor necrosis factor alpha (TNF␣), which is produced in response to infection. Toll-like receptors (TLRs) are cell surface receptors on phagocytes and other cell types that signal the activation of cells in response to microbial products, via the innate immune response. A constitutively active mutant of the first human Toll-family protein described (TLR4) was shown to induce the expression of cytokines and co-stimulatory molecules on antigen-presenting cells. Methods and Results: 18 paraffin-embedded temporal artery biopsy specimens from 14 patients with GCA that were PCR-positive for Cpn were examined by double immunohistochemical staining, using the anti-TNF␣ and anti-S-100 mAb. On consecutive tissue specimens anti-TNF␣ was used in combination with anti-CD68. The same number of consecutive specimens from patients with GCA and 12 temporal artery specimens from 10 controls without vasculitis were examined by in situ hybridization for presence and localization of TNFa expression. TNFa production and expression was found in DCs and macrophages predominantly in granulomatous infiltrates and in endothelial cells of the vasa vasorum dispersed in the temporal artery adventitial layer. No control specimens showed TNF␣ expression. In the second part of the study, double immunohistochemical staining was performed using an anti-TLR4 polyclonal Ab in combination with anti-S-100 mAb. Results showed that ⬎95% of DCs were TLR4-positive. Macrophages and endothelial cells localized almost exclusively in the adventitia showed TLR4 production. Conclusions: TNF␣ is highly expressed in granulomatous infiltrates of temporal artery specimens from patients with GCA. Since TLR4 is produced in the same cell types predominantly in the adventitial layer of the temporal artery, we speculate that these receptors are coupled to signal transduction pathways that may control TNF␣ expression.

Background: GCA is characterized by an intense systemic inflammatory response although remarkable differences may be observed among patients. We have previously reported a correlation between the intensity of the acute phase response and circulating levels of TNF␣ and IL-6 in GCA patients (Hernandez-Rodriguez et al, A&R 2000;43:S124). IL-10, IL-13 and TGF␤ have been considered to have anti-inflammatory properties because they inhibit the synthesis of pro-inflammatory cytokines by T cells and macrophages. The putative role of these cytokines in regulating the intensity of the inflammatory response in GCA has not been investigated. Objective: To determine plasma concentrations of IL-10, IL-13 and TGF␤ and their relationship with the intensity of the systemic inflammatory response in patients with GCA. Patients and Methods: Circulating levels of TGF␤ , IL-10 and IL-13 were determined in 56 untreated patients with biopsy proven GCA and in 15 healthy controls. Four parameters were used to evaluate the intensity of the systemic inflammatory response (fever, weight loss, ESR ⱖ 85 mm, and Hb ⬍ 11 gm/dL). Patients were considered to have a weak inflammatory response when had 2 or less inflammatory parameters (group 1) and a strong inflammatory response when 3 or 4 parameters were present (group 2). Results: Twenty-three patients had a weak (group 1) and 23 a strong (group 2) initial systemic inflammatory response. No differences in IL-10 levels among GCA patients and controls were observed, but IL-10 concentrations were higher in group 2 (4.2 ⫾ 3.1 pg/mL) compared with group 1 (1.4 ⫾ 2.5 pg/mL), p⫽0.002. Circulating TGF␤ levels were significantly higher in GCA patients (962 ⫾ 589 pg/ml) than in controls (744 ⫾ 791 pg/ml) (p⫽0.04). Although TGF␤ levels in group 1 were lower (866 ⫾ 574 pg/mL) than in group 2 (1100 ⫾ 596 pg/mL), differences were not significant (p⫽0.1). Circulating IL-13 levels were undetectable in most patients and controls. Conclusions: GCA patients with a weak systemic inflammatory response do not have higher concentrations of anti-inflammatory cytokines TGF␤, IL-10 and IL-13 than patients with a strong acute phase reaction. The limited increase in TGF␤ and IL-10 levels observed in GCA patients suggest that these cytokines do not significantly down-regulate the intensity of the systemic inflammatory response in this disease. Disclosure: FIS 98/0443, FIS 00/0689, Fundacio Pedro Pons, Hospital Clinic Research Award

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DO CHLAMYDIA PNEUMONIAE AND MYCOPLASMA PNEUMONIAE PLAY A ROLE IN GIANT CELL ARTERITIS (GCA) ? Chantal Dumoulin, Sabine Pereyre, Maithe Clerc, Janine Rivel, Xavier Puechal, Christiane Bebear, Joel Dehais, Bertille de Barbeyrac, Thierry Schaeverbeke Bordeaux, France, Metropolitan; Bordeaux; Bordeaux and Le Mans

HLA-B*52 ASSOCIATION WITH TAKAYASU ARTERITIS IN TURKEY. Muge Bicakcigil, Aytul Uyar, Sevil Kamali, Murat Inanc, Kenan Aksu, Izzet Fresko, Eker Doganavsargil, Yasar Karaaslan, Sedat Kiraz, Huseyin Ozer, Eftal Yucel, Haner Direskeneli, Guher Saruhan-Direskeneli, Turkey

Chlamydia pneumoniae (Cp) was suggested to play a pathogenic role in GCA, as Cp DNA has been detected in artery specimens from patients with GCA. As the pathogenicity of Cp is very closed to that of Mycoplasma pneumoniae (Mp), this bacterium might also be implicated in GCA. Objective: To seek Cp and Mp in temporal artery biopsies (TAB) and peripheral blood mononuclear cells (PBMC) from patients with GCA and other disorders. Methods: All patients who underwent TAB for GCA suspicion were recruited during a one-year period. Histological examination and microbiological study were performed on TAB. Peripheral blood specimens were collected for serological tests and PBMC isolation. PCR was performed on DNA extraction from PBMC and TAB with specific sets of primers chosen within 16SRNA gene for Cp and omp1 gene for Mp. In situ hybridization (ISH) was performed on TAB with a DNA probe chosen within omp1 gene of Cp. The diagnosis was firmly established according to histological examination of TAB and chart review. The patients were classified into 4 groups: 1) GCA (TAB⫹) when GCA was histologically proven, 2) GCA (TAB-) when the clinical presentation was highly suggestive for GCA, but the biopsy was negative, 3) PMR when the clinical presentation was more suggestive for isolated polymyalgia rheumatica (PMR) and the biopsy was negative, 4) Controls when another diagnosis was finally established. Results: 31 patients were included, 25 female and 6 male, aged 72.3 ⫾ 7 yr. 4 patients were classified as GCA (TAB⫹), 12 as GCA (TAB-), 7 as PMR and 8 as controls.

GCA (TAB⫹) (N⫽4) GCA (TAB-) (N⫽12) PMR (N⫽7) Controls (N⫽8)

PCR Cp PBMC

PCR Mp PBMC

PCR Cp TAB

PCR Mp TAB

ISH Cp TAB

Cp antibodies

Mp antibodies

2 2 2 1

0 1 0 0

0 1 1 3

0 0 0 0

2 0 0 2

3 3 2 0

0 2 0 1

Introduction: HLA alleles are implicated as important genetic predisposing factors for Takayasu arteritis (TA), a systemic large-vessel vasculitis of unknown etiology. They are known to be polymorphic and show significant ethnic and geographic variabilities in allele- and haplotype frequencies. This study aimed to investigate HLA-B alleles, which are previously shown to be associated with TA, in patients from Turkey. Materials and Methods: DNA samples isolated from 70 patients with TA and 191 healthy controls were studied. DNA typing for HLA-B specificity was investigated by using PCR and sequence specific oligonucleotide probe (SSOP) analysis. Results: Among the HLA-B alleles only B*52 has shown a significant association with TA (13/70 vs. 13/191, p⫽0.009, OR: 3.1).The comparison between subtypes according to angiographic classification did not reveal any subtype association with any allele. In this cohort, out of 17 patients with renal artery stenosis only 2 had B*39. Conclusion: In this study, the previously reported association of TA with B*52 in Japanese and other populations was confirmed in patients from Turkey, whereas no association with B*51 and B*39 is observed. Repeatedly demonstrated association with B*52 might have implications about the functional relevance of this molecule in TA pathogenesis, which has to be further explored. Disclosure:

Conclusion : No association was found between the detection of Cp or Mp and GCA. So, our results do not support the hypothesis of a pathogenic role of Cp and Mp in GCA. Larger cohort studies and standardization of molecular methods may be required to conclude properly.


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DETECTION OF PARVOVIRUS B19 IN TEMPORAL ARTERITIS/POLYMYALGIA RHEUMATICA. Carlo Salvarani, Bruno Casali, Fabrizio Cantini, Ignazio Olivieri, PierLuigi Macchioni, GianLuigi Bajocchi, Enrico Farnetti, Davide Nicoli, Maria Grazia Catanoso, Luigi Boiardi Reggio Emilia, Prato and Potenza, Italy

A SUPERANTIGEN IN LACTOBACILLUS CASEI CELL WALL EXTRACT INDUCES CORONARY ARTERITIS: AN ANIMAL MODEL FOR KAWASAKI DISEASE. Trang Duong, Earl Silverman, Martindale Bissessar, Barry Myones, Rae Yeung Toronto, ON, Canada and Houston, TX

Objective: Epidemiological studies have suggested that parvovirus B19 may play a role in the pathogenesis of giant cell arteritis (GCA). Furthermore, a significant association between histological evidence of GCA and the presence of B19 DNA in temporal artery biopsy (TAB) tissue was found in one study. Aim of this study was to determine wether B19 DNA is more likely to be present in temporal arteries of patients with GCA or polymyalgia rheumatica (PMR) than in arteries of control subjects. Methods: We examined the following artery samples for the presence of B19 DNA using two nested polymerase chain reaction (PCR) analyses : 1) fresh TAB tissue from 30 patients with biopsy-proven GCA, 44 patients with “pure” PMR, and 17 age-matched controls with a diagnosis other than PMR/GCA; 2) fresh aorta or carotid artery surgical specimens of 18 patients with atherosclerotic disease; 3) fresh samples of 14 fetal aortas obtained by autopsy. The C-terminal region of the non structural protein of 6 isolates of B19 (3 GCA patients and 3 aorta surgical specimens) were sequenced to identify B19 subtypes (Fukada et al, J Med Virol 2000; 62:60-69). Results: B19 DNA was detected in 19/30 (63.3%) GCA TAB samples, 35/44 (79.5%) PMR TAB samples, 11/17 (64.7%) control TAB samples, 12/18 (66.7%) aorta/carotid surgical specimens and 0/14 fetal aorta samples. The differences between PMR/GCA patients and age-matched controls were not significant. All 6 isolates belonged to subtype A. Conclusions: In this study, we found that B19 DNA can be detected in artery tissue of the elderly by PCR analysis. Subtype A was isolated. No association between the presence of B19 DNA in artery specimens and histological evidence of GCA was found.

Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis is currently the best animal model for Kawasaki disease (KD), which is now the leading cause of acquired heart disease in children of the developed world. In this study, we show that responses to LCWE possess all the hallmarks of a superantigen (SAg)-mediated response. LCWE-induced activation of naive T lymphocytes requires antigen presentation but not processing by accessory cells. As in the case of other known SAgs, the response to LCWE is MHC non-classically restricted, and there exists a hierarchy of differing class II MHC in the ability to present LCWE. Furthermore, the most compelling evidence of antigenic activity of LCWE is its ability to induce the characteristic activation and proliferation of T cells expressing specific T cell receptor variable families 2, 4, 6 and 14, followed by the deletion of these reactive T cells. Most importantly, superantigenic activity of LCWE correlates with its ability to induce coronary vasculitis upon injection into mice. Only LCWE preparations possessing superantigenic properties mediate cellular infiltrations in the cardiac tissue leading to coronary arteritis in vivo. Taken together, these findings demonstrate that LCWE contains a novel superantigen, the activity of which may lead to disease pathogenesis. Since LCWE-mediated coronary arteritis in mice is currently the best animal model of KD, understanding of the mechanisms leading from immune activation to coronary artery lesions in this model will be of great benefit in designing new treatment strategies, reducing vascular damage, and, more importantly, the significant morbidity and mortality associated with KD.


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THE ROLE OF T LYMPHOCYTES IN THE PATHOGENESIS OF KAWASAKI DISEASE IN AN ANIMAL MODEL. Wesley C Chan, Melanie Tsang, Rae S Yeung Toronto, ON, Canada

ENHANCED PRODUCTION OF IL-15 IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH BEHCET’S DISEASE IN RESPONSE TO HUMAN HEAT-SHOCK PROTEIN 60. Kyung-Su Park, Myung-Soo Lee, Chong-Hyeon Choi, Jin-Jung Choi, Wan-Uk Kim, Do-June Min, Yeon-Sik Hong, Jun-Ki Min, Sung-Hwan Park, Chul-Soo Cho, Ho-Youn Kim Seoul, Republic of Korea

Kawasaki disease (KD) is the most common cause of multisystem vasculitis in children in the developed world. Although KD is widely believed to be the result of an infectious agent, its etiology is still unknown. Past research has reported the possible role for bacterial superantigens in the induction of KD. Our laboratory has characterized a novel superantigen found in Lactobacillus casei cell wall extract (LCWE) which is responsible for triggering the immune response leading to KD-like coronary vasculitis in injected mice. In this study, we investigated the T helper subsets responsible for disease induction by studying expression of signature cytokines. LCWE was injected into susceptible mouse strains and RT-PCR and immunohistochemistry was performed on peripheral lymphoid tissue at set time-points. T lymphocyte activation and cytokine production was determined. Furthermore, peripheral cytokine levels and T cell activation was investigated and correlated to that in the heart end organ. Semi-quantitation of cytokine mRNA expression demonstrated a Th1 predominated phenotype in the periphery at days 1 to 3 post LCWE injection (IFN␥). This was followed by Th2 cytokine upregulation after day 3 (IL-4). 28 days after LCWE injection, a Th3 phenotype was predominant (TGF␤). In the heart, IFN␥ expression was found to be abnormally upregulated 38 days after LCWE injection. Infiltrates identified as T lymphocytes were seen as early as 3 days post LCWE injection and observed in all latter time points (up to 6 months). Different T lymphocyte subsets, at different times, are able to mediate the immunologic responses leading to inflammation. Dissecting this immune response to LCWE in mice gives important clues to the etiology of Kawasaki disease in humans.

OBJECTIVE : Heat-shock protein is thought to act as an self-antigen in Behçet’s disease (BD) and IL-15 is a growth factor for gd T cell which is known to be deeply involved in BD. We tried to evaluate the effect of human heat-shock protein 60 on the production of IL-15 by peripheral blood mononuclear cells (PBMCs) from patients with Behçet’s disease. PATIENTS and METHODS : We stimulated PBMCs from BD patients (n⫽12) and healthy controls (n⫽12) with 10 mg/ml of recombinant human heat shock protein 60 (rhHSP60), 10 mg/ml of LPS and 5 mg/ml of PHA, and then measured IL-15 level in culture supernatants. Serum levels of IL-15 and IL-12 were also determined by ELISA in BD patients (n⫽31) and healthy controls (n⫽34). BD patients were divided into active (n⫽16) and inactive (n⫽15) groups according to clinical manifestations and the differences in serum IL-15 and IL-12 levels between two groups were studied. RESULTS : The production of IL-15 from PBMCs was increased in response to rhHSP60, LPS, and PHA in BD patients and healthy controls compared to unstimulated PBMC. rhHSP60-stimulated IL-15 production was higher in BD patients than in healthy controls (132.5 ⫾ 26.0 versus 82.0 ⫾ 23.8 pg/ml, P ⬍ 0.05), whereas neither LPS- nor PHA-stimulated IL-15 production make any difference between the two groups. Additionally, the serum levels of IL-15 and IL-12 were higher in BD patients than in healthy controls (IL-15 ; 115.0 ⫾ 53.9 pg/ml vs. 57.9 ⫾ 27.2 pg/ml, P ⬍ 0.01, IL-12 ; 92.2 ⫾ 72.4 pg/ml vs. 50.0 ⫾ 48.9 pg/ml, P ⬍ 0.05 ). The serum level of IL-15 and IL-12 tended to be higher in active BD group compared to inactive BD group. CONCLUSION : Our data suggest that elevated IL-15 production may be involved in the pathogenesis of BD, which is probably mediated by HSP 60.


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FREQUENCY OF IL-12 RECEPTOR BEARING T CELLS IS A SIMPLE MARKER TO MONITOR THE DISEASE ACTIVITY IN BEHCET’S DISEASE. Mitsuhiro Takeno, Hiroko Nagafuchi, Noboru Suzuki, Takahide Matsuda, Tsuyoshi Sakane Kawasaki, Kanagawa, Japan

INFLUENCE OF INTERFERON (IFN) ␣ TREATMENT ON ALTERED LYMPHOCYYTE SUBPOPULATIONS IN BEHCET’S DISEASE (BD)- A PILOT STUDY. Michaela Treusch, Nicole Stuebiger, Ilhan Guenaydin, Christopher C Amberger, Silvia Koch, Lothar Kanz, Manfred Zierhut, Ina Koetter Tuebingen, Germany

There is accumulating evidence that preferential activation of Th1 cells is involved in clinical exacerbation of Behcet’81fs disease. The Th1 polarization is associated with an increased plasma level of IL-12 and excessive IL-12 synthesis by peripheral mononuclear cells (PBMC) in BD patients. Because there are qualitative and quantitative differences in IL-12 receptor and responsiveness to IL-12 between Th1 and Th2 cells, we here studied expression of IL-12 receptor (IL-12R) on peripheral T cells in patients with BD. We used a monoclonal antibody, TOS, which recognizes the conformational epitope of IL-12R in this study. IL-12R positive T cells expressed mRNA of Th1-related proteins such as T-bet, Txk, and IL-12R beta 2 chain and secreted large amounts of IFN-gamma when compared with IL-12R negative cells, indicating that TOS preferentially recognizes Th1 cells. Flowcytometric analysis revealed that IL-12R positive cells in circulating T cells was significantly higher in active BD patients (43.9⫹7.9%, mean⫹SEM) than healthy controls (12.9⫹0.9%), inactive BD patients (11.3⫹2.5%), and RA patients (14.8⫹3.6%). Concordantly, expression of Th1-related proteins and IFN-gamma produced by PHA and PMA-stimulated T cells were also higher in active BD patients than any other groups. A longitudinal study of individual patients showed that IL-12R expressing T cells as well as IFN-gamma production were increased in acute attacks of symptoms and decreased during remission. Thus, increased IL-12R expressing cells are closely associated with Th1 polarization during clinical exacerbations. Collectively, IL-12R expression is a simple marker to monitor the disease activity of BD and the IL-12/IL-12R system is a possible therapeutic target of the disease.

Introduction:In Behcet’s Disease, a systemic leukocytoclastic vasculitis of unknown origin, standard treatment comprises immunosuppressive drugs. We successfully treated 50 patients with BD with IFN-a2␣ which is surprising, because it is immunostimulatory and induces autoimmune diseases such as SLE and RA. The aim of this study was to evaluate the immunomodulatory effects of IFN-a2␣ in BD. Materials and methods:Nine patients with BD were examined before and under IFN treatment (6 Mill. iU daily, maintenance dosage 3 Mill. iU 3x/week) together with 5 healthy age and sex matched controls. PBMC were stained with monoclonal antibodies (CD3, CD4, CD8, CD16, CD56, CD19, CD20, CD14, CD45RA/RO, CD25, ␣␤,␥␦-T-cellreceptor), conjugated with four colour (FITC, PE, PerCP, APC) fluorescent dyes and measured by FACS (Becton Dickinson). Statistical analysis was performed by Wilcoxon rank test. Results:All patients entered complete remission. Compared to the controls, CD8⫹␥␦⫹ T-cells were elevated (median 8,5% (range 1,9-12,3%), controls median 1,8% (range 0,9-11%), ns), as were CD4⫹RO⫹RA- T-cells (median 23,5% (0,2-58,4%) vs. controls median 18,8% (7,1-22,7%), ns), and NK cells (CD16/56⫹) (median 12,8% (4,7-15,4%), controls median 3,3% (1,8-21,1%), ns). CD4⫹RO-RA⫹ T-cells (median 26,6% (1-56,5%), controls median 46,3% (36,4-71,9%), p⫽0,016) were lowered. Under IFN-␣, CD8⫹␥␦⫹ T-cells decreased (median 8,5% (1,9-12,3%), week 24 median 5,9% (0,9-7,6%), ns), as did NK cells (median 12,8% (4,7-15,4%), week 24 median 6,1% (1,6-10,7%), p⫽0,06, ns). B cells (CD19/20) and monocytes (CD14) rose (median 22,1% (7-44%), week 4 median 31,4% (10-55%), ns). Discussion:Compared to healthy controls, there was a clear tendency towards higher numbers of NK-cells and CD8⫹␥␦⫹ T-cells which decreased under IFN-␣ treatment. This did not reach significance due to the small sample size. Naive CD4⫹RA⫹ T-cells were significantly lowered in the patients, but remained unchanged. This implicates a participation of NK cells and CD8⫹␥␦⫹ T-cells in the pathogenesis of BD and may be one mechanism by which IFN-␣ exerts its therapeutic effects. The IFN-␣ induced elevation of B cells and monocytes could explain some of the side effects as autoimmune phenomena. Further analysis on larger patient and control groups is under way.

Disclosure: This work is supported by Behcet’s Disease Research Committee of Japan, sponsered by Ministry of Health, Wealfare, and Labor.

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ENHANCED EXPRESSION OF CHEMOKINES IN BEHCET’S DISEASE. Sung-Hwan Park, Mi-La Cho, Myung-Soo Lee, Kyung-Soo Park, Young-Il Seo, Wan-Uk Kim, Jun-Ki Min, Chong-Hyeon Yoon, Youn-Sik Hong, Chul-Soo Cho, Ho-Youn Kim Seoul, Republic of Korea

POLYMORPHISMS IN THE ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE ASSOCIATED WITH BEHCET’S DISEASE. Jumana A Karasneh, Ahmet Gul, Ali H Hajeer, Jane Worthington, William ER Ollier Manchester, United Kingdom and Istanbul, Turkey

Disclosure: This work is supported by a grant from catholic research institutes of medical science

Background : Behcet’s disease (BD) is a recurrent acute systemic vasculitis with a chronic course and unknown cause. A major feature of BD is endothelial damage resulting from high levels of oxygen superoxides and intermediates produced by neutrophils. Endothelial nitric oxide (eNO) is a potent vasodilator and inhibitor of monocyte and platelet adhesion, produced enzymatically from L-arginine by nitric oxide synthase (NOS). The -786 T3 C polymorphism in eNOS promoter has been reported to reduce activity in a reporter gene assay, and the 27bp VNTR in intron 4 has been associated with reduced circulating NO levels. Aim : To investigate the role of eNOS gene polymorphisms in BD. Methods: A case control study was carried out using 160 Turkish BD patients fulfilling the international study group (ISG) criteria and 105 healthy Turkish controls. Fifty six of the cases have a family history of BD. The -786 T3 C polymorphism and the 894 G 3 T polymorphism in exon 7 were genotyped by PCR-RFLP using MspI and BanII restriction enzymes respectively. The 27 bp VNTR ((a) allele is 4 repeat and (b) allele is 5 repeat) was genotyped using PCR and agarose gel electrophoresis. Results: -786 T3C Genotype frequency (n)

BD families BD cases Controls

27bp VNTR Genotype frequency (n)

TT

TC

CC

bb

ba

aa

70% (39) 54% (84) 42% (43)

29% (16) 41% (64) 43% (44)

2% (1) 6% (9) 15% (15)

88% (44) 77% (115) 64% (66)

10% (5) 22% (33) 33% (34)

2% (1) 1% (2) 3% (3)

Significant differences were found between cases and controls in eNOS -786 polymorphism for allele (p ⫽0.01) and genotype frequencies (p⫽0.02). More significant results were obtained when family history was taken in consideration (p ⫽ 0.0002, 0.001) for allele and genotype frequencies and for the intronic marker (p ⫽ 0.0047, 0.003 respectively). No difference in allele and genotype frequencies were found between cases and controls in the exonic marker. Haplotype investigation revealed that both the promoter and the intronic markers are in linkage disequilibrium and the TTbb genotype is significantly higher in patients than controls (p⬍0.00001 ). Conclusion: The eNOS gene promoter and intronic markers are associated with BD. Individuals having the genotype *TT -786 / *bb VNTR are at higher risk to develop BD (OR ⫽2.6, 95%CI (1.53-4.48)). Disclosure:

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Objective: To investigate the chemokine expression in patients with Behcet’s disease (BD) and to access the usefulness of circulating chemokine levels as the disease activity marker. Methods: C-X-C chemokine (IL-8) and C-C chemokines (RANTES, MIP-1␣ , and MCP-1) were measured by ELISA in sera and culture supernatants of peripheral blood mononuclear cells (PBMC) obtained from patients with BD and healthy controls (HC). Supernatants were collected from PBMC culture unstimulated or stimulated with interferon gamma for 72 hr. The activity of the disease was clinically evaluated by the number of actively involved organ systems registered on the day of blood sampling. Results: The serum levels of C-X-C and C-C chemokines were significantly higher in patients with BD (n⫽67) than in age and sex matched HC (n⫽44) (p⬍0.001 for all chemokines). Also, increased IL-8 serumlevels were found in patients with active disease (n⫽21) compared to inactive disease (n⫽46)(305.6 ⫹/- 88.6 versus 245.6⫹/- 90.7 pg/ml, p⫽0.020). Patients with active disease showed higher circulating levels of C-C chemokines than those with inactive disease, but differences did not reach statistical significance. The serum levels of IL-8 strongly correlated with C-C chemokine levels (MIP-1␣, r⫽0.444, p⬍0.001; RANTES, r⫽0.475, p⬍0.001; MCP-1, r⫽0.464, p⬍0.05) and serum levels of IL-15 (r⫽0.555, p⫽0.005). The chemokine productions by interferon gamma-stimulated PBMC were significantly augmented in BD (n⫽25) compared to healthy controls (n⫽11) (p⬍0.05 for all chemokines). Futhermore, the IL-8 production was more enhanced in active stage of BD (n⫽ 8) than those with inactive stage (n⫽ 17) (266.2 ⫹/- 115.0 versus 175.3 ⫹/- 61.9 pg/ml, p⫽ 0.027). Conclusion: Circulating levels of both C-X-C and C-C chemokines are elevated in BD, which seems to be related with increased production of chemokines by PBMC in response to interferon gamma. Serum levels of chemokines, especially, IL-8 may be a useful marker of disease activity in BD.

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PRESENCE OF AUTOANTIBODIES IN RELATIVES OF PATIENTS WITH PRIMARY SYSTEMIC VASCULITIDES (PSV). Luis F Flores-Suarez, Carlos E Mendez Probst, Ricardo Ramirez, Lirio Lopez, Javier Cabiedes, Antonio Villa, Jorge Alcocer-Varela, Donato Alarcon-Segovia Mexico, DF, Mexico

BLOCKADE OF ANTINEUTROPHIL CYTOPLASMIC ANTIBODY (ANCA) ACTIVATION OF NEUTROPHILS BY A THERAPEUTIC PEPTIDE. Sumiaki Tanaka, Jeffrey C Edberg, Georgio Fassina, Robert P Kimberly Birmingham, AL and CE, Italy

The presence of autoantibodies in relatives of patients with PSV has been occasionally reported. OBJECTIVE: To evaluate the prevalence of different autoantibodies in relatives of a PSV cohort. PATIENTS AND METHODS: Three groups were studied: Group I: 145 first-degree relatives (mother-20, father-16, sister-55, brother-34, son-3, daughter-17) of 57 PSV patients. Group II: 57 PSV patients (Wegener’s granulomatosis-27, Behcet’s disease-8, Takayasu arteritis-7, microscopic polyangiitis-5, polyarteritis nodosa-4, Henoch-Schonlein purpura-4, giant cell arteritis-1, cutaneous PAN-1). Group III: 37 sex- and age- matched-to-case controls. The presence of the following autoantibodies was evaluated: antinuclear and anticytoplasmic, anti-ds-DNA, rheumatoid factor, anticardiolipin (IgG, IgM, IgA), anti-ß2-GPI, antiproteinase 3 (anti-PR3) and anti-myeloperoxidase (anti-MPO). Statistical analysis: X2 with Yates correction or two-tailed Fisher exact test. The probability of having PSV according to the presence of each autoantibody was calculated as odds ratio (OR). RESULTS: Differences are shown in the table; for the rest of the antibodies tested there were no differences between patients and their first-degree relatives.

Antineutrophil cytoplasmic antibodies (ANCA) may directly activate neutrophils resulting in mononuclear cell recruitment and vascular injury. ANCA IgG ligates target molecules by its F(ab⬘)2 end and trigger neutrophil activation. To study the relative contributions of Fc␥R-dependent and independent pathway(s) of ANCA induced activation, we used the synthetic peptide (TG19320) that binds the Fc-region of IgG and blocks IgG-Fc region binding to Fc␥R both in vitro and in vivo (Marino, M. et. al., Nat Biotechnol 18:735, 2000). We quantitated ANCA stimulated up-regulation of CD11b, an important adhesion molecule and constituent of neutrophil granules, on neutrophils in washed whole blood after stimulated with human or murine monoclonal cANCA (anti-PR3) in the presence or absence of various concentrations of TG19320. Cell surface expression of CD11b was significantly upregulated by both human polyclonal and murine monoclonal cANCA (% increase of MFI relative to control incubation: 148.1⫾8.8% (n⫽7) and 137.7⫾7.3% (n⫽8) respectively, mean ⫾ SEM, p⬍0.001 in both cases). TG19320, but not a scrambled variant of TG19320, inhibited the cANCA-induced upregulation of CD11b expression by both human polyclonal and murine monoclonal cANCA in a dose dependent manner, with complete inhibition at 1000 mg/ml TG19320 (%inhibition of ANCA induced CD11b: 88.3⫾9.0% (n⫽5) and 92.6⫾9.4% (n⫽4) for human and murine monoclonal cANCA respectively, p⬍0.001). These results support a model of ANCA activation of neutrophils in which activation is fully dependent on ANCA-Fc␥R engagement. The use of a stable soluble peptide inhibitor of IgG-Fc␥R binding offers the possibility of a new strategy in the treatment of ANCA associated vasculitis syndrome.

Autoantibody

RF anti-PR3 anti-MPO anti-b2-GPI

Prevalence PSV 12/56 (21.4%) 12/55 (21.8%) 4/55 (7.3%) 5/55 (9.1%)

Relatives 10/142 (7%) 3/137 (2.2%) 1/137 (0.7%) 47/143 (33%)

OR

95% CI

p

3.6 12.46 10.66 0.204

1.46-8.9 3.36-46.2 1.17-97.7 0.08-0.55

⬍ 0.011 ⬍ 0.0009 ⬍ 0.025 ⬍ 0.002

When compared to healthy subjects, anti-␤2-GPI was the only antibody increased in relatives (33% vs 0%; p ⬍ 0.009). CONCLUSIONS: The prevalence of anti-␤2-GPI was higher in relatives of patients with PSV than that seen in patients and healthy non-related individuals. The low OR suggests a lesser probability to develop PSV. In contrast, for RF, anti-PR3 and anti-MPO, a high OR may indicate an increased probability for the development of PSV in relatives in whom these autoantibodies are present. The significance of the presence of these autoantibodies in relatives of PSV patients is unknown. Follow-up of these subjects is being performed.

Disclosure: Dr. Fassina is an employee of Tecnogen. Tecnogen has provided the TG19320 peptide for these studies.

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PTX3 PLASMA LEVELS REFLECT DISEASE ACTIVITY IN SMALL VESSEL SYSTEMIC VASCULITIDES. Fausto Fazzini, Maria Grazia Sabbadini, Giuseppe Peri, Giacomo Dell’Antonio, Andrea Doni, Elena Dal Cin, Alberto Mantovani, Angelo A Manfredi, Patrizia Rovere Querini Milano, Italy

ANTINEUTROPHIL CYTOPLASMIC ANTIBODIES (ANCA) INDUCE RELEASE OF HUMAN NEUTROPHIL ␣-DEFENSINS: T CELL AND DENDRITIC CELL RECRUITMENT FACTORS. Sumiaki Tanaka, Jeffrey C Edberg, Giorgio Fassina, Robert P Kimberly Birmingham, AL and CE, Italy

Small vessel vasculitides are characterized by the immune mediated inflammation of small vessels in the absence of antinuclear autoimmunity. Elevated serum levels of the short pentraxin C reactive protein (CRP), which is produced by the liver under the control of IL-6, reflect the systemic involvement. CRP has been described to bind to chromatin and to small nuclear ribonucleoproteins. Un-scavenged apoptotic neutrophils accumulate in and around the wall of involved vessels. The long pentraxin PTX3 is produced in peripheral tissues by endothelial cells and macrophages under the control of primary pro-inflammatory signals, including LPS, IL-1b and TNF-a. We have recently characterized the ability of PTX3 to bind to late apoptotic cells, preventing their engulfement by professional antigen presenting cells1. In this study we evaluated 53 patients with small vessels vasculitides. 12 of them had leukocytoclasia. 22 patients had active disease. PTX3 produced in locally inflamed tissues behaved as an acute phase reactant, reaching a concentration of 6.8 ng/ml (the mean value of PTX3 in the sera of 15 healthy donors was 1.5 ng/ml). Detection of PTX3 in the sera associated to the detection of PTX3 in discrete sites of disease involvement, as assessed in skin biopsies. The serum levels of PTX3 and CRP did not correlate. In newly diagnosed patients PTX3 levels decreased shortly after steroid administration. In contrast, CRP decreased with slower kinetics. The role of PTX3 levels as an early marker of relapse is now actively investigated. The feature was selective for vasculitides, since PTX3 did not rise in the plasma of patients with active Systemic Lupus Erythematosus, Rheumatoid Arthritis or CREST Syndrome. 1. Rovere P et al., The long pentraxin PTX3 binds to apoptotic cells and regulates their clearance by antigen-presenting dendritic cells. Blood. 2000 96:4300-4306.

ANCA are typically observed in patients with Wegener’s granulomatosis (WG) and can directly activate neutrophils (PMN) in an Fc␥RIIa and Fc␥RIIIb-dependent manner. Granuloma formation is also characteristic of WG, but a mechanism linking ANCA-induced PMN activation and granuloma formation with recruitment of T cells and macrophages has not been established. Human a-defensins, cationic antimicrobial peptides, are found in PMN azurophilic granules, have potent chemotactic activity for T cells, dendritic cells and monocytes, and may provide a link between ANCA-induced PMN activation and granuloma formation. In this study, we quantitated the release of a-defensins (HNP 1-3) in supernatants by ELISA after targeted Fc␥R cross-linking (XL) in washed whole blood. Fc␥R XL (either homotypic XL of Fc␥RIIa or Fc␥RIIIb or heterotypic XL of both receptors) resulted in significant release of ␣-defensins. Furthermore, Fc␥RIIa⫹IIIb XL on TNF␣primed PMN from donors homozygous for the NA1 allele of Fc␥RIIIb resulted in significantly greater release of ␣-defensins relative to donors homozygous for the NA2 allele of Fc␥RIIIb (% release of ␣-defensins relative to control incubation: 253.8⫾37.7% vs. 209.7⫾37.2% for NA1/1 vs. NA2/2 donors, p⫽0.004). Finally, both human polyclonal and murine monoclonal ANCA (anti-PR3) induced the release of ␣-defensins (% release of ␣-defensins relative to control incubation: 252.5⫾41.7% and 265.6⫾33.3% for human and murine monoclonal cANCA respectively, p⬍0.05, n⫽4-11). This release was completely inhibited with the IgG Fc-region binding peptide TG19320 that blocks IgG-Fc region binding to Fc␥R. These observations demonstrate that ANCA can induce release of ␣-defensins in an Fc␥R dependent manner and that this Fc␥R activation occurs in an Fc␥RIIIb-allele dependent manner. Based on the chemotactic potential of human ␣-defensins, ␣-defensin release by PMN may be important in modulating the acquired immune responses through recruitment of mononuclear cells thereby contributing to granuloma formation in patients with WG.

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Disclosure: Dr. Fassina is an employee of Tecnogen. Tecnogen has provided the TG19320 peptide for these studies.

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GENETIC POLYMORPHISMS IN TNF, IL-1, IL-6 AND CYTOTOXIC T LYMPHOCYTE-ASSOCIATED ANTIGEN 4 (CTLA-4) IN WEGENER’S GRANULOMATOSIS (WG). Yihua Zhou, DeRen Huang, Gary S Hoffman Cleveland Ohio

INTERFERENCE OF PR3-ANCA WITH THE ENZYMATIC ACTIVITY OF PR3. Agnieszka A Rarok, Ymke M van der Geld, Anton T-J Tool, Masha de Haas, Pieter C Limburg, Cees GM Kallenberg, Dirk Roos Groningen and Amsterdam, Netherlands

Background: Although the precipitating event(s) that trigger WG are unknown, evidence for genetic predisposition has been suggested by the occurrence, albeit infrequent, of familial cases. Cytokines and co-stimulators control the quality and intensity of immune responses. Thus they are relevant candidates for the study of immune dysregulation in WG, that may have either an acquired or inherited basis. Patients and Methods: Using PCR-based, genomic DNA genotyping, this study investigated the polymorphisms located in the genes encoding proinflammatory cytokines such as TNF-␣, IL-1 and IL-6, and CTLA-4 (a negative co-stimulator for T cell activation), in 117 American patients with WG and 123 ethnically matched healthy controls. Results: A significantly lower percentage of patients homozygous for the shortest allele 86 in the microsatellite polymorphism (AT)n located in the 3⬘-untranslated region of exon 3 of CTLA-4 was found as compared with healthy controls (47% versus 70%, p ⫽ 0.0005). Examination of a bi-allelic exonal polymorphism in CTLA-4 did not show skewing in patients. Significant differences in the allelic and genotypic frequencies of polymorphisms in the other proinflammatory cytokine genes studied were not found between patients and controls. Conclusion: The CTLA-4 (AT)n 86 allele has been previously demonstrated to be crucial for maintenance of normal levels of CTLA-4 expression and T cell activation. Our results confirm findings from a Scandinavian cohort, suggesting that this observation is not limited to WG patients who are ethnically unique. The (AT)n polymorphism in the CTLA-4 gene may represent a WG-related susceptibility mutation and account for the increased T cell activation and clonal expansion in WG patients. Blockade of T cell costimulation using CTLA-4Ig might be a useful therapeutic intervention.

Introduction: Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegener’s granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity. Functional effects of these antibodies in vitro have been suggested to correspond better to the clinical disease activity. Methods: To further investigate the relation between functional effects of PR3-ANCA and disease activity, we tested the effect of IgG samples from sera of 43 WG patients and four controls for their capacity to interfere with the proteolytic activity of PR3. Blood was drawn either during active disease or during remission of WG. The enzymatic activity of PR3 was determined with a small synthetic substrate (MeSuc-AAPV-pNA) as well as with casein as the substrate and by the complexation of PR3 with its natural inhibitor ␣ 1-antitrypsin (␣ 1-AT). Results: Most of the IgG samples from WG patients inhibited the proteolytic activity of PR3 and inhibited the complexation of PR3 with ␣ 1-AT. A difference in the capacity to interfere with the proteolysis of casein and with the complexation of PR3 with ␣ 1-AT was observed between samples taken during active disease and during remission of WG, but this was not observed for the hydrolysis of MeSuc-AAPV-pNA. PR3-ANCA titers giving fifty percent inhibition of the proteolytic activity of PR3 for the hydrolysis of MeSuc-AAPV-pNA were lower for remission samples compared to samples during active disease. Similar results were obtained for the inhibition of the PR3/␣ 1-AT complexation. A correlation between the PR3-ANCA titers and the inhibitory activity was observed both for patients with active disease and for patients during remission. Conclusion: With a fixed amount of IgG, PR3-ANCA containing IgG from patients with active disease had a higher inhibitory capacity towards the proteolytic activity of PR3 than did PR3-ANCA containing IgG from patients during remission of WG. However, when correcting the results for the PR3-ANCA titer, PR3-ANCA of patients during remission had a relatively higher inhibitory capacity towards the proteolytic activity of PR3 than did PR3-ANCA of patients during an active phase. These results may indicate that PR3-ANCA of patients with active disease recognize different epitopes on PR3 than do PR3-ANCA of patients during remission of WG, which may have relevance for the pathogenicity of the antibodies. Disclosure:

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SEQUENTIAL EPITOPE MAPPING OF PROTEINASE-3 IN WEGENER’S GRANULOMATOSUS. Donny M Wynn, Judith A James Oklahoma City, OK

CHLAMYDIA TRACHOMATIS INFECTION OF CELLS ACTIVATES BOTH HUMAN NK CELL CYTOTOXIC ACTIVITY AND CYTOKINE PRODUCTION. Catherine Elizabeth Hook, Malgosia Krystyna Matyszak, John Stanley Hill Gaston Cambridge, United Kingdom

Antineutrophil cytoplasmic antibodies (ANCA) are commonly found in over 90% of patients with Wegener’s Granulamatosus (WG). These autoantibodies appear to play a significant role in WG based upon their near universal presence and the modulation of these autoantibody titers with disease activity. Proteinase-3 (PR-3), an enzyme commonly found in neutrophilic granules, is the major target of this autoimmune response. PR-3 has many substrates, but one protein, the soluble endothelial protein-C receptor (s-EPCR) has been found to bind PR-3 external to its active site. Normally, EPCR is a key component of a coagulation pathway involving protein C. These proteins have also been shown to have an inhibitory role in inflammation. The interaction between PR-3 and EPCR could lead to novel epitopes. In addition, the interruption of this normal protein interaction could lead to increased inflammation as seen in the vasculitis of WG. This serves as a potential hypothesis for a role of anti-PR-3 in this pauci-immune disease. This study seeks to determine the sequential common antigenic targets of c-ANCA on proteinase-3 through sequential epitope-mapping. Overlapping octapeptides of PR-3 have been synthesized on derivatized, polyethylene solid phase supports. Ten WG patients, previously determined to have c-ANCA by IF and anti-PR-3 by ELISA, have been tested for reactivity with the PR-3 octapeptides. The average binding of all ten patients to the proteinase-3 protein revealed that c-ANCA reactivity to the proteinase-3 protein is concentrated at two epitopes spanning amino acids 34-44 and 45-55. In addition, five other common antigenic regions are significantly bound by the ten patients. Interestingly, the two main epitopes at amino acids 34-44 and 45-55 surround the PR-3/s-EPCR binding site, occurring at amino acids 80-91 on PR-3, when the protein is in its tertiary structure. In addition, a monoclonal antibody which also binds this region inhibits the interaction between these two proteins. This data supports the existence of autoantibodies with sequential specificities to the PR-3 antigen and that WG patient sera contain antibody specificities which target the area of PR-3 which interacts with sEPCR.

CT is an obligate intracellular bacterial pathogen infecting ocular and genital mucosae. Work in murine models has suggested a role for NK cells in the control of infections, particularly NK cell cytokine production. We hypothesized that infection of cells with CT would lead to alterations which would be recognised by human NK cells, resulting in activation of one or more NK cell effector functions. CT-infected cells are reported to be resistant to the action of perforin and granzyme. We found them to remain sensitive to the actions of NK cells, however, as highly NK susceptible NK targets (K562 and 721.221) were not protected from lysis following infection. Infection of SiHa and HeLa cervical epithelial cell lines with the L2 strain of CT led to a significant increase in lysis of the cells by polyclonal NK cell lines from healthy individuals. Timecourse studies showed that NK-cell mediated lysis was not increased until around 22 hours post-infection. UV-inactivated CT did not induce the same phenomenon, nor was it seen when CT protein synthesis was inhibited with Rifampicin. Inhibition of host-cell protein synthesis with cycloheximide did not inhibit the increase in lysis following infection. Manipulation of the CT replication cycle with Penicillin also did not alter the increase in lysis following infection. To investigate the mechanisms for the increased lysis of the CT-infected cells, we examined the expression of known NK cell ligands on infected cells. Class I MHC was reduced on cells infected with live CT but was unaltered on cells treated with UV-inactivated CT. We also investigated NK cell cytokine production following exposure to supernatants from different infected cell types. Treatment of polyclonal NK cell lines with rhIL-18 and rhIL-12 led to production of IFN-␥. It has been reported that CT-infected HeLa cells produce IL-18, and we have shown that CT-infected dendritic cells are a source of IL-12. Experiments are underway to ascertain if combination of these supernatants can stimulate NK cell IFN-␥ production, and preliminary results suggest that the HeLa supernatants are able to substitute for the rhIL-18 in stimulating NK cell IFN-␥ production. In summary, we have shown that CT-infection of cells was able to induce both NK cell cytokine production and cytotoxic activity. Disclosure:

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REPEATED INFECTIONS INCREASE PATHOLOGY IN MURINE CHLAMYDIA-ASSOCIATED REACTIVE ARTHRITIS (ReA): SEROLOGIC CORRELATES OF RE-EXPOSURE. Judith A Whittum-Hudson, Sylvette Bas, H Ralph Schumacher Detroit, MI; Geneva, Switzerland and Philadelphia, PA

INDUCTION OF A PERSISTENT INFECTION OF CHLAMYDIA TRACHOMATIS BY DOXYCYLIN IN VITRO. E-M Fliedner, U Dreses-Werringloer, I Padubrin, H Zeidler, L Koehler Hannover, Germany Background: The persistence of Chlamydia trachomatis (CT) as a viable bacterium in monocytes within the joint is thought to initiate and maintain the inflammatory process. A clinical trial reporting a partial response to tetracyclin have not been confirmed by others. It was the aim of the present study to investigate the efficacy of doxycylin for the elimination of CT in vitro. Methods: Human epithelial HEp-2 cells were infected with CT at a multiplicity of infection of 0.05. The number of infective Chlamydia was determined by titration of the cell lysates on HEp-2 cells, the number of inclusion bodies by immunofluorescence microscopy and primary chlamydial rRNA transcripts by RT-PCR. Results: At first minimal inhibitory concentration (MIC) was determined by simultanous addition of CT and doxycyclin to the HEp-2 cells. The MIC was 0.06 mg/ml. Addition of doxycylin (0.06 ug/ml) 2 days post infection (p.i.) to CT infected HEp-2 cells resulted in a loss of infectious chlamydia at day 8 p.i.. Inclusion bodies were not observed later than day 8 p.i.. To assess the vitality of the intracellular chlamydia RT-PCR analysis of primary 16S rRNA transcripts which are only produced in viable, metabolically active bacteria were performed. These transcripts were detectable until day 14-20 p.i..

Purpose: Chlamydia-associated ReA presents as a spectrum of clinical severities. It is not known whether re-exposure to this organism, duration after exposure, or a combination of these and other factors influences occurrence of disease or the variety of clinical patterns. A murine model of chlamydial ReA has been used to test whether re-exposure to the same or a new chlamydial serovar differentially influences joint inflammation and whether heterogeneous anti-chlamydial antibody (Ab) responses reflect different exposures. Methods: C3H/HeN or BALB/c mice (n⫽5/group) were genitally infected once or twice with K or E urogenital serovar of C trachomatis (homologous re-infection), or once with each serovar (K⫹E or E⫹K) at 3-4 wk intervals. Appropriate controls were included (ie, 0⫹K, 0⫹K, E⫹0, 0⫹E, or 0⫹0); all treatment groups were masked until completion of analyses. Four wks after the second infection mice were sacrificed, blood collected for serum, and tissues for histopathology. Sera were tested by ELISA for Ab to peptides mimicking epitopes of chlamydial major outer membrane protein (MOMP) variable domains I and IV. OD with signal to noise ratios of ⬎3 were considered positive. Knees were scored for histopathology. One experiment tested homologous re-infection, and two, both homologous and heterologous re-infection. Results: Re-infection of C3H and BALB/c mice with the same or different human serovars produced increased joint pathology compared to single exposure to either chlamydial serovar, eg, for C3H, K⫹K vs 0⫹K: 1.5⫾0.3 vs 0.4⫾0.4. Anti-MOMP peptide responses to single K serovar were similar to levels after repeated exposure. When BALB/c mice exposed to E⫹K serovars, anti-peptide responses were significantly higher than those observed for a single E serovar infection (p⫽.007; Student’s t-test). Similar trends were seen in all 3 experiments. Conclusions: Our results with this experimental model of ReA support the hypothesis that re-exposure to chlamydia, particularly to new strains/serovars, increases synovial pathology. The diversity and intensity of antichlamydial Ab responses also correlate with repeated exposure. Whether repeated exposure produces a combination of persistent and acute joint infection remains for future study.

Conclusions: Doxycyclin suppressed the chlamydial growth in HEp-2 but induced a persistent infection of viable, metabolically active chlamydia. Thus, doxycylin only provides a bacteriostatic effect on chlamydia. From these in vitro data and clinical data doxycyclin can not be recommanded for the treatment of Chlamydia-induced arthritis.

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IL-10 PROMOTES BACTERIAL CLEARANCE THEREBY AMELIORATING STAPHYLOCOCCUS AUREUS INDUCED ARTHRITIS. Inger Gjertsson, Andrej Tarkowski Goteborg, Sweden

EXPRESSION OF NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1) IN HUMAN MONOCYTES/MACROPHAGES DURING SALMONELLA ENTERITIDIS INFECTION. Yi Liu, Qiushui He, Johanna Makinen, Jan Kaslin, Fumio Kishi, Kaisa Granfors Turku, Finland and Yamaguchi, Japan

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We have shown that intracellular elimination of Salmonella enteritidis is impaired in HLA-B27-transfected human monocytic U937 cells and in peripheral blood monocytes from HLA-B27 positive persons with history of reactive arthritis (ReA). To elucidate the mechanisms that might be involved in the host-microbe interaction in HLA-B27 positive cells, we studied the possible expression of Nramp1 gene, product of which is supposed to inhibit the intracellular replication of Salmonella, in both cell lines and human monocytes/macrophages upon Salmonella infection. We used a real-time LightCycler RT-PCR to detect the expression of Nramp1 in U937 cells transfected with HLA-B27 and HLA-A2 genomic DNA, and in human peripheral blood monocytes and macrophages from HLA-B27 positive and negative persons with or without ReA in the past. Models for the in vitro infection of cell lines, monocytes and macrophages were the same as we have previously reported. The expression of Nramp1 mRNA was detected by the melting curve analysis of the specific PCR product (further confirmed by gel electrophoresis) and normalized to that of b-actin. Colocalization of intracellular bacteria and Nramp1 was evaluated by confocal microscope. Salmonella infection stimulates the expression of Nramp1 in cell lines, human monocytes and macrophages. In cell lines, HLA-B27 transfected U937 cells, in which the elimination of Salmonella was impaired, tend to express lower amount of Nramp1 at early stage of Salmonella infection comparing with the HLA-A2 transfected cells. Moreover, the relatively low expression of Nramp1 in monocytes from HLA-B27 positive persons with ReA in the past was found from the early stage of ex vivo infection to the end of three days when compared with the HLA-B27 negative controls. Under confocal microscope, Nramp1 was located around the bacteria-containing vacuoles and not associated completely with lysosomal membrane protein 1. Our result indicates that the real-time LightCycler RT-PCR can be used for specific detection of the expression of Nramp1. Relatively low level of Nramp1 expression in HLA-B27 positive monocytic cells suggests that HLA-B27 molecule may be involved in the modulation of Nramp1 expression during Salmonella infection, implicating its role in the pathogenesis of ReA. Disclosure:

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S. aureus induced infections often result in high mortality and permanent joint destruction despite adequate treatment with antibiotics. IL-10 is known to be an anti-inflammatory cytokine which is produced by both T- and B-cells, as well as macrophages and shifts the immune response towards Th2, thereby impairing the Th1 response and macrophage function. It is known that IL-10 protects against superantigen induced shock and TNF induced apopotosis. In this study we investigated the roll of IL-10 in S. aureus induced arthritis and sepsis. Material and methods: IL-10 deficient mice were inoculated in one of the tail veins with a superantigen-producing (toxic shock syndrome toxin-1, TSST-1) S. aureus strain. Results: The IL-10 deficient mice had significantly higher bacterial load in blood and kidneys. Suprisingly the serum level of TNF late during the infection (day 11) was significantly lower in the knock-outs than in the control group. As expected, we found a more frequent and destructive arthritis in the IL-10-/- mice as compared to controls when evaluated with histopathology. The phagocytic capacity and intracellular killing by macrophages was equal between the mouse groups when tested in vitro. Conclusion: Our results indicate that IL-10 is essential for efficient elimination of bacteria and thereby for protection against septic arthritis. We believe that the protective effect of IL-10 is related to its anti-apoptotic properties in the phagocytic cell population.

Infectious yield Inclusion bodies Primary rRNA transcripts

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ACIDIFICATION OF SALMONELLA-CONTAINING PHAGOSOMES IN THE HLA-B27 TRANSFECTED HUMAN MONOCYTIC U937 CELLS. Xiao Li, Yi Liu, Markus Penttinen, Kaisa Granfors Turku, Finland

RHEUMATOLOGICAL AND GASTROINTESTINAL SYMPTOMS AFTER INFECTION WITH CAMPYLOBACTER AND ENTEROTOXIGENIC E. COLI. Henning Locht, Karen A Krogfelt Copenhagen S, Denmark

In search for the mechanisms behind impaired elimination or fast replication of Salmonella enteritidis in HLA-B27 transfected U937 cells, we studied the acidification of Salmonellacontaining phagosomes. U937 transfectants (HLA-B27 genomic DNA and HLA-A2 genomic DNA) were first stimulated with PMA for 24 hours to be differentiated to more mature macrophage-like cells. After 15 min treatment with bafilomycin A1 (a specific inhibitor of vacuolar proton-ATPases, which inhibits the acidification of phagosomes), cells were infected with S. enteritidis. Intracellular survival of S. enteritidis was determined at different time points up to 3 days (CFU). Bafilomycin A1 pretreatment decreased the number of live Salmonella in both transfectants. There were significantly fewer bacteria in bafilomycin A1 pretreated cells than in untreated cells at 1 and 4 hours postinfection. Thereafter, the elimination of S.enteritidis was impaired in bafilomycin A1 pretreated cells compared to untreated cells. It was significantly poorer in HLA-A2 transfectants pretreated with bafilomycin A1 than in untreated cells at 24 hours postinfection. This may suggest that the S.enteritidis began to adapt to the neutralized phagosomes. It seemed that bafilomycin A1 had stronger effect on the elimination of S.enteritidis in HLA-A2 transfectants than in HLA-B27 transfectants. Our results show that bafilomycin A1 pretreatment affects the elimination and/or replication of intracellular S. enteritidis. Bafilomycin A1 pretreatment has stronger effect in HLA-A2 transfected cells than in HLA-B27 transfected cells suggesting that the impaired elimination of Salmonella in HLA-B27 transfected cells may be, at least partially, due to disturbances in phagosome acidification.

Objective To estimate the incidence of reactive joint complaints after Campylobacter enteritis. To compare gastrointestinal symptoms, antibiotic treatment and antibody levels among patients with versus without joint symptoms. Method A cohort of 210 patients with Campylobacter enteritis verified by stool culture was followed for 2 years. Serum for anti-Campylobacter antibody determination was collected 3 weeks, 3 months, 6 months, and 2 years after infection. At the end of the follow-up period all patients were mailed questionnaires inquiring about gastrointestinal symptoms and occurrence of joint symptoms in previously healthy joints within 4 weeks of debut of diarrhea. The same questionnaire was sent to a control group with an equal number of patients from the same time period with enteritis caused by enterotoxigenic E. coli (E. coli (ETEC)). Results Answers were obtained from 173 (82%) patients with Campylobacter of which 27 (16%) fulfilled predefined criteria for reactive joint symptoms. In the E. coli group 177 (84%) patients answered the questionnaire and 10 (6%) reported joint symptoms (p⫽0.004). Among the Campylobacter patients median duration of diarrhea was 13 days for patients with arthralgias and 7 days for those without joint pain (p⫽0.0058). When duration of diarrhea was compared between patients without arthralgias E. coli with a median of 14 days differed from Campylobacter with a median of 7 days of diarrhea (p⫽0.0005). Fifty-nine per cent of Campylobacter patients with joint pain had received antibiotic treatment because of enteritis compared to 26% of those with enteritis only (p⫽0.03). Campylobacter species and serotypes were equally distributed in both groups and there was no difference in anti-Campylobacter antibody levels between the groups. Conclusion There was a significantly increased risk of developing joint symptoms after contracting Campylobacter infection compared to E. coli. Patients with joint pain had more severe gastrointestinal symptoms and longer duration of diarrhea. Antibiotic treatment does not seem to prevent reactive joint symptoms and levels of anti-Campylobacter antibodies were equal in both groups.

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MHC CLASS I MOLECULES AFFECT IRAK EXPRESSION AND MODULATE LPS-INDUCED SIGNAL TRANSDUCTION IN U937 HUMAN MONOCYTIC CELLS. Markus A Penttinen, Ville Hietakangas, David TY Yu, Lea Sistonen, Kaisa Granfors Turku, Finland and Los Angeles, California

KLEBSIELLA IS NOT THE DOMINANT MICROBIAL TRIGGER IN FAMILIAL ANKYLOSING SPONDYLITIS. Millicent A Stone, Ursula Payne, Vicki Lapp, Proton Rahman, Robert D Inman Toronto, Ontario, St.John’s and Newfoundland, Canada

We have previously reported that LPS-induced NF-␬B activation is enhanced in HLA-B27 transfected U937 human monocytic cells compared to the cells transfected with HLA-A2 or with the resistant vector alone. In search for the reason for this phenomenon, we studied the expression of interleukin receptor associated kinase (IRAK), which has been shown to control LPS-induced NF-␬B, p38 and JNK activation in monocyte-macrophages, in different U937 transfectants. We also studied NF-␬B, p38 and JNK activation, and TNF-␣ production upon LPS stimulation in these cells. U937 transfectants (three cell lines transfected with either HLA-B27 or HLA-A2 genomic DNA or with the resistant vector pSV2neo alone and three cell lines transfected with either HLA-B27 cDNA, HLA-A1 cDNA or with the resistant vector RSV5neo alone) were first stimulated with PMA for 24 hours to maturate them to express LPS-receptor CD14, and were then stimulated with LPS. Activation of NF-␬B was measured by electromobility shift assay, expression of IRAK was determined by Western blotting, activation of p38 and JNK was determined using phosphospecific antibodies against respective kinases, and production of TNF-␣ from culture supernatants by ELISA. Results indicate that all transfected MHC class I molecules affect IRAK expression. In HLA-A1 and HLA-A2 transfected cells the expression of IRAK was downregulated compared to their respective vector controls, whereas in HLA-B27 cells it was slightly upregulated compared to the respective vector controls. In HLA-A1 and HLA-A2 transfected cells NF-␬B, p38 and JNK activation, and TNF-␣ production were more modestly induced upon LPS stimulation compared to the respective vector controls. In HLA-B27 cells NF-␬B activation was more strongly induced, but the activation of p38 or JNK was not clearly affected compared to the controls. Our results indicate that all studied MHC class I molecules modulate LPS-induced signaling in human monocytic cells. These modulatory effects might have important implications in LPS-induced inflammatory diseases.

The contribution of infection to AS has been controversial. In particular, a role for Klebsiella pneumoniae (Kp) has been implicated using serological methods, but those studies have not included cellular immune responses. This study is unique in its approach to the question in its utilization of AS families where the unaffected members serve as controls. We analyzed proliferation of peripheral blood lymphocytes (PBL) as measured by 3H-thymidine incorporation to Kp, Salmonella typhimurium (St), Yersinia enterocolitica (Ye) and Chlamydia trachomatis (Ct). In addition, IgG and IgA antibodies against the respective organisms were measured by standard ELISA methodology. There were 25 families studied, in whom there were 51 individuals affected (A) and 32 unaffected (U) individuals. In addition, both cell proliferation and antibody studies were performed on 17 normal healthy subjects. There were no differences between groups in mean baseline counts (A: 1932 cpm vs U: 2712cpm) nor in response to PHA (A 98503 cpm vs U: 72202 cpm). With respect to specific antigen stimulation the percentage of patients responding maximally to the microbial antigens were as follows. A: Ct 41%, Kp 28%, St 23% Ye 7.7% and U: Ct 24%,Kp 48%,St 4% and Ye 24%. For St alone the difference between A and U groups reached significance (p⬍0.05). Patients who responded preferentially to St tended to be younger while those responding to Kp tended to be older. Within the group, there was no association of a particular pathogen reactivity with age, extent of axial involvement, IBD, iritis, HLA-B27 status, disease activity, functional impairment or quality of life. There was no correlation in either A or U groups between cellular proliferation and serology for any of the target antigens. With respect to serology, Kp was the dominant microbial antigen for IgG in both A (66%) and U (82%), however there was no difference between these groups. However, a majority of normal healthy controls (77%) also exhibited a preferential IgG response to Kp. These serological results serve to emphasize the importance of control populations in interpreting antibodies against common pathogens. In summary, the specificities of host immune responses to microbial antigens in AS do not support a role for Kp. St may be a contributor to the etiology of AS in some patients, perhaps as a late sequela to reactive arthritis. Disclosure:


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DIRECT ANALYSIS OF CHLAMYDIA TRACHOMATIS (CT) INFECTED HUMAN PERIPHERAL BLOOD MONOCYTES BY FLOW-CYTOMETRY. Karen Schnitger, Andreas Thiel, Andreas Radbruch, Jens G Kuipers, Henning Zeidler Hannover and Berlin, Germany

A NEW MOUSE MODEL FOR STUDYING THE INTERACTION OF THE LYME DISEASE SPIROCHETE, BORRELIA BURGDORFERI, WITH HUMAN SKIN. Rodica M Van Solingen, Jeffrey S Schechner, Jennifer M McNiff, Linda K Bockenstedt New Haven, CT

Background:Persistence of Ct in the joint is thought to be the major cause of reactive arthritis following urogenital tract infection. The resulting changes of host cell antigen- and cytokineexpression are not precisely understood. It was the aim of the present study to develop a cytometric method for detecting Ct-infected monocytes. This technique could be useful to probe surface antigen changes or changes in the expression of intracellular molecules such as cytokines. Methods: Infectious elementary bodies (EB) of Ct serovar K were labelled by incubation with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). To optimize the staining procedure different CFDA-SE concentrations (5, 10, 20, 50 mmol/l) and staining periods (30 min up to 3 h) were used. Then human mononuclear cells were infected with the labelled EB. After 1, 2 and 3 days the cells were analyzed by flow-cytometry. Results: Staining was most effective when CFDA-SE concentrations of 20mmol/l and a staining duration of 2.5-3h were used. CFDA-SE labelled EB efficiently infected human monocytes. The infected mononuclear cells remained detectable by flow-cytometry for at least 3 days post infection. Conclusions: CFDA-SE allows the labelling of chlamydial EB. CFDA-SE-labelled EB remain infectious. CFDA-SE labelling allows the identification of chlamydial inclusions and atypical persistent Ct in human mononuclear cells by flow-cytometry. Our results demonstrate that labelling EB with CFDA-SE may become a valuable tool for studying the interaction between Ct and the host cell.

Purpose: The Lyme disease spirochete Borrelia burgdorferi first establishes infection in mammalian skin before disseminating hematogenously to cause systemic disease. In this study, we evaluated the utility of a chimeric human skin-severe combined immunodeficient (SCID) mouse model to study in vivo the interaction of B. burgdorferi with human skin components. Methods: Normal adult human skin specimens were obtained from reduction mammoplasty/abdominoplasty procedures, cut into 7 mm2 split thickness segments, and transplanted onto the bilateral flank areas of C.B-17 SCID mice from which 5 mm2 skin segments had been previously excised. Earlier studies (Proc. Natl. Acad. Sci. USA 91, p.9146, 1994) have shown that four weeks after engraftment, the human skin has a dual blood supply derived from well-demarcated anastomoses of the remnant human microvascular arcades with mouse microvessels, and from the ingrowth of new mouse vessels. Mice with mature grafts were inoculated intradermally with 104 B. burgdorferi strain N40 or sham inoculated at a site distant from the human skin grafts. One week after the infection, mice were sacrificed for analysis and the grafted human skin removed. Results: At 1 week of infection, spirochetes had disseminated from the original inoculation site to the blood, urinary bladder and human skin as assessed by culture. Dieterle staining of the grafted skin revealed the presence of spirochetes only in specimens derived from B. burgdorferi-infected but not sham-infected mice. An inflammatory infiltrate consisting of neutrophils and macrophages was visualized by hematoxylin and eosin staining in the human skin grafts, but not in control skin grafts. Summary: We have shown that spirochetes adapting to survival within the mouse will also infect human skin engraftments and incite acute inflammation, an immune response not found in normal murine skin. Conclusion: We propose that the chimeric human skin-SCID mouse model can be used to study the in vivo interaction of mammalian-adapted spirochetes with human skin and dermal blood vessels. This model provides a new avenue to explore the molecular basis of human Lyme disease pathogenesis.

Disclosure: Supported by a grant from the German Kompetenznetz Rheuma and DFG KU1182/1-3.

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INDUCTION OF HOST METALLOPROTEINASES BY BORRELIA BURGDORFERI. Juliane K Franz, Catrin Unsicker, Ute Ungethum, Olaf Fritze, Susanne Priem, Jaspar Neidel, Gerd R Burmester, Andreas Krause Berlin, Germany

DETECTION OF BACTERIAL DNA IN LATIN AMERICAN PATIENTS WITH CHRONIC SPONDYLOARTHROPATHY BY POLYMERASE CHAIN REACTION (PCR) AND SEQUENCING. Raquel S Cuchacovich, Armando Calvo, Luis Vega, Ranju Singh, Miguel Cuchacovich, Abraham Gedalia, Wenqun Huang, Luis R Espinoza New Orleans, LA; Santiago, Santiago, Chile and Lima, Peru

Although Borrelia burgdorferi (B. b.), the causative agent of Lyme borreliosis has been known for many years now, many aspects of the pathogenesis are not fully understood. Thus, it is not clear how the spirochetes can disseminate within the host organism. Therefore, the potential induction of metalloproteinases (MMPs) by B. b. was studied applying a novel three-dimensional in-vitro model of Lyme arthritis. Explant cultures of human synovial tissue were infected with B. b. and co-cultered for up to 72 hrs. The concentrations of collagenase-1 (MMP-1), stromelysin (MMP-3) and of tissue inhibitor of metalloproteinases-1 (TIMP-1) were measured by ELISA. The activities of gelatinase A (MMP-2) and B (MMP-9) were assessed by zymography. Moreover, a semiquantitative RT-PCR was used to determine the induction of MMP-1, MMP-2, MMP-3, MMP-9, and TIMP-1 mRNA. In the infected cultures there was a significant increase of MMP-1 levels compared to the uninfected cultures, whereas no increase of MMP-3 concentrations was observed. Of note, there was a reduction of TIMP-1 levels in the infected cultures. These findings were paralleled by the RT-PCR results. By zymography, a slightly increased activity of MMP-2 and -9 in the infected cultures could be demonstrated. Spirochetes themselves did not produce MMPs or TIMP. B. b. is able to induce proteolytic enzymes of the host. Moreover, the spirochetes reduce the TIMP-1 production, pointing towards an imbalance between the proteases and their inhibitor. This may reflect a mechanism potentially operative in-vivo that enables the microorganism to penetrate host barriers. In addition, the local release of MMPs within the joint may contribute to the pathophysiology of Lyme arthritis like in other inflammatory arthritides such as rheumatoid arthritis. Disclosure:

Spondyloarthropathies and reactive arthritis are relatively rare in Latin American countries. This may be due to the higher prevalence of dysenteric disease and a lower prevalence of HLA-B 27. Objective: The aim of this study was to determine the presence of bacterial DNA in peripheral blood mononuclear cells (PBMC), synovial fluid (SF), and synovial tissue (ST) using the 16 S rRNA-PCR method, and to further identify the species of the bacteria by means of sequencing analysis from a group of patients with chronic spondyloarthropathy. Patients and Methods: 5 synovial fluid (SF), 15 synovial tissue (ST) and 21 PBMC were collected from 33 Chilean and Peruvian patients (mean age 39.6⫾ SD 3.1 years; mean disease duration 7.4 ⫾SD 6.5 years) with ankylosing spondylitis(AS), reactive arthritis(ReA), and undifferentiated spondyloarthropathy(US), and also from 8 age and sex matched healthy controls. All samples were analyzed for the presence of bacterial DNA with the use of a Nested-PCR with universal 16 S rRNA primers. Positive samples were further analyzed with two couples of Salmonella sp primers, and automative sequencing and comparative data analysis (GEN Bank) were performed to identify the species. Routine bacteriologic cultures including for mycoplasma were also performed. Results: Bacterial DNA was detected in the ST of 5/15 (33%) patients including 4 ReA and 1 AS; in 3/5 (60%) SF including 2 ReA and 1 AS; and in 7/21 (29%) PBMC including 4 AS, 1 ReA, and 2 US. Sequence analysis identified multiple species, most of them were Gram negative intestinal flora, although Chlamydia trachomatis was identified in 3 ReA ST. Other species identified included Clostridium sp, Lactobacillus sp, Pseudomonas sp, Salmonella sp, and N. meningitides serogroup B. Conclusion: This study confirms the presence of bacterial DNA in approximately 30% of patients with longstanding spondyloarthropathy. Data presented supports an important role for the GI tract as a portal of entry in the pathogenesis of spondyloarthropathies in some Latin American countries. Disclosure:

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MOLECULAR TYPING OF BORRELIA BURGDORFERI SENSU LATO IN PATIENTS WITH LYME ARTHRITIS IN A HIGHLY ENDEMIC AREA FOR BORRELIA VALAISIANA. Sebastian Schnarr, Andrea Wahl, Tania Bunke, Gabriele Liebisch, Henning Zeidler Hannover and Grossburgwedel, Germany

EVALUATION OF AMPLICOR CHLAMYDIA PCR AND LCX CHLAMYDIA LCR TO DETECT CHLAMYDIA TRACHOMATIS IN SYNOVIAL FLUID. Jens G Kuipers, Jan Andresen, Lars Koehler, Sebastian Schnarr, Nils Putschky, Henning Zeidler, Juergen Wollenhaupt Hannover and Hamburg, Germany

Background: Three genospecies of Borrelia burgdorferi sensu lato (Bbsl) are known to be pathogenic for humans. According to molecular studies B. burgdorferi sensu stricto (Bbss) predominantly causes Lyme arthritis (LA) whereas B. garinii (Bg) is involved in neuroborreliosis and B. afzelii (Ba) in cutaneus manifestations. A fourth genospecies B. valaisiana (Bv) was detected in 58% of Ixodes ricinus ticks in Northern Germany and was recently described in 2 cases of human erythema migrans. The aim of this study was to analyze the pathogenetic significance of Bv in LA and to investigate the genospecies predominantly causing LA in Northern Germany. Methods: As our Bbsl-PCR targeting the OspA- plasmid was unable to detect Bv due to genetic diversity, we developed a Bv-PCR optimized for the detection of Bv in synovial fluid (SF) targeting parts of the 16S-rRNA sequence. SF of patients with LA (n⫽18, all specimens containing Bbsl-DNA according to the OspA-PCR results), undifferentiated oligoarthritis (UOA) (n⫽20) and urine of 20 patients with UOA were tested by Bv-PCR. DNA-purification was done by Proteinase-K-incubation, CTAB-extraction and the Qiaex™ gel extraction kit. Positive PCR results were confirmed by hybridisation. Direct sequencing of the PCR product was performed to determine the Bbslgenospecies. Results: The mean sensitivity for detection of Bv in SF serial dilutions was 1-10 bacteria per PCR. As the PCR was optimized for Bv the sensitivity for other genospecies was tenfold lower. Bbsl-DNA was detected in SF of 8/18 patients (44%) with LA. No Bbsl-DNA was detected in SF or urine of UOA patients. Direct sequencing of the PCR-products did not show any Bv-DNA but revealed Bbss(n⫽3), Ba- (n⫽3) and Bg- (n⫽2) DNA despite the lower sensitivity of the assay for these genospecies. Conclusion: Although Northern Germany is a highly endemic area for Bv we did not find evidence for a pathogenetic role of Bv in LA. Bbss-, Bg-, and Ba- DNA were detected in SF of LA patients indicating that all these genospecies can cause LA. These data are in contrast to the assuption that mainly Bbss is related to LA.

Objectives: By PCR C. trachomatis (CT) DNA has been successfully detected in synovial samples. However, each laboratory has developed in-house PCRs making interlaboratory comparison difficult. To allow for standardization we evaluated two commercially available amplification systems originally designed for the examination of urogenital samples (Roche Amplicor Chlamydia PCR and Abbott LCX Chlamydia LCR) for analysis of spiked synovial fluid (SF) or clinical SF-samples from reactive arthritis (ReA-), undifferentiated arthritis (UA-), and rheumatoid arthritis (RA-) patients and compared the sensitivity for the clinical SF-samples with our in-house developed CT-specific nested-PCR. Methods: SF was spiked with purified CT-elementary bodies (EB) and analyzed by the commercial assays. Clinical SF samples from ReA- (n⫽21), UA- (n⫽79) and RA- (n⫽50) patients were examined by the commercial assays and an in-house PCR. Results: Using SF samples spiked with defined numbers of CT-EB, the sensitivity of the commercial tests was high and similar to published PCR-sensitivity. Also in clinical SF-specimens the commercial assays allowed the detection of CT however the in-house PCR was more sensitive: Out of 10 PCR-positive SF-samples Amplicor tested positive in only 4/10 and LCX in only 3/10. Conclusion: Commercial amplification assays for detection of CT allow detection in clinical specimens however with lower sensitivity than optimized PCR. Disclosure: Supported by grants of the Federal Ministry of education and research (BMBF: 01 VM 9395 and 01 GI 9950). Reagents for commercial amplifications assays were kindly provided by the manufacturers.

Disclosure: This work was supported in part by BMBF grant #01VM9395

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COMPARISON OF RECULTURE AND PCR FOR THE DETECTION OF BORRELIA BURGDORFERI IN CELL AND TISSUE CULTURES AFTER ANTIBIOTIC TREATMENT. Susanne Priem, Torben Klimberg, Juliane Franz, Udo Schneider, Jasper Neidel, Gerd R Burmester, Andreas Krause Berlin, Germany

PARVOVIRUS B19-INDUCED APOPTOSIS IN NONPERMISSIVE CELLS. Brian D Poole, Stanley J Naides Hershey, PA Parvovirus B19 Infection has been implicated in a variety of rheumatologic diseases, including rheumatoid arthritis. B19 productively replicates only in erythroid precursors. However, there is evidence that a nonproductive infection can be established in nonpermissive cells, with production of B19 nonstructural protein, NS1. Homologous nonstructural proteins may induce apoptosis in animal parvovirus infections. We therefore undertook to determine the effects of nonproductive infection of nonpermissive cells. The hepatocellular carcinoma cell line hep-G2 was synchronized with 1 mM hydroxyurea for 16 hours, and then washed with media three times. After washing, the cells were infected with 10, 100, or 1000 viral genomes/cells in the form of diluted viremic serum. As negative controls, cells were untreated or mock infected with non-viremic serum. As a positive control, hep-G2 cells were treated with TNF-␣125 ng/ml, and actinomycin D 5 mM. After six days of infection, cell lysates were analyzed for apoptosis by western blotting. Poly(ADP-ribose) polymerase (PARP) is cleaved by caspase 3 in the late stages of apoptosis and cleavage fragments were probed with a mouse monoclonal antibody, followed by goat anti-mouse IgG conjugated to horseradish peroxidase, and detected by chemiluminescence. Apoptosis was indicated by the presence of a PARP cleavage band at 85 KD. We found 85 KD bands of increasing density at the 100 viral genomes per cell, 1000 viral genomes per cell and positive control lanes, respectively, after six days of infection. No bands were detected at 85 KD in the control or uninfected lanes. Our results indicate that infection of hep-G2 cells by parvovirus B19 induces these cells to undergo apoptosis. These results may explain previously reported B19 involvement in fulminant liver failure. B19 induced apoptosis of nonpermissive cells suggests a possible mechanism for disruption of cellular interactions in other B19 induced diseases, including arthropathy.


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Polymerase chain reaction (PCR) for the specific detection of Borrelia burgdorferi (B.b.) DNA is an important tool in the diagnosis of Lyme borreliosis. Moreover, PCR is increasingly being used to evaluate the efficacy of antibiotic treatment of patients with Lyme borreliosis. However, it is unclear how long PCR may remain positive after successfull therapy and whether a positive PCR indicates a persisting borrelial infection. To determine the significance of a positive PCR after antibiotic treatment and to compare the sensitivity of PCR and culture, we used a novel threedimensional human synovial tissue culture model of Lyme arthritis as well as conventional human synovial fibroblast monolayer cultures. Six cultures each were infected with different B.b. sensu lato strains. After 24 hours ceftriaxone (1␮g/ml) was added and tissues and culture supernatants were examined for at least 6 weeks in regular intervals by B.b. DNA-specific PCR. In parallel, it was tried to re-culture B. b. from supernatants and tissue samples in borrelia-specific BSK-H medium. The results showed that two days after the addition of ceftriaxone in none of the samples live borreliae could be detected by darkfield microscopy. All attemps to reculture B.b. were negative except for one experiment. In contrast, B.b. specific DNA could be detected until four (monolayer) and six (tissue cultures) weeks after antibiotic treatment. These results were reproducible and no difference between the several B. b. strains and species tested could be observed. In conclusion, the experiments show that ceftriaxone is able to readily kill B.b. in tissue cultures. However, PCR can remain positive for several weeks. A positive B. b. DNA PCR after antibiotic treatment therefore does not proof the persistence of live borrelia. These results are of major importance for the interpretation of positive PCR results in the diagnosis and therapy monitoring in patients with Lyme borreliosis.

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PRESENCE AND EXPRESSION OF HUMAN ENDOGENOUS RETROVIRUSES (HERV) IN PATIENTS WITH PSORIATIC ARTHRITIS (PsA). Raquel S Cuchacovich, Miguel Cuchacovich, Ranju Singh, Wenqun Huang, Robert Garry, Marta L Cuellar, Andrea Holluige, Luis R Espinoza New Orleans, LA and Santiago, Santiago, Chile

OSTEARTICULAR MANIFESTATIONS OF A BRUCELLOSIS OUTBREAK IN AN ENDEMIC AREA. Veronica C Perez-Guijo, R Jimenez, C Natera, Jose M Kindelan, Eduardo Collantes Cordoba, Cordoba, Spain


Psoriasis and its related arthritis and immunodysregulation are prevalent in individuals infected with known human retroviruses such as human immunodeficiency virus (HIV-1). In addition, increasing number of reports suggest that expression of human endogenous retroviruses (HERV) and their proteins play an important role in the induction of autoimmune diseases. Objective: The aim of this study was to determine the presence of HERV clones 3-1 and 4-1 DNA and the expression of m RNA HERV clones 3-1 and 4-1, in peripheral blood mononuclear cells (PBMC) from psoriatic patients, and also to compare them with healthy controls. Patients and Methods: 34 Patients with PsA (mean age 50.1 plusminus SD 14.9 years, disease duration skin 14.3 and articular 7.1 years) were recruited from our Rheumatology clinics; all fulfilled Moll and Wright’s criteria for PsA .PBMC were collected from 34 patients with Ps A , and frozen at [-]70 C until tested. The clinical features were: polyarticular 18, oligoarticular 16, axial 9, skin 21 and nails 9. Treatment: Methotrexate 9, Sulphasalazine 2, NSAID 7, no treatment 1. Nine control samples were obtained from age and sex matched normal healthy volunteers. Samples were analyzed for the presence of DNA and RNA with the use of a one step RT-PCR with adequate primers of clones 4-1 gag region and 3-1pol region. To examine the contamination of proviral DNA, samples containing m RNA without RT were also amplified under the same conditions. Results: DNA of clones 3-1 and 4-1 was detected in all Ps A and controls. In contrast, mRNA expression of clones 3-1 and 4-1 was undetectable in all of the Ps A patients regardless of disease activity and treatment received. In the control group m RNA clone 3-1 was expressed in all of them, and m RNA clone 4-1 was expressed in 4/9. Conclusion: Lack of mRNA expression of HERV clones 3-1 and 4-1 in PsA patients may be the result of the underlying immune dysregulation seen in these patients, and may adversely influence the outcome of infections with the same HERV or exogenous retroviruses, or other microorganisms.

OBJECTIVE: To determine prevalence and osteoarticular manifestations in an outbreak of brucellosis in an endemic area. METHODS: The case histories of all patients with active brucellosis (hospitalized and outpatients) during a brucellosis outbreak (December-98 to December-99) in Cordoba (Spain) were reviewed. Diagnosis of brucellosis was established by one of the following criteria: isolation of Brucella spp. in blood or other fluids or tissue samples; or a clinical picture compatible with brucellosis in the presence of raised titers of specific antibodies by seroagglutination (ⱖ1:160) or Rose-Bengal plate agglutination tests. RESULTS: 45 of the 216 patients (47% males and 53% females) diagnosed with brucellosis in this outbreak, needed specialized assistance at hospital. The osteoarticular inflammatory manifestations were evidenced (clinical, Rx and isotopic techniques) in 17 patients (table): CONCLUSIONS: Brucellosis developed mostly in women, sacroiliitis and monoarthritis being the most frequent manifestations in these outbreaks. The prevalence of inflammatory osteoarticular manifestations in a single brucellosis outbreak was different from those found over a longer period of time.

Peripheral arthritis Monoarthritis Oligoarthritis Poliarthritis Sacroilitis Spondylitis

Number of cases hospitalized

Average age ⫾ SD

Gender (m/f)

8 (17.7%) 50.7 ⫾ 10.56/2 4 (8.8%) 3 (6.6%) 1 (2.2%) 8 (17.7%) 1 (2.2%)

50.7 ⫾ 10.5 49.2 ⫾ 14.5 54.6 ⫾ 5.51 45 35.2 ⫾ 22.69 78

6/2 3/1 2/1 1/0 3/5 1/0

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IS ETANERCEPT (ENBREL) EFFECTIVE IN THE TREATMENT OF REACTIVE AND UNDIFFERENTIATED ARTHRITIS? Robert Joseph Meador, Elizabeth C Hsia, Tassanee Kitumnuaypong, H Ralph Schumacher Philadelphia, Philadelphia

LONG TERM TREATMENT OUTCOME OF SALMONELLA SEPTIC ARTHRITIS. Ratanavadee Nanagara, S Suwannaroj, A Mahakkanukrauh, H Ralph Schumacher KhonKaen, Thailand and Philadelphia, PA

PURPOSE: Etanercept, a tumor-necrosis-factor (TNF) inhibitor, has ben shown to be effective in the treatment of rheumatoid arthritis. TNF, a pro-inflammatory cytokine, has been found in synovial fluid of patients with reative and undifferentiated oligo-arthritis, providing rationale that etanercept may be an appropriate therapeutic agent for use in these patients. However, persistence of possibly viable bacteria in joints of patients entering the study was a potential concern. METHODS: An open-label clinical trial assessed the efficacy and safety of etanercept (25 mg twice-weekly subcutaneous injection) in 5 patients with undifferentiated or reactive arthritis. Synovial biopsies were performed on all patients prior to treatment. Polymerase chain reaction (PCR) analysis was performed on all biopsies, and showed nucleic acids of chlamydia trachomatis in 4 patients. Outcome measures included improvements in tender and swollen joint counts, patient and physician global assessment, pain and stiffness on 10-point visual analog scales (VAS), as well as quality of life through the Health Assessment Questionnaire (HAQ). FINDINGS: Before etanercept, 3 patients were taking minocycline, 2 were taking azulfidine, and one was taking methotrexate. All had taken prednisone prior to initiating etanercept. Over a 6 month treatment period, all 5 (100%) patients developed marked improvement. Mean HAQ score declined from 0.55 initially to 0.22 at 3 months and 0.01 at 6 months. Mean tender joint count score declined from 10.3 initially to 1.3 at 3 months and 1 at 6 months; mean swollen joint count score declined from 4.3 initially to 0.33 at 3 months; physician VAS declined from 5.2 initially to 1.8 at 3 months and 1.7 by 6 months. Mean erythrocyte sedimentation rate declined from 41 initially to 26 by 3 months and 1 by 6 months; mean c-reactive protein declined from 7.4 initially to 0 at 3 and 6 months. By 6 months, tender and swollen joint counts were 0 in 2 patients. None of the patients experienced any serious adverse effects, despite frequent evidence of chlamydia on PCR analysis prior to initiating etanercept. CONCLUSION: Etanercept appears to be well tolerated and provides therapeutic benefit in patients with reactive and undifferentiated arthritis, offering a new option for treatment of these conditions.

Salmonella septic arthritis often has a protracted course or relapse despite the fact that antimicrobial agents are effective against the organism in vitro. Our previous study showed effective intracellular killing of this organism in joints at the early phase with either quinolone compounds or cotrimoxazole. The long term treatment outcome with these antibiotics has not been evaluated. Clinical data on 16 patients with 23 episodes of culture proved group D or B Salmonella septic arthritis were reviewed. Long term outcome was evaluated after at least 1 year follow up. All patients had underlying diseases, with SLE the most common. Fifteen patients were on immunosupressive drugs. Acute arthritis in hips or knees was most common. Chronic infection mostly occurred in patients with partial response to the first line antibiotic. Septic polyarthritis was found in 20%. Arthroscopic drainage or arthrotomy was performed in all but one septic knee, treated by repeated arthrocenteses. All patients were initially given at least 2 weeks of parenteral antibiotic. There were no relapses after 31 months mean follow up (1-5 years) in 8 infections which were initially treated with parenteral ciprofloxacin for 2 weeks, followed by oral ofloxacin or ciprofloxacin for another 10 months. Dramatic response at the early phase was also seen in 9 of 10 episodes treated with parenteral cotrimoxazole. Three patients in this group were lost to follow up. In 6 patients treated with cotrimoxazole for 3 months, 4 cases relapsed usually within 4 months after antibiotic was stopped. One patient who was initially treated with cotrimoxazole followed by ofloxacin did not relapse. In the beta-lactam (ampicillin or ceftriaxone) treated group,only 2 out of 5 infections were cured without relapse; progressive joint destruction was found even early in the other 3 cases. Thus, we found better clinical outcome of Salmonella septic arthritis in patients who were treated with a 3 month course of a quinolone. An alternative is parenteral cotrimoxazole followed by an oral quinolone compound for three months. Long term treatment with cotrimoxazole alone frequently resulted in relapse. Treatment with beta-lactam antibiotics yielded the worst outcome.

Disclosure: Etanercept was provided free of charge by Immunex Corporation.

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OSTEOARTICULAR BRUCELLOSIS: ARE WE IN NEED TO REVISE OUR THERAPEUTIC POLICY? Yasser M El Miedany, Maha M El Gafary, Ihab Ahmed, Manal M Baddour Cairo and Alexandria, Egypt

CHANGES IN THE DISTRIBUTION OF ORGANISMS RESPONSIBLE FOR SEPTIC ARTHRITIS OVER A 20 YEAR PERIOD. Jean Jacques Dubost, Martin Soubrier, Anne Tournadre, Jean Louis Bussiere, Bernard Sauvezie

Objective: To Identify risk factors of relapse among patients with osteoarticular brucellosis. Methods: In a prospective cohort study, we investigated 90 patients diagnosed to have brucellosis. Diagnosis of brucellosis was established by clinical picture and at least 4 folds rise in antibody titer. Osteoarticular involvement was defined by inflammatory signs and x ray changes. 21 patients received 2 drugs therapy (rifampicin⫹co-trimoxazole or doxycycline) while 69 patients received 3 drugs therapy (streptomycin⫹rifampicin⫹ doxycycline). Follow up was done monthly both on clinical and laboratory basis (seroagglutination, IgG & IgM antibody titers). Recovery of the patients was considered on showing clinical improvement in addition to reaching seroagglutination antibody titer⬍1:80, as well as negative results for IgG and IgM antibody titers. Incidence of relapse was recorded over 2 years after finishing the course of treatment. Results: None of our patients received treatment for only 6 weeks as previously recommended. Relapse occurred in 59.3% in patients who received treatment for duration less than 5 months, while relapse occurred in 7.9% among those who received treatment for more than 5 months (p⬍0.001). 60% of the patients who received 2 drugs combination therapy had relapse while there was no relapse in patients who received 3 drugs therapy (p⬍0.001). Logistic regression analysis identified IgG as the significant predictor for relapse; the predictivity of the model was 85.56%. Conclusion: 3 drugs combination therapy and extending treatment for longer duration than previously recommended (6 weeks) gave significantly lower incidence of relapse than 2 drugs combination therapy or short courses of treatment. IgG antibody, in addition to, seroagglutination antibody titers are useful for the serological follow up of patients with brucellosis. IgG antibody is the best predictor of relapse.

Objective : To assess changes in the distribution and resistance of the pathogens responsibles for septic arthritis over a 20 year period in patients admitted to the same hospital unit. Patients and methods : Retrospective study of the hospital records of patients admitted between 1979 and 1998 for septic arthritis with positive microbiological diagnosis after blood and/or joint cultures. Results : 303 cases of septic arthritis were studied, 141 in the period 1979-88 and 162 in the period 1989-98. The frequency of occurrence between the two period did not vary significantly for the staphylococci (63% vs 68%), streptococci (20% vs 15%) and Gram-negative bacilli (10% vs 6%). Tuberculous infections decreased from 9% to 4% (p.0.04). No gonococci were isolated in the second 10-year period. Among the staphylococcal species, there was an increase in the number of coagulase-negative staphylococci (10 cases vs 21 p.0.05) between the two periods. There was no significant difference in the frequency of occurrence of methicillin-resistant pathogens (12.6% vs 16.6%). The number of streptococcal B infections increased (2 vs 10 cases) and there was emergence of betalactamin-resistant pneumococci. In the second 10-year period, patients were older and were more likely to have co-existing disease, particularly tumoral growth, and less rarely on dialysis. Localization of joint infection was comparable except an increase in prosthetic knee infections. Conclusion : The distribution and sensitivity of pathogens causing septic arthritis changed little over a 20 year period. Disclosure:

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PAST RADIOTHERAPY AND SEPTIC ARTHRITIS. Jean Jacques Dubost, Valerie Chanet, Martin Soubrier, Bernard Sauvezie Clermont-Ferrand France

A LONGITUDINAL STUDY OF RHEUMATIC COMPLICATIONS IN PATIENTS INFECTED WITH HIV: THE INFLUENCE OF HIGHLY ACTIVE ANTIRETROVIRAL THERAPY. Jazibeh A Qureshi, Elizabeth Sykora, Lee S Lee, Leonard H Calabrese

Aim : To establish the frequency and characteristics of septic arthritis developing in a joint in patients whose past-history included irradiation for adjacent cancer. Method : Retrospective analysis of patients hospitalized over the past 20 years for septic arthritis documented by joint aspiration and/or blood culture (282 cases, 109 in females) exclusion criteria being joint injection and/or rheumatoid arthritis. Results : 10 cases of septic arthritis developed 3 to 34 years (median 16) after radiotherapy whose field had included the affected joint. The patients had had breast cancer (n⫽9) or cancer of the cervix (n⫽1). The location was the shoulder joint (n⫽6, 5 on the right side, among 23 cases of shoulder septic arthritis in the 109 women), the sterno clavicular joint (n⫽3, the only 3 cases of septic arthritis in this joint in the 109 women) and the hip joint (n⫽1, 11 cases in the 109 women). Ten women with radiotherapy induced arthritis (mean age 69), had no other condition promoting infection. Breast cancers were treated by surgery and radiotherapy which had entailed lymphoedema and radiodermitis. S. aureus was identified in 7 cases and streptococcus A, B or Enterococcus in 1 each. Compared to 23 cases of shoulder septic arthritis (both sexes) without past radiotherapy, the first patients above had fever and hyperleukocytosis less often (60% vs 87%, 25 vs 70% respectively) and a longer diagnosis delay. longer antibiotherapy (11 vs 3 months) did not prevent extensive destruction of the humerus head (80% vs 6%) and relapse (40% vs 12%). Conclusion : Radiotherapy promoted the occurrence of septic arthritis years after its end. The diagnosis is difficult, owing to preexisting local changes and mitigated signs and symptoms. Extensive destruction and relapse are not fully accounted for by diagnosis delay. Irreversible radio lesions of bone marrow should be considered. Disclosure:

Objective: The advent of highly active antiretroviral therapy (HAART) in 1995 has dramatically influenced the patterns of HIV related morbidity and mortality. Although the rheumatic disorders have been well documented in patients with HIV infection, the incidence of these disorders under the influence of this therapy has not been reported. The present study evaluates the frequency of rheumatic symptoms in a cohort of HIV patients, assessing risk factors associated with development of rheumatic symptoms and determining whether introduction of HAART influenced temporal trends in development of rheumatic disorders. Methods: Beginning in September of 1989, all patients presenting to HIV clinic were enrolled in the study of potential rheumatic manifestations. Each patient underwent baseline rheumatic evaluation as well as examination, review of systems and indicated laboratory evaluation at a minimum of twice yearly. All patients were examined by the same investigator(LHC). Results: 395 patients have been evaluated. Mean duration of follow up was 53 months (SD⫽47). At last follow up, 123 (34%) of patients were known to be alive, 115(29%) died and 146(37%) have lost to follow up. Cummulatively, 30(8%) have been documented to have noninfectious inflammatory arthritis, of which 8(27%) had reactive arthritis, 3(10%) with psoriatic arthritis, and 19(63%) had various forms of oligoarticular/monoarticular or polyarticular arthritis. In addition 5(1%) have developed AVN. 124 patients have been treated with HAART since 1995. Of those experiencing noninfectious rheumatic complications, 44 of these were identified in patients not exposed to HAART and only 9 were found in patients currently exposed to HAART (p⬍0.026). Conclusions: 1. Non infectious inflammatory rheumatic complications are relatively infrequent but well documented complications of HIV infection. 2. The advent of HAART therapy has resulted in a decreased incidence of certain inflammatory rheumatic disorders in this population. 3. Immune reconstitution secondary to HAART may account for this protective effect. Disclosure:

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COMBINED SPIN-TRAP AND ANTIBIOTIC TREATMENT IS EFFECTIVE AT COMBATING S. aureus INDUCED ARTHRITIS. Egidija Sakiniene, Vincent Collins Gothenburg, Sweden

HIV-ASSOCIATED POLYMYOSITIS: A LONG TERM PROSPECTIVE STUDY. R W Johnson, F M Williams, S Kazi, M Dimachkie, J D Reveille Houston and Dallas, TX

Bacterial arthritis causes rapid joint destruction with permanent loss of function in up to 50% of patients with this disease. Despite early antibiotic treatment to eradicate bacteria joint destruction often ensues as a result of host inflammatory response. The spin trap compound phenyl-N-tertbutylnitrone (PBN) has been shown to have both anti-oxidant and multiple anti-inflammatory effects, e.g., in downregulating the production of proinflammatory cytokine genes, such as TNF-␣, IL-1 and IFN-␥. The aim of the study was to assess the effect of systemic administration of PBN combined with antibiotic treatment on the development of S. aureus arthritis. Materials and methods. NMRI mice were injected i.v. with S. aureus LS-1. Three days after i.p. inoculation of bacteria, daily treatment with cloxacillin and PBN was started. The mice were followed up individually. Arthritis was evaluated clinically and histopathologically. Results. The frequency and severity of arthritis in mice treated with PBN plus antibiotic was lower than that in mice treated with antibiotics alone. The cure rate in the combined treatment group was significantly higher (P⬍0.03) compared to the antibiotic-alone treated animals. Histological evaluation of the joints revealed nearly twice as severe synovitis and cartilage and/or bone destruction in the antibiotic-alone treated group as compared to the mice receiving combined treatment. Conclusions. Systemic administration of PBN in conjunction with antibiotic treatment ameliorated the course of experimental S. aureus arthritis as evidenced by increased cure rate. The outcome indicates a potential approach to the management of bacterial arthritis which simultaneously reduces bacterial load and downregulates pro-inflammatory mediators.

HIV-associated polymyositis, which is clinically and histologically indistinguishable from autoimmune polymyositis, is a relatively rare disorder for which the pathogenesis, clinical course, and appropriate therapy remains unclear. We longitudinally followed for a median of 48 months 13 HIV infected patients with an inflammatory myopathy. Methods: 64 patients attending a county outpatient HIV/AIDS facility were referred to our rheumatology clinic for muscle weakness or elevated creatine kinase (CK). After exclusion for zidovudine therapy, drug/alcohol use, metabolic/ endocrine disorders, or other infections (echovirus, coxsackievirus, HTLV-1, and Toxoplasma gondii), remaining patients underwent electromyography (EMG) and muscle biopsy. Patients were prospectively followed and those who were symptomatic received immunosuppressive therapy. Results: 13 patients (20.3 %) had biopsy proven myositis. At the time of diagnosis, 6 patients (46%) had AIDS (per CDC criteria). The median duration of HIV infection prior to diagnosis of myositis was 4.3 years (⫾ 3.4 years). Whereas progressive muscle weakness was present in 70% of the patients, 4 patients had only elevated CK and myalgias or sicca symptoms. 6 patients (46%) had concomitant diffuse infiltrative lymphocytosis (DILS). The median CK was 2484 IU/L (⫾ 1787 IU/L) without a significant correlation to severity of weakness. The mean CD4 was 381/mm3 (⫾ 263/mm3) and the mean CD8 was 1329/mm3 (⫾ 791/mm3). The stage of HIV infection did not correlate with muscle strength or CK. Only 61% of patients had a characteristic EMG. 9 patients received prednisone (CS) at 60 mg/day. 5 patients attained complete resolution of myositis and were able to discontinue CS in a mean duration of 9 months (⫾ 3 months). 3 patients who had partial response to CS received IVIG but without benefit. 4 patients (30.7%) had spontaneous resolution of their myositis. No patient was disabled as a consequence of the polymyositis and the mean survival time after diagnosis was 48 months (⫾ 25 months). Conclusion: In this longitudinal study, HIV-associated polymyositis appears to be a milder variant of autoimmune polymyositis. It occurs at any stage of HIV infection with no correlation to CD4 or CD8 counts and may occur in the presence of DILS. HIV-associated polymyositis responds well to CS, does not beget disability, and appears to have no adverse outcome on the course of the HIV infection.

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SOFT TISSUE RHEUMATIC LESIONS AND HIV INFECTION IN ZAMBIANS. Panganani D Njobvu, Paul E McGill Lusaka, Zambia

LOCAL AND SYSTEMIC REACTIONS AFTER INTRAVESICAL BACILLUS CALMETTE-GUERIN (BCG) IMMUNOTHERAPY FOR BLADDER CANCER: THE FRENCH EXPERIENCE. Hughes Blangy, Damien Loeuille, Jean Yves Jouzeau, Charles Benoit Ghiringhelli, Henri Debois, Patrick Netter, Pierre Gillet Nancy and Lyon, France Aim & Methods. Intravesical therapy of BCG is effective in the treatment of superficial bladder cancer. This beneficial effect is unfortunately counterbalanced by rare detrimental ancillary immunological or infectious side-effects. In France, BCG (Immucyst, Connaught strain) is marketed since 1996, and, to date, 22 600 patients underwent BCG therapy for superficial bladder cancer. We have undertaken a 3-years descriptive retrospective study for the reported side effects, to depict hypothetical risk factors. Results. 97 serious ADRs were recorded as local (12), regional (12) or systemic (73) reactions. Loco-regional toxicity consisted in febrile cystitis, contracted bladder, epidymitis, prostatitis, renal abscess or peritonitis. Systemic reactions, infectious (46) or not (27), were reported: high-grade fever, acute renal failure, pulmonary miliary, granulomatous hepatitis, osteomyelitis, and sepsis. Dysimmune reactions were identified as 18 reactive arthritides, 5 patients being HLA B27⫹. Risks factors in treatment schedule may result in systemic BCG absorption: too recent or traumatic catheterization, concurrent cystitis, immunodeficiency, previous pelvic radiotherapy. Intravascular devices or prosthesis may facilitate septicopyohemia. Conclusions. Occurrence of such rare ADR (0.35%) has conducted Aventis Pasteur to revise the Summary of Products Characteristics of Immucyst and to inform the precribers. Physicians have now to distinguish and prevent normal therapy symptoms (in prescribing oral hyper hydratation after instillations), from adverse effects, especially with respect to previous instillation tolerance, delay after biopsy, and sterile urine status. Suspected severe infections complications may lead to mono or combined antituberculous therapy. Disclosure:

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In everyday clinical practice in much of subSaharan Africa the HIV test is not available.There is a great need therefore for simple clinical and/or laboratory algorithms to identify patients with a high liklihood of HIV infection.This is particularly so in patients in the early stages of HIV infection who have no clinical sign of the disease. We have previously described the association of spondyloarthopathy and HIV infection in Zambians.We have also observed patients with enthesitis or sacroiliitis who are HIV⫹.However the link with specific soft tissue syndromes remains unclear in the subSaharan HIV pandemic. METHODS.Prospective 2 yr study of all patients ⬎17 yr attending a rheumatic clinic in a large teaching hospital setting.Routine bloods in all and Xray when indicated.HIV by ELISA and clinical staging by WHO criteria.Patients with isolated sacroiliac pain, enthesitis or other soft tissue lesion selected for analysis.For HIV positive patients,only those in WHO clinical stage 1(asymptomatic or persistent generalised lymphadenopathy) were selected.Patients with septic soft tissue lesions and inflammatory myositis were excluded. RESULTS.120(41m,79f;age 23-75 yr)satisfied inclusion criteria.Diagnosis and number(%HIV⫹)as follows:Sacroiliitis 14(100%):heel pain,14(100%):costochondritis 3(100%):Polyenthesitis(⬎4 sites),20(100%):carpal tunnel syndrome,8(63%):rotator cuff syndrome,18(30%): tendonitis(insertional)8(25%):,sciatica/cervical spondylosis,12(16%):sacroiliac strain,7(0%):DeQuervains tenosynovitis,16(0%):Overall HIV seroprevalence 54%: 74%in those under 45 and 17% over 45 yr.Population prevalence in Lusaka ⬃30% in 30-40yr age range.Within each subgroup the mean ESR was significantly higher in HIV⫹ patients. CONCLUSION:A young age and a raised ESR are both good indicators of HIV infection in Zambian patients with soft tissue rheumatic disorders.Enthesitis is a distinct HIV related phenomenon which may be an early form or a forme fruste of HIV related spondyloarthropathic disease.

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MYCOPLASMAE PNEUMONIAE(MP) REACTIVE ARTHRITIS IN CHILDREN. Donald P Goldsmith, Carrie A Edelman Philadelphia, PA

OVERLAP OF THE DIFFERENT ETIOLOGIC FACTORS ASSOCIATED WITH CRYOGLOBULINEMIA: ANALYSIS OF A SERIES OF 443 PATIENTS. Mario Garcia-Carrasco, Olga Trejo, Manuel RamosCasals, Jordi Yagu ¨ e, Francisco Lopez, Alfonso Garcia-Carrasco, Ricard Cervera, Josep Font, Miguel Ingelmo Barcelona, Spain and Puebla, Mexico

Transient polyarthralgias coincide with acute MP infection in up to 20% of patients. As many as 2%, however, develop frank arthritis most often thought to be reactive. We report four children with self limited arthritis without other apparent cause and with serologic evidence of recent MP infection. (Table) These chldren presented during late fall and winter (11/99 to 3/00) with the relatively acute onset of synovitis and a clinical pattern resembling reactive arthritis. None had preceeding gastrointestinal or genitourinary symptoms, mucosal involvement, or skin changes. The ESR was elevated in 2 of 4 (pt# 2,3) and the peripheral WBC increased in 1 of 4 (pt#2). There was no evidence of recent infection with streptococcus, parvovirus, or Borrelia burgdorferi. Enzyme-linked immunosorbent assays (ELISA) for MP showed elevated IgM in all four children, indicating recent exposure. MP infection is very common in younger individuals, with up to 97% of 17 year olds having serologic evidence of exposure. Culture techniques remain difficult, taking as long as ten days, and are not usually offered in a routine diagnostic microbiology laboratory. The ELISA assay is a highly specific, sensitive, and rapid method to confirm recent MP infection. Only one of these children experienced significant respiratory symptoms. This is consistant with previous reports that 20 to 40% of patients with extra-respiratory manifestations of MP infection have not had a recognizable respiratory illness. The duration and pattern of arthritis in these children did not suggest a diagnosis of juvenile rheumatoid arthritis (JRA), but for many years the role of various mycoplasmas in the pathogenesis of chronic arthritis has been questioned. In Manatoba, Canada, a significant correlation was found between the incidence of JRA and the # of MP infections between 1985 and 1992 (J Rheumatol 1995;22:745). Even without respiratory symptoms, if children present with a clinical pattern suggesting reactive arthritis, particularly in the fall and winter, MP should be considered as a possible etiology. Age onset (yrs)/gender

7/M

8/M

12/F

3/F

Affected Joints Duration of arthritis (wks) Assoc Resp Symptoms Syn Fluid WBC/% Polys HLA B-27

ankle, knees 8 n nd neg

knee 6 n 13,500/90 neg

elbows, wrists, knees, ankles 6 y nd nd

hip 2 n 58,000/75 neg

OBJECTIVE. To study the main etiological factors associated with cryoglobulinemia in a universitary hospital, to determine the percentage of patients with no evidence of associated disease (“essential” cryoglobulinemia) and to analyze the amount of overlapping between the different etiologies. PATIENTS. We analyzed 443 consecutive patients with cryoglobulinemia, who tested positive for circulating cryoglobulins in our Department of Immunology between 1991 and 1999. Of these 443 patients, 258 (58%) were women and 185 (42%) men (mean age 54 years, range 14 to 91). RESULTS. Infectious diseases were detected in 331 (75%) patients, autoimmune diseases in 94 (24%), hematological disease in 33 (7%) and essential cryoglobulinemia in 49 (11%). Patients with autoimmune diseases showed a cryocrit higher than 5% (26%) more frequently compared with those with other etiologies (10%, p⫽0.003). HCV infection was found in 321 (73%) patients, HBsAg in 15 (3%) and HIV antibodies in 29 (7%). Forty (9%) patients had Sjogren’s syndrome (SS), 30 (7%) systemic lupus erythematosus (SLE), 7 (2%) periarteritis nodosa and 6 (2%) progressive systemic sclerosis. Finally, 16 (4%) patients had a NHL, 3 chronic lymphocytic leukemia, 3 multiple myeloma and 2 Hodgkin’s lymphoma. When the overlapping of more than one etiological factor was analyzed, we found that most patients with HBsAg or HIV infection were also HCV positive. In addition, we observed a strong overlap between HCV and some autoimmune (50% in SS, 67% in PAN) or hematological (50% of NHL) diseases. CONCLUSION. HCV infection was the main etiological factor identified, being present in 73% of patients with cryoglobulinemia. Of the systemic autoimmune diseases, SS and SLE were those more frequently associated to cryoglobulinemia, and NHL was the most frequent hematological process found. No associated disease was found in 11% of patients (essential cryoglobulinemia). Finally, we report a strong overlap between certain etiological factors (HCV, SS and NHL).

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CLONAL B-CELL POPULATIONS IN THE BLOOD AND LIVER OF PATIENTS CHRONICALLY INFECTED BY THE HEPATITIS C VIRUS. Laurent Vallat, Pascale Ghillani, Yves Benhamou, Vincent Thibault, Pierre Hausfater, Yvan Sterkers, Jean Charles Piette, Thierry Poynard, Helene Merle-Beral, Frederic Davi, Patrice Cacoub Paris, France

NATURAL HISTORY AND TREATMENT IMPACT ON FATIGUE IN PATIENTS WITH CHRONIC HEPATITIS C: A FOLLOW UP IN 425 PATIENTS. Patrice Cacoub, Vlad Ratziu, Pascale Ghillani, Vincent Thibault, Frederic Charlotte, Jean Charles Piette, Joseph Moussali, Anne Mercadier, Thierry Poynard Paris, France

Numerous studies have suggested a possible role of the hepatitis C virus (HCV) in the development of B-cell lymphoproliferations. If the association of HCV with type II mixed cryoglobulinemia (CG) is well established, there is some discrepancy concerning its presence in B-cell lymphoma. High prevalences of HCV infection (up to 40%) have been reported in italian series, but have not been observed in other american and european studies, including two large french surveys. Aim: To determine the frequency of circulating and liver infiltrating monoclonal B-cells in patients infected with HCV. Patients and methods: 108 HCV-positive patients,(mean age 51 yrs), were prospectively studied. They had not received anti-HCV therapy for at least 6 months. CG were isolated and characterized by immunoblotting at 37°C (positive if ⫽ .05 g/l at least on 2 determinations). Clonality of B-cells was determined by DNA amplification of the heavy chain immunoglobulin gene (IgH) rearrangements followed by polyacrylamide gel electrophoresis (sensititivity⫽5%). Results: A clonal B-cell population was detected in the blood of 20/108 (19%) patients. Among the 99 patients analyzed for the presence of CG, 34 were positive and 9/34 (26%) had a circulating clonal B-cell population. A clonal B-cell population was found in the blood of 9/22 (41%) patients with type II CG, but also in 8/65 (12%) patients without detectable CG. No clone was observed in the 12 patients with type III CG. For 69 patients, a liver biopsy was similarly analyzed: a clonal IgH rearrangement was detected in 12/14 (86%) cases having a clonal circulating population, and in 7/55 (13%) cases with polyclonal circulating B cells. Three of the 20 patients with a circulating clonal B-cell population developped a definite B-cell malignancy (Waldenstrom’s macroglobulinemia ⫽ 2, marginal zone lymphoma ⫽ 1). Conclusion Clonal B lymphocytes can frequently be detected in the blood and the liver of patients infected with HCV, in the absence of overt B-cell malignancy. These clones are usually, but not always, associated with the presence of type II CG. In rare patients, these clonal populations may evolve into a true B-cell neoplasia. Disclosure:

Fatigue has never been studied prospectively in a large cohort of patients (pts) infected with HCV before, with, without and after treatment. Aim: to assess the prevalence and severity of fatigue in a large data base of pts with chronic hepatitis C, before, with, without and after treatment. Methods: 1614 consecutive pts of a one-center prospective cohort were investigated before any treatment. Fatigue has been graded in 3 classes: none, moderate, and impairing activity. A questionnaire including 128 items was filled out every 6 months. Inclusion criteria were pts followed for at least 18 months with 4 visits. Analyses used variance analysis and logistic regression. Results: 425 pts were included (not different than the non included) 56 % men, mean age 49⫾13 years, 28 % extensive fibrosis or cirrhosis, 46% with cryoglobulin. 72 were not treated, 82 were sustained responders, 47 relapsers and 224 non responders. A total of 253 pts (60 %) complained of fatigue including 84 pts with impaired activity (20 %).The prevalence of fatigue has been analysed at baseline and at 18 month follow-up among groups. The percentage of pts without fatigue at 18 month was lower in responders (69%) vs all the other groups (42% in non treated, 40% in relapsers, 46% in non responders)(p⬍0.01). Among sustained responders, patients with (moderate or severe fatigue) at 18 month (n⫽26) compared to those without fatigue (n⫽56) were older 50⫾14 vs 47⫾13 years (p⫽0.05), more often female (62 vs 36%, p⫽0.03), less often i.v. drug abusers (4 vs 23%, p⫽0.03); only one patient was depressed. There was no difference for cryoglobulin, alcohol intake, extensive fibrosis and body mass index. Conclusions: In patients with chronic hepatitis C, fatigue is improved among sustained responders in comparison to controls and non responders. Despite undetectable HCV-RNA, one third of sustained responders still complained of fatigue.


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NATURAL HISTORY AND TREATMENT IMPACT ON EXTRAHEPATIC MANIFESTATIONS IN PATIENTS WITH CHRONIC HEPATITIS C: A FOLLOW-UP IN 425 PATIENTS. Patrice Cacoub, Vlad Ratziu, Nathalie Costedoat, Pascale Ghillani, Frederic Charlotte, Vincent Thibault, Jean Charles Piette, Anne Mercadier, Thierry Poynard Paris, France

ANIONIC ANTIPHOSPHOLIPID ANTIBODIES IN HEPATITIS C INFECTION PATIENTS: Adriana Molano, Francisco Medina, Luis Felipe Flores, Javier Caviedes, Margarita Dehesa, Alberto Juarez, Antonio Fraga Mexico, DF, Mexico

Extra-hepatic manifestations (EHM) are frequent in chronic HCV infection but have never been studied prospectively in a large cohort of patients (pts) before and after treatment. Aim: To assess the prevalence of EHM in a large data base of HCV pts before, with, without and after treatment. Methods: 1614 consecutive HCV positive pts of a one-center prospective cohort were investigated before any anti-HCV treatment. A questionnaire including 128 items was filled out every 6 months. Pts followed for at least 18 months with 4 visits were included. Main clinical (arthralgia, myalgia, paresthesia, sicca syndrome) and biological (cryoglobulin) EHM were studied. Fibromyalgia was defined as the presence of fatigue and myalgia⫾arthralgia. Analyses used variance analysis and logistic regressions. Results: 425 pts were included (not different than the non included) 56 % men, 49⫾13 yrs, 28 % with extensive fibrosis or cirrhosis, 46% with cryoglobulin. 72 pts were not treated, 82 sustained responders (persistent negative viremia after stopping HCV treatment), 47 relapsers (negative viremia during HCV treatment, but positive viremia in the following months), 224 non responders (positive viremia during treatment). In non treated and in relapsers, the presence of at least one of clinical EHM noted at baseline in 43% and 23% pts, and cryoglobulin in 44% and 59%, respectively, did not change at 18 month. Among sustained responders, there was a significant decrease of the prevalence of cryoglobulin (50 vs 6%, p⬍0.001) and fibromyalgia (29 vs 13%, p⫽0.02). In sustained responders, patients with compared to those without fibromyalgia at 18 month were more often female (22 vs 7%, p⫽0.04) and older 55⫾12 vs 44⫾12 yrs (p⫽0.01). There was no difference for alcohol intake (⬎50 g/d), extensive liver fibrosis, body mass index and depression. In non responders, although HCV viremia remained positive, there was a decrease of the prevalence of cryoglobulin (58 vs 33%, p⫽0.01) and fibromyalgia (30 vs 15%, p⬍0.001), whereas the presence of at least one clinical EHM did not change (33 vs 29%). Conclusions: Cryoglobulin and fibromyalgia improved in all treated patients. In sustained responders, it remained a cryoglobulin in 6 %, a fibromyalgia in 13% and one extra-hepatic manifestation (arthralgia, myalgia, paresthesia, sicca syndrome) in 24 %.

Anticardiolipin (aCL) and anti-␤2-glycoprotein I (anti-␤2- GPI) antibodies have been reported in up to 60% of patients with chronic hepatitis C infection. However, contrary to other chronic infections (HIV, syphilis), these patients present manifestations of antiphospholipid syndrome (APS, thrombotic phenomena, abortion).OBJECTIVE: To study the frequence of antibodies to phospholipids: antiphosphatidilserine (aPS), antiphosphatydil-inositol (aPI), antiphosphatydilethanolamine (aPE), anti-phosphatydil-choline (aPC), anti-sphingomyelin (aSM), aCL and anti-␤2GPI in patients with chronic hepatitis C virus (CHCV) infection, as well as its correlation with APS.METHODS: Consecutive CHCV patients documented by RIBA II and/or histopathology. We applied a structured questionnaire and physical examination to identify APS manifestations. Fasting blood sample was drawn at 8:00 AM to determinate hepatitis B (HBsAg), HIV (ELISA), antinuclear antibodies (ANA, HEp-2 cells), aPS, aPI, aPE, aPC, aSM, aCL and anti-␤2-GPI (ELISA).RESULTS: We studied 60 patients, 35 females and 25 males, with a mean age of 46.2 ⫾11.63 years (25-73), 35 treated with interferon ␣2b (IFN␣2b). Five patients had recurrent abortions, five cases presented thrombotic episodes (4 deep venous thrombosis and pulmonary thromboembolia) and four had myocardial infarction. A false-positive VDRL was found in 33/60 (55%). Thrombocytopenia was found in 17/48 (35%), the lupus anticoagulant in 6/48 (12%), ANA in 38/60 (63%), aPS-IgG 30/60 (50%) aPS-IgM 12/60 (20%), aPI-IgG 2/60 (3%), aPI-IgM 23/60 (38%), aPE-IgG 9/60 (15%), aPE-IgM 16/60 (26%), aPC-IgG 1/60 (1.6%), aPC-IgM 23/60 (38%), aSM-IgG 2/60 (3%), aSM-IgM 26/60 (43%), anti-␤2-GPI-IgG in 7/60 (12%) Anti-␤2-GPI-IgM in 6/60 (10%), aCL antibodies were positive in 11/60 (18%), IgG in 7, IgM in 4. Spearman’s correlation showed correlation between thrombosis and aCL IgG (p⫽0.003) and IgM (p⫽0.0001). There were no differences between antibody levels and IFN␣2b treated and not treated patients.CONCLUSIONS: The present study suggests a correlation between antiphospholipid antibodies and thrombosis in hepatitis C patients (not observed in other infectious diseases). We also found a high prevalence of anionic phospholipid antibodies that may explain these thrombotic phenomena. Further cohort studies are needed to confirm these findings.

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ENTEROVIRUS IN CHRONIC FATIGUE SYNDROME (CFS). N-T Jou, J K-S Chia, J S Louie, M R Liebling Torrance, CA

INCREASED FREQUENCY OF THE TNFA-308A ALLELE IN POLY-AND DERMATOMYOSITIS. Ingrid E Lundberg, Adla B Hassan, Leonid Padyukov Stockholm, Sweden

Ongoing infection with enterovirus has been proposed as one etiology for CFS. The role for enterovirus in this syndrome has been controversial with both positive and negative results reported. Extrapolating from the chronic enteroviral model in mice, we hypothesized that evidence for ongoing infection in humans might be found by exploring the possibility of a mononuclear cell reservoir. To examine this hypothesis, we developed a powerful single-tube, nested, RT-PCR using primers directed at the highly conserved 5’ untranslated region of the enterovirus genome. Peripheral blood mononuclear cells (PBMC) and plasma were simultaneously collected from patients who fulfilled CDC criteria for CFS following a flu-like illness but tested negative for serologic evidence of CFS-associated pathogens (other than enterovirus). Controls included healthy individuals without a history of fatigue or recent respiratory or gastrointestinal symptoms, patients with CFS who were positive for antibodies to C. pneumoniae but negative for enteroviral antibodies, and patients with fatigue accompanying SLE. Samples were blinded by coding prior to RT-PCR. On a molecular level the assay was found to be quite sensitive, detecting as little as 5.79 attomoles of coxsackie B3 RNA. At the time of submission, 26 CFS patients with symptoms for 8 months to 20 years and 12 controls were enrolled in the clinical portion of the study. The sensitivity and specificity of the assay using RNA extracted from PBMC was 70 and 100%, respectively, while the sensitivity and specificity employing plasma as a source of RNA was 30 and 75%, respectively. No patient whose PBMC were negative was found to have a positive plasma sample. Three of the samples with positive amplification products were sequenced and showed a minimum of 98.5% sequence identity with the same region of coxsackievirus B3. Intriguingly, of 11 patients with multiple samples, 6 converted to negative, some while taking antiviral therapy. Correlation with clinical status is not yet available. No patient has as yet converted from negative to positive. These preliminary data suggest that an ongoing enteroviral infection may exist in some patients with CFS, that mononuclear cells may serve as one reservoir for this infection and that a sensitive RT-PCR assay is capable of detecting enteroviral RNA in a significant proportion of these patients. Disclosure: Supported by the John Tu Rheumatology Research Fund and personal funds.

Aim Polymorphisms of the regulatory regions of cytokine genes have been considered as potential markers for disease susceptibility. Some of these polymorphisms are proven to have a functional role such as the polymorphism within the tumor necrosis factor gene, -308TNFA. Substitution of G to A in the -308TNFA position is associated with high TNF-alpha production. This could be an important mechanism in the pathogenesis of polymyositis (PM) and dermatomyositis (DM) in which increased expression of the proinflammatory cytokines such as TNF-alpha and interleukin IL-1 has been observed in muscle tissue. The aim of our study was to investigate whether -308 TNFA gene polymorphism associates with PM and DM as well as with certain clinical and immunological parameters in these disorders. Patients and Methods 63 patients with probable or definite PM or DM according to the Bohan and Peter criteria were genotyped for -308 TNFA alleles and compared with a control group from the same population. Genotyping was performed by gene specific PCR with restriction endonuclease mapping. Results The frequency of the TNFA-308A allele was significantly increased in myositis patients compared to the controls with 35% of the -308A allele and 65% of the -308G allele in the myositis patients compared to a frequency of 19% of the -308A allele and 81% of the -308G allele in the controls (p⬍0.01). There were 5 patients with homozygous TNF -308A/A (8%), 34 with heterozygous -308A/G (54%) and 24 with homozygous -308G/G (38%). In the control group 3% of the 308A/A, 17% of the -308A/G and 80% of the -308G/G were found. There was no difference between the DM and PM groups in allele or genotype frequencies. Conclusion The TNFA-308A allele was significantly more frequent in poly- and dermatomyositis patients compared to the controls. This finding may help to explain mechanisms by which TNF production is increased in myositis patients. Disclosure:

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DETECTION OF TROPHERYMA WHIPPELII IN UNEXPLAINED ISOLATED CASES OF ARTHRITIS. Xavier Puechal, Thierry Schaeverbeke, Jean Sibilia, Alain Saraux, Jean Domininique Poveda, Reseau Rhumato

INTERSTITIAL LUNG DISEASE IN INFLAMMATORY MYOPATHIES: INFLUENCE ON CLINICAL PATTERN AND MORTALITY. Carmen Torres, Francisco J Gomez-Reino, Juan C Bermell, Agustin Gomez-Camara, Ana Cabello, Paloma Ferrando, Patricia E Carreira Madrid, Spain

Whipple disease usually affects men and is usually preceded by arthritis for years. It has been hypothesized that PCR testing for Tropheryma whippelii will make it possible to detect the disease at an earlier stage, thereby being able to prevent the development of severe and sometimes fatal systemic forms. Objective. The present study was undertaken to investigate the presence of T. whippelii in synovial samples from male patients with unexplained arthritis. Patients and Methods. Forty five male patients with seronegative, isolated, undifferentiated oligo- or polyarthritis were evaluated in a cross-sectional PCR study. Fifty six frozen synovial fluid and/or tissue were tested by PCR for T. whippelii DNA as previously described. Results. The mean mean age of the patients was 45 years (range 14 to 81 years) with a median duration of articular symptoms of 36 months (range 2 to 348 months). Thirty two patients had undifferentiated oligoarthritis, 9 had unexplained seronegative polyarthritis and 4 had chronic unexplained monoarthritis. Twenty patients had a relapsing course of arthritis. PCR detected T. whippelii DNA on three different runs in only one of the 56 specimens studied, this was from a patient with no other evidence of Whipple disease during the 30-month follow-up period. Conclusion. Whipple disease could not be diagnosed in a series of male patients with isolated unexplained seronegative oligo- or polyarthritis by the PCR test on joint fluid or tissue specimens. T. whippelii does not seem to be frequently involved in unexplained cases of arthritis and PCR assay on joint fluid or tissue specimens does not seem to be helpful in this setting. However, T. whippelii may be very occasionally detected in joint samples in the absence of Whipple disease.

Objective: To analyze the influence of interstitial lung disease (ILD) in clinical pattern, response to treatment and mortality of patients diagnosed of inflammatory myopathy (IM). Methods: A cross sectional study based on a consecutive sample of patients with IM (Bohan and Peter criteria), seen in Rheumatology in a University Hospital between 1980 and 2000. Demographical data (age at initial symptoms and diagnosis, gender, death), clinical data (Raynaud, articular, cardiac and GI involvement, ILD), serological features (ANA, antisyntethase antibodies), treatments received and response to treatment were obtained from the charts. ILD was defined as respiratory insufficiency with diffuse interstitial infiltrate in chest X-ray or CT scan, in absence of infection/cardiac failure. For statistics, univariate and multivariate analysis with logistic regression were performed, using Odds ratio with 95% CI to measure the strength of association between variables. Results: We found 68 IM patients (29M, 48F) with 35 (2-70) years at initial symptoms and 6,6 (1,5-19) years of follow up. Forty-two (62%) had skin involvement. ILD was present in 19 (28%). Twelve patients, 7 with ILD, died. In univariate analysis ILD was less frequent in patients with disease onset below 18 years (OR⫽0,09; 95%CI 0,01-0,69; p⫽0,007) and was associated with Raynaud (OR⫽5,3; 95%CI 1,7-16,9; P⫽0,027), arthritis (OR⫽4,1; 95%CI 1,2-14,4; p⫽0,027), cardiac involvement (OR⫽13,2; 95%CI 3,1-57,8; p⬍0,0001) and antisyntethase antibodies (OR⫽13,2; 95%CI 1,4-123,3; p⫽0,013). ILD patients needed more azatioprine to control disease symptoms (OR⫽4,9; 95%CI 1,5-15,9; p⫽0,013), tended to have poorer muscular response to treatment (OR⫽0,2; 95%CI 0,05-0,9; p⫽0,047) and had higher mortality (OR⫽5,6; 95%CI 1,4-21,9; p⫽0,025). Multivariate analysis confirmed the association between ILD and age at onset (OR⫽1,6; 95%CI 1,07-2,3; p⫽0,019, for every 10 years stratum), cardiac involvement (OR⫽30,6; 95% CI 3-317,9; p⫽0,004) and arthritis (OR⫽19; 95%CI 2-184,2; p⫽0,01). Mortality of IM patients was associated with cardiac involvement (OR⫽14,4; 95%CI 3,1-66,9; p⫽0,0007). Conclusions: In inflammatory myopathies, ILD frequency increases with age at disease onset, and is associated with articular and cardiac involvement, and higher mortality. Mortality of IM patients is also highly associated with cardiac involvement.

Disclosure: This work was supported by Pharmacia Corporation, Guyancourt, France. Pharmacia has not been involved in any part of the study including conception, design, analysis, logistic and publication.

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ASSOCIATION OF A PROMOTER POLYMORPHISM OF TUMOR NECROSIS FACTOR-␣ (TNF␣) WITH ADULT DERMATOMYOSITIS. Victoria P Werth, Jeffrey P Callen, Kathleen Sullivan Philadelphia, PA; Louisville, KY

ANALYSIS OF RELAPSE RATE OF INTERSTITIAL PNEUMONIA IN PATIENTS WITH POLYMYOSITIS/DERMATOMYOSITIS . Masaomi Yamasaki, Hidehiro Yamada, Hiroshi Niimi, Yasuyuki Kurihara, Shoichi Ozaki Kawasaki, Kanagawa and Kawasaki, Kanagawa, Japan

We recently reported that the -308A TNF promoter polymorphism is associated with SCLE and mediates an exaggerated response to ultraviolet B (Werth et al., J Invest Dermatol 115:726-730, 2000). We now hypothesized that this polymorphism may also be important in adult dermatomyositis (DM), a photosensitive disorder with some features in common with SCLE. 37 patients with DM and 316 race-matched controls were examined for the -308A TNF polymorphism and the more common 308G allele. The frequency of the -308A allele was 0.27 in the entire DM group, vs. 0.14 for all controls (p⫽0.005, ␹2 2x2 table). Caucasians were the only racial/ethnic group in our study large enough to allow separate statistical analysis (33 DM, 205 controls). The frequency of the -308A allele was 0.25 for DM and 0.15 for the controls (p⫽0.05). Caucasians are known to exhibit a linkage dysequilibrium between -308A and HLA-DR3. We previously reported that 53% of controls but 100% of SCLE patients with at least one -308A allele also carry the DR3 allele (J Invest Dermatol 115:726-730, 2000). In contrast, we found no increase in the association of -308A and HLA-DR3 in DM over control (p⫽0.762 by Fisher’s exact test), and the association of these two genes in DM was significantly less than we reported for SCLE (p⫽0.009). The three homozygous -308A patients with DM had exceptionally severe skin disease in a photodistribution that continued after resolution of myositis, requiring continued combination antimalarials and immunosuppressive therapy. We conclude that the TNF-308A polymorphism is associated with DM, which suggests a pathophysiologic contribution from UV-induced TNF␣. The difference in HLA-DR3 linkage and several divergent clinical features suggest a specific mechanistic difference between DM and SCLE.

Background: Interstitial pneumonia (IP) is one of the major causes of death in patients with PM/DM. Despite the improvement of IP after the initial treatment with high dose steroid, some patients show a relapse of IP and require a further aggressive therapy. Objective: To evaluate the relapse rate of IP in patients with PM/DM and to identify the factors that contribute to the relapse of IP. Methods: Thirty-three patients with PM/DM-associated IP who were treated at our institute during 1994-2000 were retrospectively analyzed after collecting clinical information from medical records. Results: Nine patients who did not respond to the initial treatment and died within 3 months were excluded from further analysis. Among the rest of 24 cases (PM7/DM15), 15 patients were initially treated with high dose steroid alone and 9 patients were treated with immunosuppressant plus steroid at the initial presentation. Fourteen out of the 24 cases showed a relapse of IP after 14.6 ⫹/- 13.9months of the initial treatment. The other 10 patients did not show a relapse during a mean follow up period of 31.3 months. Kaplan-Meier life table analysis showed that the relapse rate at 2 years after the initial therapy was 51.5%. Log-rank test indicated that patients treated with immunosuppressant had a significantly lower relapse rate than those treated with steroid alone (p⬍0.005). There was no statistically significant difference of the clinical parameters at the initial presentation between 2 groups. There were no other factors that influenced significantly on the relapse rate among tested including age, sex, PM/DM, severity of IP, dose of steroid and the presence of anti-Jo-1 antibody. Multivariate logistic regression analysis revealed that the mode of the initial treatment alone had an independent contribution to the relapse of IP (odds ratio of the treatment with steroid alone 28.1(95%CI; 1.76-448.6)). Conclusion: PM/DM-associated IP had a high rate of relapse, especially the case initially treated with steroid alone. Concomitant use of immunosuppressant at the initial presentation may be required for the prevention of the late relapse of IP.

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CONSENSUS AND ECONOMIC ADVANTAGES IN USE OF ABBREVIATED MANUAL MUSCLE TESTING (MMT) IN THE ASSESSMENT OF IDIOPATHIC INFLAMMATORY MYOPATHIES (IIM). J Hicks, R Wesley, D Koziol, B Feldman, K Whitney, E Adams, M Villalba, M Jain, C Lindsley, S Wright, L Pachman, P Plotz, E Giannini, F Miller, L Rider Bethesda, MD

INTRAVENOUS IMMUNOGLOBULINS AS TREATMENT FOR OESOPHAGEAL DISORDERS OF INFLAMMATORY INCLUSION BODY MYOSITIS. Patrick Cherin, Sophie Pelletier, Antonio Teixeira, Pascal Laforet, Anne Simon, Serge Herson, Bruno Eymard Paris, France

We previously reported that unilateral subscores of 6 (MMT6) and 8 muscles (MMT8), which combine representative axial, proximal, and distal muscles, are comparable to 24 muscles (MMT24) tested bilaterally in juvenile IIM. We confirmed these findings in 2 adult IIM therapeutic trials (15 DM, 30 PM): the best 8 abbreviated subscores (1 MMT6 and 7 MMT8) demonstrated internal reliability (rs with MMT24 ⫽ 0.81-0.91), consistency (Cronbach’s ␣ 0.70-0.91), responsiveness (relative efficiency 0.9-1.3 vs. MMT24) and construct validity (rs 0.25-0.61 with MD global activity and damage, ADL, MRI, muscle enzymes, P⬍0.041). To reach consensus on which MMT subscore is optimal for therapeutic trials and clinical studies, 11 clinicians with myositis expertise used nominal group technique to assess the face validity, ease of testing, functional impact, and limitations of the 8 leading subscores compared to MMT24, and ranked them:

Introduction: We describe four patients with sporadic IBM and severe dysphagia with dramatic response with intravenous immunoglobulin (IVIg) therapy. Case 1: A 59-year-old man was hospitalised in July 1995 with severe dysphagia and aspiration pneumonia. He had complained of muscle weakness for 4 years. Muscle biopsies revealed s-IBM Percutaneous endoscopic gastrostomy (PEG) tube, IVIg (2 g/kg/cure monthly) for six months and prednisone were started, with dramatic improvement after the third IVIg infusion. After four years the patient remains free of oesophageal manifestations. Case 2: A 72-year-old man with a 5-years history of weakness was hospitalised in June 1996. Muscle biopsy revealed s-IBM. In May 1998, he complained of severe dysphagia. Enteral feeding with a nasogastric tube was started, with IVIg (2 g/kg/infusion per month) for six months. Swallowing disorders dramatically improved after the third IVIg infusion and then disappeared totally. After two years the patient remains asymptomatic for oesophageal manifestations. Case 3: A 64-year-old woman was referred in 1992 for muscle weakness. Muscle biopsy showed s-IBM. In October 1996, she was hospitalised for severe dysphagia and infectious aspiration pneumonia. Enteral feeding with a nasogastric tube, prednisone and IVIg were started. Swallowing disorders improved gradually. In February 1997, dysphagia disappeared. IVIg therapy was stopped in April 1997. After three years the patient remains free oesophageal symptoms. Case 4: A 66-year-old man was hospitalised in October 1997 with a 6-year history of limb weakness and falls. Examinations revealed mild dysphagia. Muscle biopsy revealed s-IBM. Prednisone was started without benefit. IVIg were started in March 1998. Swallowing disorders improved after the second IVIg infusion. IVIg therapy was continued for eight months. After 18 months, the patient remains free of oesophageal manifestations. Discussion: In all cases, oesophageal manometry revealed low pressure in the upper sphincter and the absence of peristalsis, with regression of abnormalities after IVIg. The oesophageal manifestations dramatically improved in all patients after the initiation of IVIG alone, or associated with prednisone, allowing a return to normal oral feeding. Our findings suggest IVIG should be considered as a choice treatment for severe dysphagia of IBM.

MMT subscore Neck flexor, deltoid, trapezius, wrist extensor, gluteus maximus, gluteus medius, quadriceps, ankle dorsiflexor Neck flex, deltoid, trapezius, wrist flexor, gluteus maximus, quadriceps, iliopsoas, ankle dorsiflexor Neck flexor, deltoid, biceps, wrist flexor, gluteus maximus, gluteus medius, quadriceps, ankle dorsiflexor Neck extensor, deltoid, biceps, wrist extensor, gluteus maximus, quadriceps, iliopsoas, ankle dorsiflexor

Group Vote (rank) 18 (1) 16 (2) 14 (3) 11 (4)

Completion time for the top MMT8 subscore vs. MMT24 was examined in 20 pts (13 juvenile, 7 adult DM; age 6.3-70 yrs; mean MMT24 score 230) for a cost savings analysis. The MMT8 was completed on average in 1.9 min (range 0.95-3.1) vs. 5.6 min for MMT24 (range 3.2-9.7, P⬍0.001). Medicare costs for MD/therapist time, clinic overhead, and US Labor Dept hourly wages were used to calculate the cost savings of MMT8, assuming MMT is assessed 9 times in the first year. Potential savings were $700-$870/patient in the first year of illness, depending on MMT speed, patient/parent occupation, and use of clinic vs. office facilities. QOL savings, including possible decreased patient fatigue, improved reliability, and reduced absence from work/school, were not assessed. We conclude that a subset of 8 muscles tested unilaterally containing representative axial, proximal and distal muscles is valid, responsive, and reliable in juvenile and adult IIM. This approach to MMT has achieved consensus from myositis experts for use in clinical studies and therapeutic trials, and is economically advantageous.

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DIFFUSE MUSCLE AND JOINT CONTRACTURE WITH MONOCLONAL GAMMOPATHY. J Desmond O’Duffy, Richard L Ehman, Robert A Kyle, Thomas A Gaffey, Andrew G Engel

INTEREST OF INTRAVENOUS IMMUN GLOBULIN INFUSIONS IN OESOPHAGEAL DISORDERS OF REFRACTORY MYOSITIS : AN OPEN STUDY WITH 15 ADULTS PATIENTS. Patrick Cherin, Antonio Teixeira, Thierry Genereau, Olivier Benveniste, Sophie Pelletier, Anne Simon, Pascal Laforet, Bruno Eymard, Serge Herson Paris, France

Inflammatory disorders that may lead to fixed contracture include arthritis, scleroderma, dermato-myositis and fasciitis. The latter includes eosinophilic fasciitis, Spanish toxic oil syndrome and the eosinophilia myalgia syndrome. We present a distinct syndrome, in 3 men, of severe and progressive painful contracture of limb, trunk and facial muscles associated with joint contracture, and in all cases,a monoclonal serum protein. PATIENTS: Three patients all men, aged 49, 29 and 45 years attended with painful contractures affecting their hands, elbows, shoulders and lower extremity joints. In 2 patients the neck, masseter and abdominal muscles were involved. Local tenderness, usually with edema, would precede in a stepwise fashion the spread of contractures to new sites. The duration of symptoms had been 1.5 to 9 years. Two patients had experienced a painful fasciitis of the penis that led to penile collapse and impotence. All had been diagnosed and treated as having “arthritis”, and were severely disabled in gait and hand function. Examination showed joint contractures, averaging 50% restriction of involved joints. Manual atempts to correct the contractions were painful. Two patients had penile length of 5 cms. MRI revealed foci of abnormal signal in affected muscles. Xray showed no erosion or calcinosis. Muscle biopsy showed increased epimysial, endomysial and perimysial fibrous connective tissue with chronic lymphocytic infiltration.One had capsulitis in the wrist and elbow joints. There was no myonecrosis...All had a monoclonal serum protein by SPEP and IEP; IgG kappa in two, and IgG lambda in one. Disease modifying drugs had failed to affect the progression to new muscle contractures. Patient no.1 died of malnutrition related to severe trismus, patient no.3 was treated with melphalan and prednisone with stabilization of symptoms. CONCLUSION: We describe a unique syndrome of progressive painful contracture of limb and trunk muscles associated with a mono-clonal serum protein . It progresses to crippling limb muscle and joint contracture but also may involve the trunk and facial muscles as well as the penile fascia.

PURPOSE : Polymyositis (PM) and dermatomyositis (DM) are rare inflammatory muscular diseases of unknown cause. Corticosteroids and immunosuppressive drugs are therapies of first choice, but not always effective and sometimes responsible for serious side-effets. A many studies showed the interest of polyvalent intravenous immunoglobulin (IVIg) in myositis. We perform an open prospective study to evaluate the efficacy of IVIg in subjects with PM or DM with oesophageal disorders refractory to traditional treatment. PATIENTS AND METHODS : Fifteen adult caucasian patients (9 females, 6 males, mean age 39.4 (SD 12.0) years), with chronic refractory PM (11 cases) or DM (4 cases) and severe oesophageal disorders, were treated by high doses of IVIg, because of the failure of traditional treatments : prednisone (15 cases), methotrexate (10), azathioprine (6), cyclosporine (2). Oesophageal disorders were evaluated by clinically and manometry investigations. There were no treatment changes in the two months before the introduction of IVIg therapy and no increases in dose during this treatment. We used preparations of polyvalent human IVIg with increased intact IgG. The patients received 1 g/Kg daily for 2 days each month. The mean course of treatment was 4 to 6 months. RESULTS : IVIG therapy was effective in esophageal disorders for 11 of the 15 patients (8 / 11 PM and 3 / 4 DM). Mild dysphagia disappeared in 5 of the 6 patients. Six of the 9 patients with life threatening esophageal involvement were dramatically improved after IVIg infusions (5 / 7 PM and 1 / 2 DM). This efficacy resulted in return to normal oral alimentation and ablation of feeding tubes in these 6 patients. Significant clinical improvement of muscle power was observed in 11 of the 15 patients treated. Minor IVIg side-effects were noted in 2 patients. This benefit allow a reduction in their prednisone dosage by more than 50 percent from initial dosage in all patients responders (p ⬍ 0.05). CONCLUSION : IVIg is an interesting therapy in oesophageal disorders of refractory myositis, showing a persistent benefit in 70 percent of patients treated. Disclosure:


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TREATMENT OF INCLUSION-BODY MYOSITIS WITH THE ASSOCIATION OF CORTISCOSTEROIDS AND INTRAVENOUS IMMUNOGLOBULIN: LONG-TERM FOLLOW-UP. Patrick Cherin, Sophie Pelletier, Antonio Teixeira, Bruno Eymard, Pascal Laforet, Anne Simon, Serge Herson Paris, France

A NEW CAUSE OF PROGRESSIVE EXTRINSIC OPHTALMOPLEGIA : MACROPHAGIC MYOFASCIITIS? Patrick Cherin, Dan Milea, Pascal Laforet, Serge Rivaux, Thierry Maisonobe, Serge Herson, Phan Le Hoang Paris, France

Purpose: Sporadic Inclusion Body Myositis (s-IBM) has recently recognized as the most common acquired inflammatory muscle disease occurring above the age of 50 years. s-IBM is generally refractory to standard immunosuppressant therapies. Because of the synergistic effect of corticosteroids associated with intravenous immunoglobulin (IVIG) in polymyositis and dermatomyositis, we tried this association in patients with active s-IBM. Methods: Twenty consecutive Caucasian patients [17M / 3F, mean age 62.1 (SD 13) years], with evolutive IBM, were treated by IVIG associated with corticosteroids between January 1995 and December 1998. The average duration of s-IBM before study was 62.8 ⫾ 24.8 months. Four patients failed to respond to corticosteroid or methotrexate. We used preparations of polyvalent human IVIG with intact IgG (1 g/Kg daily for 2 days, monthly). Corticosteroids were began at the same time, using boluses of methylprednisolone (15 mg/Kg) at the first day of the study, then prednisone per os (1 mg/Kg/day for one month followed by a progressive schedule). The mean course of treatment was 12 months. We evaluated responses at baseline and at the end of each treatment period using expanded (0-88) MRC scales, a functional score, and biochemical creatine kinase values. We calculated the differences in scores from baseline to end of treatment (Chi-2, p ⬍ 0.05). Results: One patient developed an history of prostatic carcinoma and renal failure responsible for the exclusion of the study. Significant persistent clinical improvement was noted in 8 out of the 20 patients at end points (12 and 24 months). The 11 remained patients showed a stabilization of the disease progression after 2 years of evaluation, persistent over 36 months for the 12 patients evaluated at this time. No patient worsened during treatment. Clinical improvement was slowly acquired, usually noted within the 6 first IVIg infusions. The MRC evaluation showed a mean improvement of 8.6 points (20 patients). Dysphagia disappeared in the 4 patients with initial esophageal disorder. All patients with initially elevated CK levels showed biological improvement with a normalization of their CK level values (Mean initial CK value: 393 ⫾ 208 UI/l, after one year : 170 ⫾ 124 UI/l). Conclusion: The association of corticosteroids and IVIG seems to be an interesting therapeutic approach in active s-IBM, stabilizing or improving the disease with a long-term follow-up.

A new type of inflammatory myopathy, named macrophagic myofasciitis (MMF) was recently described and recorded with an increasing frequency by the GERMMAD. MMF may be a complication of aluminic vaccinations in predisposed patients. From our series of 140 MMF, we described 3 patients with MMF and ocular myopathy associated. Case 1. A 63-year-old woman complained of bilateral ptosis, without fluctuation or diplopia. There was an associated asthenia and diffuse myalgia since 1999. Ocular examination disclosed global limitation of all extraocular muscles, suggesting progressive extrinsic ophthalmoplegia. CK level, electromyogram (EMG) and encephalic MRI were normal. Orbital MRI showed diffuse thin ocular motor muscles. Extensive work-up ruled out thyroid eye disease, myasthenia or other ocular myopathy. Electro-oculographic data revealed global decreased amplitude and speed of saccades. The patient received HVB aluminic vaccination in 1994. A muscular biopsy ruled out a mitochondrial disease, but disclosed evidence for MMF. Case 2. A 56-year-old patient complained of progressive onset of binocular painless diplopia and right ptosis in 1999. There was a limited elevation of right eye, but the remainder of the neuro-ophthalmological examination was normal. Complete work-up was not contributive for an ocular myopathy and orbital and encephalic MRI was normal. Electro-oculography disclosed hypometric saccades. Follow-up disclosed progressive bilateral limitation of elevation as well as adduction of the right eye. EMG and CK levels were normal. The patient received HVB aluminic vaccination in 1994. Muscular biopsy ruled out a mitochondrial disease, showing MMF. Case 3. A 44 year-old woman had a work-up for diffuse 2 year lasting myalgia in March 1999. Recently, she was complaining of bilateral onset of ptosis, more visible on the right side, with no diplopia. There was a bilateral ocular globe elevation deficit. An MRI was normal and extensive work-up did not find any evidence for ocular myasthenia or other ocular myopathy. EMG and muscle enzyme levels were normal. The patient received HVB aluminic vaccination in 1997. Muscular biopsy disclosed characteristic MMF. Conclusion : Work-up of progressive ophthalmoplegia may lead to MMF diagnosis if myalgia and/or asthenia are present and deltoid muscle biopsy performed. Conversely, MMF may be complicated by ocular myopathy.

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AM3, AN IMMUNOMODULATOR, PROTECTS COMPETITIVE SPORTS PLAYERS FROM MUSCULAR DAMAGE. Alfredo Cordova, Francisco Martin, Eduardo Reyes, Alfredo Prieto, Erika D Bernstein, Melchor Alvarez-Mon Soria and Alcala de Henares, Madrid, Spain

MRI CRITERIA FOR DISTINGUISHING BETWEEN INCLUSION BODY MYOSITIS AND POLYMYOSITIS. Patrick Cherin, Elisabette Dion, Charles Payan, Jean Claude Fournet, Thierry Papo, Thierry Maisonobe, Eric Auberton, Olivier Chosidow, Pierre Godeau, Jean Charles Piette, Philiipe Grenier, Serge Herson Paris, France

Strenuous physical exercise of limb muscles commonly results in injury, especially when the exercise is intense, prolonged and includes eccentric contractions. Many factors contribute to exercise-induced muscle injury and the mechanism of this muscle injury is likely to differ with exercise type. Competitive sports players are highly susceptible to exercise-induced injury. The ability of AM3, as an immunomudulator drug, to prevent chronic muscular injury following strenuous exercise characterized by eccentric muscle contraction, was evaluated by a pilot double-blind randomized study. Subjects were randomized to placebo or AM3 and evaluated for biochemical indices of muscle damage before and after a thirty-day treatment with either placebo or AM3 during the peak competitive season of our subjects. Fourteen male professional volleyball players from the 1st Division Spanish League of Volleyball volunteered to participate in the study (means of physical characteristics are: 25 years old weight 86 Kg and height 190 cm). In our study, we observe that competitive eccentric exercise (as is volleyball) is accompanied by significant increases in CK, myoglobin, AST, and LDH to varying degrees after a 30-day period of maximal effort. The administration of AM3 not only inhibits these changes, but also results in a decrease from baseline in serum concentrations of CK and myoglobin. Specifically, CK-MB concentration is significantly decreased from baseline with AM3 treatment, but not placebo. Conclusions: These results show that the use of an immunomodulator, such as AM3 in elite sportsmen during a period of maximal competition significantly inhibits the enhanced serum levels of proteins associated with tissue damage.

Objective: To develop diagnostic imaging criteria for polymyositis (PM) and sporadic inclusionbody myositis (s-IBM). Methods: Two hundred twenty patients with inflammatory myopathies were investigated by MRI 5magnetic Resonance Imaging). Findings were compared with the results of clinical and biological examinations and muscle biopsy. PM and IBM were diagnosed in 25 patients each. The diagnosis of PM was established according to Bohan & Peter (23). S-IBM was confirmed by light and electron microscopy. Quantitative and qualitative MRI analysis of the three muscle groups of the two thighs included fatty infiltration, atrophy, inflammation, and the type and distribution of the lesions, were performed by 4 independent experienced radiologists blinded to clinical and histological data. Results: There were 26 men and 24 women, with a mean age of 54.9 years (range 19-77 years). MRI was abnormal in all the patients. Fatty infiltration and atrophy were more frequent in s-IBM (p ⬍ 0.05). Inflammation as the sole abnormality was preferentially encountered in PM (p ⫽ 0.05). Widespread abnormalities were more frequent in s-IBM (p ⬍ 0.01). Abnormalities in PM tended to be distributed along the fascia. Involvement of the anterior group, an asymmetrical distribution, and a distal predominance were all more frequent in s-IBM (p ⬍ 0.001). Conclusions: MRI can distinguish between polymyositis and sporadic inclusion-body myositis and can help physicians in difficult diagnosis. Disclosure:

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A PILOT STUDY OF THE EFFECT OF METHIMAZOLE, A DRUG THAT DOWN-REGULATES MHC CLASS I, ON DERMATOMYOSITIS AND POLYMYOSITIS. Imelda P Cabalar, Lourdes Villalba, Jeffrey Sherman, Cheryl Yarboro, Holly Cintas, Deloris Koziol, Leonard Kohn, Jeanne Hicks, Paul H Plotz Bethesda, MD

EFFICACY OF CYCLOSPORINE TREATMENT TO INTERSTITIAL PNEUMONITIS ASSOCIATED WITH POLYMYOSITIS AND DERMATOMYOSITIS. Kenji Nagasaka, Masayoshi Harigai, Masako Hara, Yasuyuki Yoshizawa, Takao Koike, Nobuyuki Miyasaka Tokyo and Sapporo, Japan

Methimazole, a drug used to control hyperthyroidism, has been shown to down-regulate MHC class I expression. In polymyositis and dermatomyositis, there is a strong surface expression of MHC class I, whereas normal muscle has trace or absent MHC class I. Furthermore, animal studies have shown that upregulation of MHC class I (H-2Kb) in mouse skeletal muscle leads to clinical, biochemical, histologic and immunologic features resembling myositis. In an open label pilot study, we assessed the clinical efficacy of methimazole, as measured by serum muscle enzyme levels and manual muscle testing, in patients with dermatomyositis and polymyositis as well as the expression of MHC class I in muscle by quantitative northern analysis. Patients with persistent weakness and laboratory evidence of inflammation while on a stable regimen of medication to control their muscle inflammation were entered into a pilot study of 30-60 mg/day of methimazole for 3 to 6 months. Patients were assessed before and after treatment for the primary outcome measures of strength and function, as well as MHC class I expression. Drug-related toxicities were recorded during the study and for three months after discontinuation of treatment. Of the 19 patients entered in the study, 3 patients had improvement in their activities of daily living, and 1 patient improved in muscle strength at 6 months. Neither of these is statistically significant (ADL delta% p-value⫽0.06 and MMT delta% p-value⫽0.59). Of the other measures of disease activity, CK, aldolase and physician’s global assessment of disease activity improved from baseline (p-values were 0.002, 0.004 and 0.019 respectively). Adverse effects requiring discontinuation - liver enzyme elevation, fever,infection, rash or flu-like symptoms - occurred in 6 patients. No new toxicities were observed. MHC class I was reduced during drug administration in several patients. Thus, despite the down-regulation of MHC class I, and improvement in a small number of patients in the primary outcome measures, methimazole failed to demonstrate a clinically significant benefit.

Objective. Interstitial pneumonitis (IP) associated with polymyositis and dermatomyositis (PM/DM) is a serious complication which affects the prognosis. It is often refractory to conventional corticosteroid treatment with or without various immunosuppressants. There have been some reports that cyclosporine A (CyA) was effective to corticosteroid-resistant IP associated with PM/DM. We undertook a retrospective multi-center study to examine the efficacy of CyA. Methods. Questionnaires were sent to 32 hospitals having rheumatology division in Japan to analyze the efficacy of CyA to IP associated with PM/DM. Fifty-four IP patients with PM/DM (9 PM, 45 DM) who have been treated from 1989 to 2000 were analyzed. Thirty-two CyA-treated patients (9 PM, 23 DM) were included in the study. Four parameters including subjective symptoms, ausculatory sound, chest radiographs and respiratory index (PaO2/FiO2) were serially evaluated. General evaluation was performed one month after the commencement of CyA treatment using these parameters and final outcome was also evaluated. Results. All CyA-treated IP patients took a concomitant use of corticosteroids during the treatment. All of PM and chronic IP with DM, and 53% of acute IP with DM were graded as more than partially effective in general evaluation after 4 weeks of the combination treatment. In contrast, all cases graded as progressive in general evaluation were acute IP with DM. Regarding outcome, there was a statistically significant correlation between general evaluation after 4 weeks of CyA treatment and final outcome among the CyA-treated patients (r⫽0.36, p⫽0.048). Noteworthy, in acute IP with DM, the survival rate of steroid plus CyA-treated group was significantly higher than that of steroid-treated group (p⫽0.040). Conclusion. A combination treatment of CyA with corticosteroids is effective to IP with PM/DM, and significantly improves the survival rate of acute IP with DM. Disclosure: This work was supported by grants-in-aid from The Ministry of Health, Welfare and Labour, Japan and from Human Science Promotion Foundation, Japan

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CHLOROQUINE MYOPATHY: PREVALENCE AND CLINICAL FEATURES (PRELIMINAR DATA). Jordi Gratacos, Enrique Casado, Marta Larrosa, Juli Font, J Miquel Martinez, Carles Tolosa Sabadell, Barcelona, Spain

FRACTURE DETECTION AND BMD THRESHOLDS: ARE WE MISSING OPPORTUNITIES FOR FRACTURE PREVENTION? Ethel Siris, Paul Miller, Thomas Abbott, Ya-Ting Chen, Suna Barlas, Kenneth Faulkner, Elizabeth Barrett-Conner, Marc Berger, Arthur Santora, Louis Sherwood New York; Denver; West Point; Madison and San Diego Purpose: Low bone mineral density (BMD) predicts increased risk for fracture. However, debate continues over the BMD threshold that should be used for treatment decisions. To address this issue, we compare the sensitivity for prospectively detecting osteoporotic fractures (hip, forearm/ wrist, spine, and rib combined) at different BMD (expressed as T-score) thresholds using the National Osteoporosis Risk Assessment (NORA) data. Previously we reported data from the NORA primary care arm cohort of over 150,000 postmenopausal women who had BMD measured using peripheral technology. That analysis demonstrated that over 70 % of osteoporotic fracture occurred in women with peripherally measured T scores above -2.5. In the current study, we report the sensitivity for detecting osteoporotic fractures at different thresholds of BMD measured by central technology using data from the NORA specialist arm cohort. Methods: The NORA-specialist arm enrolled approximately 1,200 postmenopausal women aged 50 years and older from the offices of 47 specialists in the management of osteoporosis. BMD was measured at the hip, spine, and either forearm or heel. Fractures occurring in the next year were identified by self-report. Results: This analysis uses the femoral neck BMD (DXA) measures of 809 Caucasian women who responded to the follow-up survey. During 1-year follow-up, 21 osteoporotic fractures were reported. Using a threshold of T-scores -2.5 and below, only 38% of fractures were captured. In contrast, using the NOF guidelines for pharmacologic intervention of T-scores -2.0 and below or T-scores below -1.5 with at least one additional risk factor, 71% of the fractures were captured. Conclusions: Over 60% of the women experience fractures at T-score (measured at femoral neck) above -2.5. Limiting treatment interventions to women with very low T-scores misses a significant portion of potentially preventable osteoporotic fractures.

Disclosure: Disclosure: Financial Support By Merck & Co., Inc.

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Purpose: To analyze the prevalence and clinical features of chloroquine myopathy in a cohort of out-patients in a Rheumatology Unit. Methods: 119 patients (84 female, 35 male), aged 57.8⫾13.7 were taken antimalarials for more than 6 months and were evaluated during 2 years. The underlying disease was RA in 69, palindromic rheumatism in 14, connective disease in 22, spondyloarthropathy in 7 and other rheumatic diseases in 7. Muscular enzymes were determined in all patients and electromyography (EMG)was practiced in patients with repeated increased determinations. Patients with altered EMG were biopsied. Results:Laboratory: 18 patients (15%) had a repeated muscle enzymes increase: LDH in 17 patients, CPK in 5 and aldolase in 1 patient. EMG: Practiced in 13 patients, showed a muscular involvement in 6. Two patients are pending and 3 were dead before the exam. Muscle biopsy: 6/7 patients had a chloroquine myopathy (optic and/or electron microscopy); 1 patient had an steroid myopathy. Two patients (RA) had an associated inflammatory myositis. Clinical features: Six patients with chloroquine myopathy (5 RA and 1 spondyloarthropathy) means a prevalence of 5 %. The duration of treatment was 36⫾21 months (group 40⫾31), with a cumulative dosis of 203⫾25 g (group 305⫾243g). All patients had weakness and had other simultaneous muscle involvement factors (1 cervical myelopahy, 2 hypothyroidism, 3 hypovitaminosis D and 1 prolonged bed rest). Conclusions: Toxic chloroquine myopathy is not an exceptional event (prevalence 5%). When an increase of LDH and/or CPK is present we should to rule out this complication. If a simultaneous factor with muscle involvement is present, probably the weakness gets worse and makes easy its clinical detection.

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ISOKINETIC AND PROGRESSIVE RESISTIVE EXERCISES IN THE TREATMENT OF KNEE OSTEOARTHRITIS. Sibel Eyigor, Simin Hepguler, Kazim Capaci

ROFECOXIB IN CHRONIC LOW BACK PAIN: EVALUATION OF ONSET OF ACTION. Nathanial Katz, David Krupa, William Ju, Diana Rodgers, Donna Walters, Kristine Johnson, Barry Gertz Boston, MA and Rahway, NJ

Objectives: To compare the efficacy of isokinetic and progressive resistive exercise (PRE) programs in patients with knee osteoarthritis (OA). Material and Method: Forty-four patients with bilateral knee OA according to the ACR criteria were included in this study. Five patients were excluded because they could not complete the study. The patients were grouped in two groups according to the radiological stage. The patients in Group I (n⫽21) performed isokinetic exercise (60, 90, 120, 180 deg/sec, 6 repetition, 3 set, Cybex) three days per week, for six weeks. The patients in Group II (n⫽18) performed progressive exercise program with De Lorme technique five days per week, for six weeks. Clinical examination, routine biochemical parameters and conventional roentgenography of the knees were evaluated before the treatment. Disease severity, pain, range of motion (ROM), morning stiffness, 15 meters walking time, WOMAC, Lequesne index and AIMS2 were compared before and after the treatments. With Cybex isokinetic test device, knee extension and flexion isokinetic muscle strength measurements at velocities of 60, 90, 120, 180 deg/sec (peak torque ⫽ PT, total work ⫽ TW, endurance), and 30, 60, 90 degree extensor isometric muscle strength measurements (PT) were performed. Results: The mean age was 53.14 (SD 6.73) years in Group I, and 51.89 (SD 0.12) years in Group II. There was no difference of sex ratio, age, BMI, BW, disease duration and radiological stage between the groups (p⬎0.05). Disease severity, pain, Lequesne, WOMAC, walking time improved with the treatment in both groups (p⬍0.05). ROM did not improve in both groups (p⬎0.05). PT, TW velocities increased for both extension and flexion in isokinetic measurement (p⬍0.05). Endurance was not significantly changed (p⬎0.05). All velocities increased for flexion, PT velocities increased for extension in PRE group (p⬍0.05). In this group, endurance improved at 180 deg/second (p⬍0.05). The comparison of all parameters was not statistically significant in both groups (p⬎0.05). In PRE group, extensors at 90-180 deg/second TW; and isometric 90 degree PT improved (p⬍0.05). Conclusion: 1. Isokinetic and PRE programs are efficient in the treatment of knee OA. 2- Statistically significant difference could not be found between the two programs. 3- PRE program, being cheaper, easy to be performed and efficient, can be preferred in the treatment of knee OA.

Background: NSAIDs are the most commonly prescribed medications for chronic low back pain (CLBP). Their efficacy for CLBP is unclear and long-term use of nonselective NSAIDs can result in serious gastrointestinal toxicity. Objective: To evaluate the onset of efficacy of rofecoxib, a selective COX-2 inhibitor, in the treatment of CLBP. Methods: Two replicate randomized, double-blind, placebo-controlled, parallel group, 4-week, multicenter U.S. studies. Patients with non-radicular CLBP were randomized 1:1:1 to rofecoxib 25 mg, 50 mg, or placebo once daily in the morning. Low Back Pain Intensity and Bothersomeness were assessed at Days 2 through 8. Pain Relief after the First Dose was assessed at 30 minutes, 1,2,3, and 4 hours, at bedtime and upon awakening following the first dose of study drug. Results:For maximal precision, data from a preplanned, combined analysis are presented. 690 patients were randomized to placebo (N⫽228), rofecoxib 25 mg (N⫽233), or rofecoxib 50 mg (N⫽229). Mean age 53.4 years, 62.3% female, mean pain duration 12.1 years, and pretreatment mean pain intensity was 76 mm on a 100-mm visual analog scale. Compared to placebo, rofecoxib 25- and 50-mg both demonstrated statistically significantly greater pain relief as early as bedtime after the first dose of study medication (p⬍0.001 for both regimens vs placebo). The 25- and 50 mg groups were not different from each other (p⫽0.102). Both rofecoxib groups demonstrated significantly greater improvements in Low Back Pain Intensity (p⬍0.001) and Low Back Pain Bothersomeness (p⬍0.001) vs placebo by morning of Day 2 of therapy. The majority of patients (60.4% and 58.4% in the rofecoxib 25 and 50 mg groups, respectively) achieved at least a 50% reduction in Low Back Pain Intensity. The 25- and 50 mg regimens were both well tolerated. Conclusions:Patients treated with 25 or 50-mg rofecoxib experienced greater pain relief than placebo-treated patients as early as bedtime after the first dose. Daily doses of rofecoxib reduced Pain Intensity and Bothersomeness more than placebo. The majority of patients on rofecoxib achieved at least a 50% reduction in Low Back Pain Intensity during the first week of the study.

Disclosure:

Disclosure: D Krupa, W Ju, D Rodgers, D Walters, K Johnson, and B Gertz are employees of Merck & Co., Inc.

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TIME TO DISLOCATION, DEEP INFECTION AND PULMONARY EMBOLUS AFTER TOTAL HIP REPLACEMENT IN US MEDICARE BENEFICIARIES. Charlotte B Phillips, Jane A Barrett, Elena Losina, Elizabeth A Lingard, Jeffrey N Katz Boston, MA; Lebanon, NH

PATIENT EXPENDITURE AND OUTCOMES FOLLOWING BILATERAL AND UNILATERAL TOTAL KNEE REPLACEMENT: VALUE FOR MONEY? Lyn M March, Marita J Cross, Kate L Tribe, Helen M Lapsley, Brett G Courtenay, Mervyn Cross, Leo Pinczewski, Myles Coolican, Frank Robertson, Peter M Brooks St Leonards, Sydney, Kensington, Darlinghurst and Crows Nest, NSW, Australia and Brisbane, QLD, Australia

Purpose: Determining the duration of risk for dislocation, pulmonary embolus (PE) and deep infection after total hip replacement (THR) informs surgeons about the optimal duration for implementing prevention strategies.Methods: Eligible subjects were Medicare beneficiaries 65 yrs and older who had elective primary or revision THR in 1995. We used Medicare claims to calculate incidence rates (and 95% confidence intervals) per 10,000 person weeks of dislocation, PE and deep infection, for 3 periods of risk (1-4 weeks, 5-13 and 14-26 weeks) following primary and revision THR. Endpoints were defined with algorithms using ICD-9-CM and CPT codes and included 90-day frequency of dislocation, infection and PE. Results: 58,521 patients had primary THR and 12,956 had revision THR. Primary and revision subjects had a similar demographic profile with a mean age of about 75 years, about 2/3rds female and 94% Caucasian. Among primary (revision) THR subjects, 3.5% (10%) had dislocation, 0.9% (0.8%) had PE and 0.3% (1.1%) had deep infection. The risk of these outcomes was highest in the 1st 4 weeks postop, diiminishing considerably in weeks 5-13 and even further after week 13. An exception is risk of infection following revision THR, which remains elevated. Conclusions: The probabilities of dislocation, deep infection and PE after THR diminish dramatically during the first 3 months, except after revision THR, where the infection rate remains elevated even after the 14th week post-op. Prevention measures are especially important in the first 1-3 months after THR.


Primary THR Dislocation PE Deep infection Revision THR Dislocation PE Deep infection

1-4 weeks postop

5-13 weeks postop

rate (95% CI) 213 (201,225) 65 (59, 72) 15 (12, 18)

rate (95% CI) 110 (102, 119) 24 (20, 28) 7.0 (4.8, 9.1)

14-26 weeks postop rate (95% CI) 39 (34, 44) 4.9 (3.0,6.8) 3.4 (1.9, 4.9)

530 (489, 570) 60 (47, 74) 52 (40, 65)

387 (351, 422) 19 (11, 26) 29 (19, 38)

140 (118, 162) 5.4 (1.1,9.7) 33 (23, 43)

Total knee replacement (TKR) has become one of the most commonly performed operations. Bilateral knee arthritis poses a particular dilemma. Our aim was to determine out-of-pocket expenditure, health services utilisation & health outcomes of patients undergoing bilateral TKR (bTKR) to compare with unilateral (uTKR). Patients were recruited from 3 hospitals & 11 surgeons. Diaries were completed for 3mths pre-op & 12mths post-op. Costs covered medications, visits to health professionals, tests, equipment, & services. Health status was assessed with the SF-36. Data from 126 primary uTKR (76% OA, mean age 67.7yrs, 58% female) & 58 simultaneous bTKR patients (97% OA, mean age 67.8yrs, 48% female)show no differences in age, comorbidities, pension status or pre-op SF-36 scores. bTKR had more post-op complications but no mortality and no difference in length of stay (uTKR 13.54 days vs bTKR 12.35 days). bTKR were more likely to be discharged to a rehabilitation facility (58% vs 30%, p⬍0.001). Average out-of-pocket expenditure for the first post-op year was AUS$477 for bTKR & AUS$718 for uTKR patients. This difference was not significant. uTKR patients made more visits to their surgeon (mean 2.3 vs 1.3, p⬍0.001), more visits to GP (4.7 vs 3.3, p⫽0.05) & physio (9.5 vs 6.3, ns) than bTKR patients during the first post-op year. By 12mths post-op, patients with bTKR reported better Physical Function, General Health, Vitality & Role Emotional whereas uTKR patients reported better Bodily Pain. CONCLUSION: Utilisation of services for both bTKR and uTKR decrease while health status improves over the first year. Despite more hospital complications bTKR patients had similar hospital stays and post-op out-of-pocket expenditure and reported better physical function & general health with fewer health care visits one year post-op. Patients appear to be receiving value for money from their simultaneous knee replacements and in the long term may be providing savings for the health care system.

Disclosure:

Disclosure:

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1811

CELECOXIB AND IBUPROFEN ACCELERATE RETURN TO NORMAL FUNCTION COMPARED TO PLACEBO IN PATIENTS WITH ACUTE ANKLE SPRAINS TREATED WITH STANDARD SUPPORTIVE CARE. Justus J Fiechtner, Evan F Ekman, John G Fort, Stephane Levy, C Orevillo East Lansing, MI; Columbia, SC and Peapack, NJ

EPIDURAL CORTICOSTEROID INJECTIONS FOR SCIATICA : A RANDOMIZED, DOUBLE BLIND, CONTROLLED CLINICAL TRIAL. Jean-Pierre Valat, Bruno Giraudeau, Sylvic Rozenberg, Philippe Goupille, Pierre Bourgeois, Valerie Micheau-Beaugendre, Martin Soubrier, Stephanie Richard, Eric Thomas France

Objectives: To compare the return to normal function/activity and tolerability of celecoxib, ibuprofen and placebo plus standard supportive care in the management of acute ankle sprains. Methods: In a 10-day, multicenter, randomized, double blind trial, 443 adults with first- or second-degree inversion ankle sprain were treated with celecoxib 400 mg/day (n⫽147), ibuprofen 2400 mg/day (n⫽155), or placebo (n⫽141). At baseline, patients had moderate to severe ankle pain on weight bearing. Patients were allowed other nonpharmacologic therapy such as RICE (rest, ice, compression, and elevation), taping/bracing, crutches, etc. Results: Injuries occurred within 48 hours of administration of the first dose of study medication (mean duration, 25.6 hours). RICE was used in 84 to 93% of patients; crutches in 33-44% of patients and strengthening exercise in 17-20% of patients. Endpoints included Patient’s Global Assessment of Ankle Injury and Ankle Pain (VAS score) at Day 4 and Day 11 and time to return to normal function/activity or function improvement by at least 2 grades (5-point categorical scale). Based on Patient’s Global Assessment of Ankle Injury, significantly more patients in the celecoxib group were classified as responders compared with placebo at Days 4 and 8 (p⫽0.017 and p⫽0.022). By Day 11, there was no statistical difference among the three groups (p⫽0.437). Based on Patient’s Assessment of Ankle Pain, mean VAS pain scores at Day 4 for the celecoxib (p⫽0.002) and ibuprofen (p⫽0.011) groups were significantly lower compared with the placebo group. Median time to return to normal function/activity or function improvement by at least 2 grades was 5 days for the celecoxib group, 6 days for the ibuprofen group, and 8 days for the placebo group (celecoxib vs. placebo, p⫽0.001). Conclusion: Celecoxib and ibuprofen were superior to placebo in treating acute first- or second-degree ankle sprain. Celecoxib also demonstrated faster recovery from ankle injury, by Day 5, as measured by return to normal function /activity or function improvement by at least 2 grades compared with placebo. Treatment of acute ankle injuries with NSAIDs and standard non-pharmacologic methods provide for a significantly faster recovery than standard supportive care alone. Celecoxib and ibuprofen were well tolerated. Celecoxib, with its platelet sparing properties, has the potential to offer an advantage over ibuprofen.

Background: Although epidural corticosteroid injections are commonly used for sciatica, their efficacy has not been established. Methods: In a randomized double blind clinical trial we administered three epidural injections (at 2 days interval) in 2 different ways : 2 ml of prednisolone acetate (50 mg) or 2ml isotonic saline to patients with sciatica presumably due to a herniated nucleus pulposus, during for more than 15 and less than 180 days. Self evaluation (recovery, important improvement, poor improvement or worse) was the main judgement criterion at day 20. Patients with recovery or important improvement were considered as success. Pain on VAS, SLR test, Schober’s test, Dallas pain questionnaire, Roland-Morris index were evaluated at day 0, 5, 20, and 35. Only analgesics were authorized, patients requiring NSAIDs during the study were considered as failure. Results: 85 patients were included, 42 in the control (C) group, 43 in the steroid (S) group. The mean duration of sciatica was 56.6⫾42.6 days in the C and 40.9⫾36.2 in the S group (p⫽0.072). On ITT analysis 15/42 (35.7%) patients in the C group and 22/43 (51.2%) in the S group (p⫽0.15) were considered as success (difference⫽15,5%, 95%CI ⫽[-5.4;⫹36.3]). Among the 48 failures 14 patients (6 C, 8 S) required NSAIDs, 3 (2C, 1 S) required surgery and 7 (3 C, 4 S) other treatments. On per protocol analysis 11 patients were excluded (2 did not satisfy the inclusion criteria, 4 did not receive the 3 epidural injections, 5 were lost from follow up). In the 74 remaining patients 12/35 (34.3%) in the C group and 22/39 (56.4%) in the S group (p⫽0.057) were considered as success (difference ⫽22.1%, 95%CI ⫽ [0.0⫹44.2]). For secondary endpoints, missing data were handled by applying the LOCF procedure. Intragroup improvement at day 20 was observed for pain on VAS (C: -17.4⫾28.5 p⬍0.001 and S: -28.3⫾28.8 p⬍0.001) but there was no difference between the two groups (p⫽0.082). The same qualitative conclusions have been drawn for other secondary endpoints as Schober’s test, SLR test, daily living activities and work-leisure subscales of the Dallas pain questionnaire, Roland-Morris Index : intragroup improvement with time was statistically significant, but intergroup differences were not. Clinically significant side-effects were 3 in the C and 2 in the S group (p⫽0.676). Conclusion: Although we cannot exclude the efficacy of isotonic saline administered by epidural route for sciatica, epidural steroid injections afford no more improvement. Disclosure:

Disclosure: Supported by Pharmacia Corporation and Pfizer Inc.

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FRACTURES AND OSTEOPOROSIS IN USERS OF HIGH-DOSE ORAL GLUCOCORTICOIDS: ANALYSIS OF A PHARMACY CLAIMS DATABASE. Susan Brenneman, Shiva Sajjan, Ya-Ting Chen, Pamela Landsman, Thomas Abbott West Point

MANAGEMENT OF OSTEOARTHRITIS (OA) WITH PATIENT-ADMINISTERED ASSESSMENT TOOLS AND/OR UNSUPERVISED HOME-BASED EXERCISES PROGRAM. A 2x2 FACTORIAL DESIGN CLUSTER RANDOMIZED CONTROLLED TRIAL. P Ravaud, B Giraudeau, I Logeart, JS Larguier, R Treves, L Euller-Ziegler, B Bannwarth, M Dougados Paris, France

Background: Osteoporosis is a common complication of long-term glucocorticoid therapy. Few data exist for patients on corticosteroids. This study determined the frequency of documented osteoporosis diagnosis and osteoporosis-related fractures in users of glucocorticoids using in a large managed care adminstrative claims database. Methods: More than 400,000 subjects enrolled in multiple health plans comprised the database supplied by Protocare Sciences. All members who were continuously enrolled with pharmacy coverage from 1997 through 1999 were eligible for the study. High-dose glucocorticoid (ⱖ7.5 mg. of prednisone or equivalent per day) users were identified. For each “user”, 4 “non-users” were matched by age and sex. Non-users had no glucocorticoid claim during the study period. The “index date” for all “users” was defined as the first date of a glucocorticoid prescription filled in 1998; non-users were assigned the same index date as their user-match. Osteoporosis diagnosis and fracture events one year prior to and one year after the index date were identified using ICD-9-CM codes. Glucocorticoid use was classified into 3 categories: “continuous users” used glucocorticoids both pre- and post-index date, “new long-term users” began use in 1998 with ⱖ2 and ⬎30 days usage, and “new short-term users” began use in 1998 with 1 script and ⬍30 days usage. Results: 4128 high-dose glucocorticoid users were identified; among whom 971 (23.5%) were continuous users, 1230 (29.8%) were new long-term users, and 1927 (46.7%) were new short-term users. A diagnosis of osteoporosis during the study period was documented in 16.0%, 8.5%, 3.9%, and 3.5% of the continuous, new long-term, new short-term, and non-users, respectively. The post-index rate of fracture per 100 patients-years was 2.37, 1.71, 1.35, and 0.84 for the continuous, new long-term, new short-term, and non-users, respectively. Conclusions: Longer duration of glucocorticoid use was associatied with higher rate of fractures and documented osteoporosis diagnosis. There is opportunity for intervention in this population to prevent fractures related to the low bone mass associatied with glucocorticoid use.

Objectives: To evaluate the short term symptomatic efficacy of patient-administered assessment tools (Tools) and home non-supervised exercises programs (Exercises) alone or in combination in OA patients. Methods: Design. A 24 weeks open cluster randomized controlled-trial with a factorial design. Setting. French rheumatologists (n⫽867) were assigned to 4 groups: Tools (n⫽220), Exercises (n⫽213), Tools ⫹ Exercises (n⫽213), no intervention (n⫽221). Patients. Each rheumatologist had to enrol 4 painful OA patients (knee OA:3, hip OA:1). Interventions. 1) Tools: weekly diary including the record of pain and disabling activities 2) Home-based exercises programs with the aid of videotape and booklet, performed daily. Concomitant therapy. All patients received rofecoxib (12.5 mg or 25 mg QD). Outcome variables. Pain (VAS 0-100), WOMAC function subscale (0 100), patient assessment of the quality of care (0-100). Results: Overall 2957 patients (2216 knee OA, 741 hip OA) were included. After 24 weeks, both pain and function improved (-17⫾27, -20⫾29, -15⫾27, -19⫾29) and (-11⫾19, -12⫾19, -10⫾19, -11⫾20) in the different groups (Tools, Exercises, Tools ⫹ Exercises and no intervention groups respectively), without statistical significant difference between groups. Patient’s assessment of rheumatologist implication to preserve their activity was 77.7 and 81.3 in the exercises groups and 63.4 and 63.5 in the other groups. Conclusion: Although the patients assessment was in favour of the exercises programs, this study failed to demonstrate short term symptomatic effect of the two non pharmacologic evaluated treatments in OA patients concomitantly receiving Cox-2 inhibitors. Disclosure: The study was in part supported by a grant from Merck-France

Disclosure: Financial Support By Merck & Co., Inc.

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POPULATION CHANGES IN BACK PAIN BELIEFS FOLLOWING A MASS MEDIA CAMPAIGN. Rachelle Buchbinder, Damien Jolley, Mary Wyatt Melbourne, Victoria, Australia

INCREASED SERUM LEVELS OF CARTILAGE OLIGOMERIC MATRIX PROTEIN AND HUMAN CARTILAGE GLYCOPROTEIN-39 IN MARATHON RUNNERS. Morton A Scheinberg, Sueli Maciel Sao Paulo, SP, Brazil

AIM: We evaluated the effectiveness of a population-based media campaign designed to alter beliefs about back pain and ultimately reduce back-related disability. METHODS: A quasi-experimental, non-randomised, non-equivalent, before-after study design was used with an adjacent state as control group. Computer assisted telephone interviews of the employed population were conducted before, two and two and a half years after campaign onset. Population beliefs were measured using the Back Beliefs Questionnaire (BBQ). The personal characteristics of the respondents were collected including age, sex, country of birth, highest level of education achieved, metropolitan or rural address, occupation and type of work, employer/ employee status, income and previous experience of back pain: ever, in the past year and the past week. Quantile-quantile (Q-Q) plots were used to assess the homogeneity of change in BBQ score between different times. RESULTS: There were 4730 surveys completed, with equal numbers in each state, and a ratio of 2:1:1 across the three time periods. Improvements in population beliefs about back pain were only observed in the state exposed to the media campaign (mean improvement in BBQ score ⫽ 1.9 (95% CI: 1.3 - 2.5), p⫽0.000 and 3.2 (2.6 - 3.9), p⫽0.000 between baseline and Survey 2 and 3 respectively). At baseline the BBQ scores in each state were distributed identically. At Surveys 2 and 3, BBQ scores were uniformly higher in the state exposed to the media campaign compared with the control state across the range of BBQ scores. Scores in the state exposed to the media campaign improved irrespective of other personal characteristics. CONCLUSION: A population-based strategy of delivery of positive messages about back pain resulted in a uniform shift in population beliefs irrespective of baseline back beliefs and other personal characteristics. Altering societal views towards this common complaint may be a highly effective primary preventive strategy to reduce the incidence of back pain related disability in the community.

Objective:Marathon runners have an increased risk of developing joint disease. Before and immediately after a 40-km run, we measured the serum levels of cartilage oligomeric matrix protein (COMP)and human cartilage glycoprotein-39 (gp-39) two markers for joint metabolism and/or damage. Methods: Forty marathon runners were split in two groups A (between 20 and 40 years of age) and group B (between 40 and 60 years of age). Blood was drawn before and immediately after a two hour marathon held in the city of Sao Paulo. Twenty five sera run on two different days derived from normal volunteers were used as controls (group C). Both measurements were performed by classical immunoassays previously reported. Results expressed as the variation ⫾ sd before and after. Statistical analysis were done using the paired t test. Results:As seen on the table below, runners had significant variations on the two markers when compared to healthy controls (p⬍0.01). Group B showed almost twice as much variation on the markers when compared to group A (p⬍0.01). Conclusions:These results may be relevant to the mechanisms associated with early development of osteoarthritis in long term marathon runners.[table] Number

20

20

25

Group gp 39 (ng/ml) COMP(ug/ml)

A 42 ⫾ 19 0.9 ⫾ 0.3

B 58 ⫾ 13 1.1 ⫾ 0.6

C 9⫾6 0.3 ⫾ 0.2

Disclosure:

Disclosure:

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THE NEGATIVE IMPACT OF OBESITY ON THE LOCOMOTOR SYSTEM OF CHILDREN. Ana Lucia S Pinto, Patricia Holanda, Ari S Radu, Sandra Villares, Fernanda Lima Sao Paulo, Sp, Brazil

PAMIDRONATE AND 1,25-DIHYDROXY-VITAMIN-D3 INHIBIT TUMOR NECROSIS FACTOR-ALPHA (TNF-␣)- AND WEAR DEBRIS-INDUCED INTERLEUKIN-6 (IL-6) RELEASE AND RECOVER SUPPRESSED TYPE I COLLAGEN SYNTHESIS IN BONE MARROW-DERIVED PRIMARY HUMAN OSTEOBLASTS. Csaba Vermes, Chandrasekaran Raman, Jozsef Dobai, Gunnar BJ Andersson, Howard An, Joshua J Jacobs, Tibor T Glant Chicago, IL

Background: Previous studies have implicated adult obesity as a risk factor for rheumatological disorders as back pain and ostearthritis but it is unknown the role of body weight in the locomotor system of the children. Aim: A case-control design was undertaken to explore the association between child obesity and osteoarticular alterations. Methods: Twenty-five boys and twenty-four girls (mean age:10.8 ⫾ 2.07 years old) with a body mass index (BMI) above 95th percentile were compared with same mean age controls (11.1 ⫾ 2.2 years-old)(p ⫽ NS) with BMI bellow 80th percentile and a mean duration of obesity of 6 ⫾ 3 years. Results: There was a statistically difference observed between the numbers of minutes/week of physical education class, obese mean ⫽ 67 ⫾ 37 mim/week versus control mean ⫽ 82 ⫾ 26 min/week (p ⫽ 0.02) and hours/day on TV and/or computer, obese mean ⫽ 5 ⫾ 2 h/day versus control mean ⫽ 3 ⫾ 2 h/day (p ⫽ 0.001). A higher frequency of at least one osteoarticular manifestation was observed in obese patients (55%) compared to the control group (23%) (p⫽ 0.001).A statistically significant association was also found between obesity and back pain, genu valgum, genu recurvatum and tight quadriceps (Table). Symptoms

Obesity group (n⫽49)

Control group (n⫽47)

P

15 (30.6%) 14 (28.5%) 1 (2%) 8 (16.3%)

1 (2%) 6 (12.7%) 0 3 (6.3%)

⬍0.001 NS NS NS

5 (10.2%) 27 (55.1%) 12 (24.48%) 17 (34.7%) 18 (36.7%) 37 (75.5%) 22 (44.9%)

9 (19.1%) 1 (2%) 1 (2%) 9 (19.1%) 13 (27%) 32 (68%) 10 (21.2%)

NS ⬍0.001 0.001 NS NS NS 0.01

Interestingly, 32/49 (65%) of the obese group presented sleep disturbances (chronic snoring, morning headaches, daytime sleepness, sleep apnea) compared with 4/47 (8.5%) in the control group (p⬍0.001). Fibromyalgia tender points (ⱖ 11) was present in similar frequency in both groups [obese: 3/38 (9%) vs control: 1/48 (2%)](p⫽NS). Conclusion: These data support the hypothesis that obesity has a negative impact on the osteoarticular health by promoting biomechanical changes in lumbar spine and lower extremities and sleep disturbances. Disclosure:

Disclosure:

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Back pain Lower extremity joint pain Upper extremity joint pain Lower extremity soft tissue pain Signs Scoliosis Genu valgum Genu recurvatum Flat feet Lumbar hyperlordosis Tighthamstrings Tight quadriceps

Periprosthetic bone loss following total joint arthroplasties is a major clinical problem resulting in implant failure. Generation of particulate wear debris from implant material activates macrophages/monocytes in the periprosthetic space and these cells release various mediators including proinflammatory cytokines (TNF-␣, IL-6 and IL-1␤). While the presence of these cytokines and wear debris leads to the activation of osteoclasts resulting in increased bone resorption, these components also alter osteoblast functions, which may further contribute to the imbalance of bone turnover. In this study we focused on the effects of particulate wear debris and TNF-␣ on bone marrow-derived primary human osteoblasts. TNF-␣- and wear debris-treated human osteoblasts released a several-folds increased amount of IL-6, a cytokine which is a key osteoclast-activating agent. Simultaneously, TNF-␣- and wear debris-challenged primary human osteoblasts exhibited suppressed procollagen a1[I] mRNA expression and type I collagen synthesis losing their capacity to replace the resorbed bone. The suppression of collagen seems to be independent of IL-6 release, because exogenous IL-6 had no effect on collagen expression, and protein synthesis inhibitor cyclohexamide blocked IL-6 release but did not have any effect on TNF-␣- or wear debris-induced suppression of procollagen ␣1[I] mRNA. The base level secretion of IL-6 was inhibited by bisphosphonate Pamidronate and 1,25-dihydroxy-vitamin-D3 (1,25(OH)2D3), whereas TNF-␣ and wear debris-induced IL-6 release was marginally affected by 1,25(OH)2D3, but abolished by Pamidronate. In contrast, 1,25(OH)2D3 seemed to be more valuable in the normalization of the suppressed collagen production in primary human osteoblasts as it could completely reverse the expression of procollagen a1[I] mRNA and type I collagen synthesis. These results provide a mechanism by which osteoblasts may play an important role in both osteoclast activation and suppressed production of bone matrix in the periprosthetic space. In addition, the combination of Pamidronate and 1,25(OH)2D3 may have an additive effect to normalize IL-6 and type I collagen production in osteoblasts leading to equilibrated osteoclast and osteoblast functions i.e. to a balanced bone turnover.

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METACARPOPHALANGEAL ARTHROPLASTY IN RHEUMATOID ARTHRITIS:WHAT DETERMINES SATISFACTION WITH SURGERY? Lisa A Mandl, Joanne P Bosch, Cynthia C George, Dina Galvin, Teal P Axt, Barry P Simmons, Jeffrey N Katz Boston, MA

FRACTURE INCIDENCE IN THE FRAIL ELDERLY. Jane M Zochling, T C Lau, Jennifer M Schwarz, Lyn M March, Robert G Cumming, Ian D Cameron, Philip N Sambrook Sydney, New South Wales, Australia

BACKGROUND: Studies of silastic spacers for metacarpolphalangeal(MCP)joint reconstruction in rheumatoid arthritis (RA) have focused primarily on objective outcomes,although some subjective measures have been investigated. We investigate which outcomes most strongly correlate with patient satisfaction. METHODS: We assembled a retrospective cohort of 26 RA patients with MCP arthroplasties. Patients answered a telephone survey using the Michigan Hand Questionnaire, plus additional questions asking about pain relief,performance of activities of daily living, and satisfaction with surgery. 18/26 patients also agreed to be examined. The strength of association between outcome variables and overall patient satisfaction with surgery was measured using Spearman correlations. RESULTS: 78% were female with a mean age of 63.5 years (39.6-83.4). The mean time since last MCP surgery was 6.0 years (1.8 -11.1). The strongest determinant of patient satisfaction was post-operative hand appearance for both the dominant hand (DH),(Spearman r⫽0.60, p-value⬍ 0.01)and the non-dominant hand (NDH)(Spearman r⫽0.70, p-value⬍ 0.01). Pain relief was also highly correlated with satisfaction with surgery (Spearman r⫽0.46, p-value⬍ 0.05 DH, Spearman r⫽0.67, p-value⬍ 0.01 NDH).The ability to perform activities of daily living was moderately correlated with patient satisfaction. (Spearman r⫽0.37 DH and r⫽0.30 NDH, p-value⬎ 0.05). Key grip was moderately correlated with patient satisfaction only in the NDH (Spearman r⫽0.37, p-value⬎ 0.05). Other measures of hand strength and range of motion, such as grip strength, 3-point pinch and active and passive MCP motion showed only minimal correlation with patients overall satisfaction with surgery(Spearman r⫽0.03-0.29, p-value ⬎ 0.05). CONCLUSION: Overall satisfaction with silastic spacer surgery in this cohort of RA patients was most influenced by post-operative hand appearance and by pain. While objective measures of surgical outcomes are valuable reflections of technical success, they are not important determinants of patient satisfaction. The criteria used to assess MCP arthroplasty results should be revised to better capture the outcomes that appear to matter most to patients.

The purpose was to prospectively determine the incidence of fractures in an elderly institutionalised population (Fracture Risk Epidemiology in the Elderly, FREE, study). Residents of a random sample of 24 nursing homes and 17 hostels in the Northern Sydney Health Area were invited to participate. Baseline assessment of fracture risks was carried out by questionnaire, medical record review and physical examination including falls risk parameters and calcaneal ultrasound. Medical records were then reviewed at a regular interval for documented falls and fractures. Fractures were confirmed by radiological report. Follow-up time ranged from 3 to 18 months, representing 986 person-years of observation. Over this time, 88 fractures have been seen in 82 individuals. Twenty-nine (35%) were hip fractures, 9 (11%) wrist fractures and 3 (4%) fractured neck of humerus. Three vertebral crush fractures were documented. Three individuals suffered two fractures from a single fall; five residents suffered one fracture on repeated occasions. Total fracture incidence was 596 per 10,000 person-years, and of hip fracture was 216 per 10,000 person-years. There were no significant differences in fracture rate between institution type or gender. Fracture rates were found to be much higher than in previous community-based studies, with the exception of vertebral fractures. This is likely to be in part due to under-investigation of back pain in the frail institutionalised elderly. Disclosure:

Disclosure: This study was funded in part by Wright Medical Technology.

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CHRONIC LOW BACK PAIN AND MODIC CLASSIFICATION. Remy Dupuy, Hadja Rakotovao, Nathalie Komosa, Nadia Mehsen, Chantal Dumoulin, Karine Bechara, Joel Dehais, Thierry Schaeverbeke Bordeaux, France

FACTORS RELATED TO PHYSICAL FUNCTION OUTCOME MEASURES IN NON - INFLAMMATORY RHEUMATIC DISEASE. Emma K Guymer, David A Williams, Jill Cunningham, Dakinah Sinkfield, Richard H Gracely, Daniel J Clauw Washington, D.C. and Bethesda, MD

Modic’s classification has been proposed from MRI observations. Although no correlation with clinical state has been evaluated yet, this classification is often used to justify lumbar spine arthrodesis. Objectives: To compare a group of patients with low back pain presenting with a discopathy confirmed by vertebral plates remodeling identified on MRI study (MODIC’s grades) with a group of chronic low back pain without MRI abnormality of the vertebral plates. Methods: Selection of the patients suffering from chronic low back pain who underwent an MRI examination of their lumbar spine. Clinical evaluation, including the VAS pain, the EIFEL and DRAD scoring. Results: 41 patients were included, distributed into 3 groups according to the results of the MRI. 12 patients without abnormality of MODIC (MODIC 0), 18 patients with MODIC 1 abnormalities and 11 patients with MODIC 2 abnormalities. Concerning pain: intensity is more important in the group MODIC 1 compared with the group MODIC 0 and with the group MODIC 2 (EVA ⫽ 53.6 mm vs 35.4 p ⬍ 0.015, and 30.9 p ⬍ 0.001), no significant difference was observed between the other groups. Concerning the functional capacity: average EIFEL score varied from 54.89 % to 57.95 % without significant difference between the groups. Concerning the quality of life: there was no significant difference between the groups on the various items of the DRAD. Conclusion: The discopathies of MODIC 1 is responsible for more intensive pain than the discopathies of MODIC 2 or Modic 0. Considering the natural evolution of the Modic 1 stage towards the Modic 2 stage in 2-3 years, the arthrodesis proposed in this indication does not seem to us necessary: a conservative treatment associating NSAIDs and lumbar brace should allow to wait for spontaneous evolution towards a less painful stage of discopathy.

Aim: To identify factors which relate to variation in self-report physical function outcome measures in individuals with non-inflammatory rheumatic disease. Methods: 211 subjects, between 50-80 (mean 65) years were recruited from the population for an “arthritis screening study”. Subjects with medical conditions that would affect function other than non-inflammatory rheumatic disease were excluded. The participants completed 3 self-report physical function measures - Roland Morris Disability Questionnaire (RM), WOMAC physical function score (WOMAC-pf) and SF36 physical function score (SF36-pf). Information regarding demographics, general health status and psychosocial factors was collected. Subjects were evaluated by dolorimetry measures of tenderness, and by lumbar and knee radiographs that were scored by a blinded rheumatologist. First order correlations between physical function measures and demographic variables, physical function, psychosocial factors and tenderness were performed. A stepwise hierarchical regression analysis using significant first order correlations was used to identify the factors predicting variance for each of the physical function outcome measures. Results: Body Mass Index (BMI) and depression (Beck Depression Inventory) were significantly correlated in first order correlations with RM, and both predicted variance in regression equations (cumulative R2⫽0.18 and 0.25 respectively). WOMAC-ps was significantly correlated with BMI, depression, tenderness, internal locus of pain control (ILPC), anxiety (Spielberger), radiological grading of spine disease (X-Ray spine) and fitness (Minnesota Heart Health Questionnaire). Unique contributors to variance in WOMAC-pf were depression (R2⫽0.15) ⫹ BMI (R2⫽0.21) ⫹ tenderness (R2⫽0.26) and X Ray spine (R2⫽0.31). SF36-pf was significantly correlated with BMI, depression, ILPC, fitness, tenderness and anxiety. Significant determinants of SF36-pf were depression (R2⫽0.17) ⫹ BMI (R2⫽0.25) ⫹ tenderness (R2⫽0.30) ⫹ ILPC (R2⫽0.34) and fitness (R2⫽0.37). Conclusions: Self-report measures of physical function are influenced by multiple independent factors. BMI and depressive symptomatology were most significantly associated with all three physical outcome measures, whereas other variables such as fitness, pain threshold and radiographic changes (albiet in the spine), predict some of the variance in the WOMAC and SF-36 physical function scores.

Disclosure:


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PATIENT QUALITY OF LIFE DURING THE 12-MONTHS FOLLOWING TOTAL JOINT REPLACEMENT SURGERY. John D FitzGerald, E John Orav, Thomas H Lee, Edward R Marcantonio, Robert Poss, Lee M Goldman, Carol Mangione Los Angeles and San Francisco, CA; Boston, MA

THE KNEE PLETHYSMOGRAPH: A NEW METHOD FOR MEASURING POSTOPERATIVE SWELLING. Mogens S Hansen, Lotte Ibsen, Bent W Jakobsen, Thomas Christiansen, Karsten Kroner, Henrik Staunstrup, Troels S Jensen, Kristian Stengaard-Pedersen Aarhus, Denmark

Purpose: To determine whether patient socio-demographic and pre-operative clinical characteristics influence health-related quality of life (QOL) outcomes at 1-, 6- and 12-months after total joint replacement (TJR) surgery. Methods: 222 patients at a university hospital with an indication of osteoarthritis undergoing primary TJR between November 1990 and March 1993 were prospectively studied. The QOL dimensions of bodily pain (BP) and physical function (PF) were assessed pre-operatively and at the 3 post-operative timepoints using the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36). Results: BP and PF improved by 37 and 32 points on a 100-point scale respectively 12-months after TJR. At 1-month after surgery, despite improvements in BP, PF deteriorated. At 1-month after surgery, patients undergoing knee replacement experienced less improvement in BP than patients undergoing hip replacement. Patients who were either married or living with someone reported greater improvement in BP and PF. Patients aged 75 years and older reported greater improvement in BP. Although, women presented with poorer pre-operative QOL, the magnitude of improvement after surgery was similar to that experienced by men. Pre-operative BP or PF status was a strong predictor of patients respective post-operative BP or PF. Conclusions: Patients experienced dramatic improvements in BP and PF after TJR. However, the decline in PF at 1-month implies significant need for prolonged informal or formal patient assistance with basic PF after surgery. In this sample, greater social support was associated with better QOL outcomes.

Objective: To develop a method, by which the postoperative swelling of the knee joint can be measured on a daily basis. Method: A new plethysmographic method was described and compared to three commonly used measuring methods. The reproducibility was tested on 21 persons, who had two volume measurements made on each knee. The measurements were made with a measuring tape method, a water displacement method, and with the new plethysmographic method. The accuracy was tested on 39 ACL patients, who had their knees measured with a spiral CT-scanning and with the new plethysmographic method the day before and two days after an ACL-reconstruction in order to quantify the amount of postoperative knee swelling. Results: The difference between two consecutive measurements (mean⫾ SD): Measuring tape: 0.08 cm⫾ 0.71 cm; Water displacement: 2.5 ml ⫾ 27 ml; Knee plethysmography: -7.1 ml ⫾ 24 ml. No difference between the first and second measurement were seen in any of the methods. The average swelling of the operated knee measured with the: Measuring tape: 8.8 cm ⫾ 3.3 cm; Knee Plethysmograph: 581 ml ⫾ 198 ml; CT-scanning (mean ⫾ SD): 526 ml ⫾ 183 ml. The difference between the volume of the swelling in the operated knee measured with the plethysmograph compared to the spiral CT-scanning (mean⫾ SD): 55.5 ml ⫾ 69.0 ml. Probability 0.1 % for equal results. Discussion: The plethysmograph provides volumetric data, involves no X-ray or exposure to water. The patient can be seated during the measurement which can be made immediately postoperatively. The method provides no information on the distribution of the swelling between the subcutaneous tissue and the intra-articular space. Conclusion: The plethysmograph has an acceptable reproducibility and accuracy compared to other used methods. It is a new method for measuring knee swelling due to postoperative oedema when frequent measures are needed on a group of patients. Disclosure:

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SHOULDER ROTATOR CUFF CHANGES IN PATIENTS WITH RHEUMATOID ARTHRITIS. Kenji Hayashida, Tetsuya Tomita, Takahiro Ochi, Hideki Yoshikawa Hirakata, Osaka and Suita, Osaka

EARLY AND AGGRESSIVE TREATMENT OF RECENT-ONSET RHEUMATOID ARTHRITIS ABOLISHES THE RELATIONSHIP BETWEEN HLA CLASS II ANTIGENS AND THE PROGRESSION OF JOINT DESTRUCTION. Leroy R Lard, Maarten Boers, Arco Verhoeven, Johanna MW Hazes, Ferdinand C Breedveld, Rene RP de Vries, Eric Zanelli, Sjef van der Linden, Tom WJ Huizinga Leiden, Amsterdam and Maastricht, The Netherlands

(Objective) Rotator cuff is very important muscle for shoulder function and is often involved in a patient with rheumatoid arthritis (RA). We examined shoulder rotator cuff changes in RA patients using MRI. (Materials and Methods) Fourty-six shoulder MRI and X-ray films of 25 RA patients were evaluated. The thickness of rotator cuff tendon (supraspinatus; SSP, infraspinatus; ISP, and subscapularis; SBS) was measured, and pannus formation was also observed. MRI and X-ray films of 15 normal shoulders were also examined as a control. (Results) The most characteristic change of the rotator cuff of RA patients was thinning, which was recognized in 36 of 46 shoulders. Regarding the thickness of the rotator cuff, the SSP tendons of RA patients were significantly thinner than the normal tendons. According to the extent of bone destruction and pannus formation, the RA shoulders were classified into three groups as follows. Group A shoulder had no pannus formation and no bone destruction (n⫽6), group B shoulder had the pannus formation and no bone destruction (n⫽24), and group C shoulder had bone destruction (n⫽12). Thinning of the cuff tendon was not seen in the group A, affected the SSP tendon was seen in the group B and all tendons were involved in the group C. The cuff tendons were significantly thinner in shoulders with pannus than in those without pannus. (Conclusion) Thinning of rotator cuff, especially SSP, precedes bone destruction, and was related to pannus formation. Disclosure:

Introduction: HLA class II antigens influence disease progression as measured by extend of joint destruction in RA. This effect is supposed to be caused by formation of autoreactive T-cells after presentation of (auto)antigens in the context of HLA class II. To investigate whether in patients with recent-onset RA early and aggressive DMARD treatment could prevent the involvement of autoreactive T cells, we analyzed the association of HLA class II and joint damage in two different RA patients cohorts treated according to different treatment strategies. Methods: The first patient cohort consisted of 216 patients which were treated according to the pyramid strategy (n⫽109, median duration of disease 4 months) or treated immediately with chloroquine or sulfasalazine (n⫽97, median duration of disease 2 weeks). The second cohort consisted of 155 patients with early RA (median duration of RA disease 4 months), which were randomly assigned combination (COBRA) treatment with step-down prednisolone, methotrexate and sulfasalazine (n⫽76) or sulfasalazine alone (n⫽79). Prednisolone and MTX were tapered and stopped after 28 and 40 weeks, respectively [Boers, Lancet ’97]. DNA isolation and DRB1 typing were performed. Extend of joint damage was measured by the modified Sharp method. Results: In the first cohort, the median increase in Sharp score in the shared epitope (SE) ⫹ patients of the pyramid group was 11.5 in contrast to 1.0 in the SE- patients after 2 years (P⫽0.002). However, in the group treated immediately, the median increase in joint damage in the SE⫹ patients was 3.0 in contrast to 2.0 in the SE- patients (P⫽0.7). In the second cohort, the median increase in Sharp score in the conventional sulfasalazine group after one year for DR4⫹ patients was 11.0 compared to 3.0 in the DR4- patients (P⫽0.006). In contrast, in the combination treatment group the median increase in the DR4⫹ patients at risk was 3.5 compared to 2.2 in the DR4-patients (P⫽0.46).These results were confirmed by multiple regression on the log-transformed scores. Conclusion: Early and aggressive anti-rheumatic drug treatment abolishes the association of HLA class II alleles with initial progression of joint damage in RA. Disclosure:

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ASSESMENT OF A BIOFEEDBACK PROGRAM TO TREAT CHRONIC LOW BACK PAIN. Jamil Natour, Karina Fonseca Sousa, Adriana Garcia Orfale, Jose Roberto Leite

ASSOCIATION BETWEEN A POLYMORPHISM IN THE MMP-3 GENE PROMOTER AND SEVERITY OF RA IN A PROSPECTIVE STUDY. Arnaud Constantin, Valerie Lauwers-Cances, Frederique Navaux, Michel Abbal, Joost PM van Meerwijk, Bernard Mazieres, Anne Cambon-Thomsen, Alain Cantagrel Toulouse, France

Introduction: Low back pain is a common problem that can affect up to 80% of adults during their lives and up to 10% become chronic. The symptoms of these chronic pain patients often include affective, cognitive and behavioural components. Various interventions heve been found to be beneficial in the rehabilitation of chronic low back pain patients, one of them is biofeedback. Objectives: Asses the efficacy of a biofeedback program for chronic low back pain patients and its impact in their pain, disabilities, and depression and anxiety symptoms. It also intended to promove higher (EMG) levels of abdominal muscles without increasing EMG levels of paraspinal muscles. Material and methods: Sixty patients with low back pain for more than 3 months were randomly assigned into a control and a treatment group.Subjects in both groups were oriented to take analgesics when necessary. The treatment group participated during 8 weeks in a biofeedback program that included: relaxation trainning and abdominal strength exercises with and without biofeedback and cognitive reestructuring techniques. The outcome measures used were Visual Analogue Scale (VAS), Roland-Morris questionnaire, Schober Index, Beck Depression Inventory (BDI) and State-trait Anxiety Inventory (STAI) in the begining and at the end of the observation period. EMG levels of abdominals and paraspinal muscles were registered in the begining, after 1 month and at the end of the observation period. Results: At the end of the observation period treatment group patients improved significantly on VAS (p⫽0,012) and STAI (p⫽0,003). Roland-Morris questionnaire improved in both groups and there were no differences between them (treatment p⫽0,0001 and control p⫽0,006). Only the control group improved significantly on BDI (p⫽0,013). There were no differences in the EMG levels of abdominals and paraspinals muscles. Conclusions: We can conclude that our biofeedback program reduced pain and anxiety symptoms of chronic low back pain patients. The life quality improved in both groups. The program didn’t interfere with depression symptoms and didn’t change the paraspinal EMG levels while contracting abdominal muscles.

Non-HLA genes contribute to the pathogenesis of RA. MMP-3 is considered as the main metalloproteinase involved in cartilage degradation. We tested the hypothesis of an association between a polymorphism in the MMP-3 gene promoter and susceptibility to, and severity of, RA. One hundred and three early RA patients (⬍1 year, ACR 1987 criteria) were included in this prospective longitudinal study. The total radiographic damage score (TRDS) was used to quantify RA severity at baseline and after four years of follow-up (Sharp/van der Heijde method). The 5A/6A biallelic polymorphism in the MMP-3 gene promoter (gene map locus 11q23) was analyzed using a fluorescent-based PCR. One hundred and twenty-seven unrelated healthy individuals were used as controls. Allele and genotype frequencies did not differ between patients and controls (Chi-square test, data not shown). The distribution of the TRDS was different across all three MMP-3 genotypes, both at baseline and after four years of follow-up (Kruskal-Wallis test, see table). The MMP-3 6A/6A genotype was associated with the highest TRDS while the 5A/5A genotype was associated with the lowest. This association concerned both the erosive and the joint space narrowing components of the radiographic damage score. The results of the present prospective longitudinal study provide the first evidence of an association between a polymorphism in the MMP-3 gene promoter and severity of RA.

Baseline TRDS, median (range) Four-year follow-up TRDS, median (range)

5A/5A genotype (n ⫽ 23)



p-value

0 (0-9) 3 (0-109)

0 (0-58) 11.5 (0-115)

4 (0-49) 17 (0-190)

0.0053 0.018

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EPIDURAL VERSUS FORAMINAL CORTICOID INJECTIONS IN DISCAL RADICULOPATHY. A DOUBLE-BLIND CONTROLLED TRIAL. Eric Thomas, Catherine Cyteval, Laurent Abiad, Francis Blotman Montpellier, France

CD154 (CD40 LIGAND) STIMULATES TNF-␣ PRODUCTION OF SYNOVIAL MACROPHAGES FROM PATIENTS WITH RHEUMATOID ARTHRITIS VIA ACTIVATION OF p38. Masayoshi Harigai, Tomoko Sugiura, Satomi Ohsako-Higami, Chikako Fukasawa, Miki Nakagawa, Hisae Ichida, Kae Takagi, Yasushi Kawaguchi, Masako Hara, Naoyuki Kamatani Shinjuku, Tokyo, Japan and Musashimurayama, Tokyo, Japan

Disclosure:

OBJECTIVE. We have previously reported that CD40 and CD154 (CD40 ligand) is expressed in synovial tissue from patients with rheumatoid arthritis (RA) and that CD40/CD154 interaction can augment tumor necrosis factor-␣ (TNF-␣) and interleukin-1␤ production of primary synovial cell culture. In the present study, we set out to explore the mechanism of CD154-induced TNF-␣ production of synovial macrophages. METHODS. Effect of CD40 ligation on TNF-␣ promoter activity was examined by luciferase assay. Briefly, HEK293 cells were cotransfected with human CD40 expression plasmid (pcDNA3-CD40) and a luciferase vector containing the promoter region of TNF-␣ (-1229/⫹129 relative to transcription start site) (pTNF-␣-luc). Phosphorylation of p38 and extracellular-signal-regulated kinase (ERK) was determined by Western blotting. TNF-␣ in culture supernatants was measured by ELISA. Recombinant soluble CD154 (rsCD154), a trimeric form of CD154, was kindly provided by Immunex Corp. Synovial macrophages were separated from primary culture of synovial cells by anti-CD14 antibody-coated magnet beads using MACS system. RESULTS. Overexpression of CD40 resulted in only 1.24-fold induction of luciferase activity from pTNF-␣-luc. Experiments with luciferase vectors containing 5⬘-deleted promoter region of TNF-␣ showed similar results. ERK, but not p38, was constitutively phosphorylated in synovial macrophages from RA patients. Stimulation of synovial macrophages from RA patients with rsCD154 and interferon-␥ (IFN-␥) induced phosphorylation of p38. rsCD154 plus IFN-␥ enhanced TNF-␣ production of synovial macrophages from RA patients. SB202190, a potent cell-permeable inhibitor of p38, significantly blocked the effect of rsCD154 plus IFN-␥ on TNF-␣ production by 61% as compared to that without SB202190. CONCLUSION. Ligation of CD40 by CD154 activated p38 phosphrylation of synovial macrophages, which contributed to TNF-␣ production. CD40 overexpression, which has similar biological effects to CD40 ligation, only weakly stimulated TNF-␣ promoter activity. These data suggest relative importance of posttranscriptional regulation in TNF-␣ production by CD40 ligation in synovial macrophages from RA patients. Disclosure:

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Epidural corticoid injections are widely used in the treatment of discal radiculopathy but their efficacy is still debated. In open studies, foraminal injections were shown to be mainly efficacious in patients with lateral herniation of a lumbar disc. However, the comparative efficacy, on discal radiculopathy, of each type of injection is not known. The present study concerned 31 patients (13 men, 18 women), aged 50.2 ⫾ 15.4 years, with discal radiculopathy lasting for less than 3 months, and confirmed by MRI or CT scans. They were randomized to receive either epidural (N ⫽ 16) or foraminal (N ⫽ 15) corticoid injection (Dexamethasone 5 mg in 2 ml). Before and 30 days after injection, the following items were assessed : radicular pain on 100 mm VAS, lumbar mobility (Schober’s index), low back handicap (Dallas’ and Rosser’s scales). At day 30, pain was improved in both groups, but more significantly in the foraminal group [mean comparison by Mann Whitney Wilcoxon test]. Lumbar mobility was improved in each group, but without significant intergroup difference. In the epidural group, all Dallas’ and Rosser’s scale items significantly and positively changed. In the foraminal group, improvement was only shown in 3 out of 4 items of Dallas’ scale and no change was seen when using Rosser’s scale. Foraminal injections seem more efficacious on pain control than epidural injections, but have not global influence on quality of life in patients with discal radiculopathy. Long term trials (⬎ 6 months) are needed.

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UP-REGULATION OF TRANSCRIPTIONAL ACTIVITY BY A SINGLE NUCLEOTIDE POLYMORPHISM AT THE 5⬘ FLANKING REGION OF SAA1 GENE THAT WAS ASSOCIATED WITH RISK FOR AA-AMYLOIDOSIS SECONDARY TO RA. Hirotaka Kaneko, Chihiro Terai, Masato Moriguchi, Yumi Koseki, Hiroshi Kajiyama, Naoyuki Kamatani Tokyo, Japan AA-amyloidosis characterized by deposition of amyloid A (AA) protein, is one of the most serious complications of RA. Serum amyloid A1 (SAA1), a predominant precursor of AA protein, is encoded by SAA1 gene on chromosome 11. Among three haplotypes of SAA1 at exon3, SAA1␥ and some of SAA1␤ associate with AA-amyloidosis in Japanese RA patients. AA-amyloidosis is linked with SAA1␣ in Caucasian where the frequency of AA-amyloidosis and SAA1␥ are very low. We found a novel SNP in the SAA1 promoter region, -13T/C, that showed stronger association with AA-amyloidosis than SAA1 exon 3 haplotypes. The -13T was found in all of 44 Japanese patients with AA-amyloidosis except one. In addition, we found tight linkage between the -13T and SAA1␥ and some of SAA1␤ in Japanese, whilst -13T was linked to part of SAA1␣ , rather than SAA1␤ in Caucasian. Thus, the SAA1 -13T is suggested to be primarily associated with AA-amyloidosis-risk. Objective: To address whether the differences in SAA1 promoter haplotypes effect on transcriptional activity of SAA1 gene. Methods: Four human SAA1 promoter haplotypes (ccg, Gcg, GcA, and cTg) defined by -61C/G, -13T/C, and -2 G/A SNPs, were cloned from genomic DNA of homozygotes of each SAA1 promoter haplotypes. The PCR fragments were cleaved with BglII and subcloned into the promoterless luciferase reporter plasmid. The pRL-Tk vector coding for Renilla luciferase was used as transfection efficiency control. The human hepatoma cell lines, HepG2 cells were transfected with one of these plasmids and stimulated with IL-1␤ (10 ng/ml) and IL-6 (10 ng/ml). Luciferase activities were determined at certain time after transfection and cytokine stimulation, with the Dual-Luciferase Reporter Assay System in a luminometer. Firefly luciferase values were normalized with the Renilla luciferase activity. Results: The relative luciferase activities of SAA1 promoter constructs were negligible without the cytokines stimulation to HepG2 cells and became maximum at 6 h after the cytokines stimulation. The activity of SAA1 promoter construct cTg containing -13T was highest and increased by 19% and 42% compared with constructs ccg and Gcg, respectively. The differences of activities between construct cTg and other constructs were significant. Conclusion: The SAA1 -13T might predispose RA patients toward AA-amyloidosis by increasing synthesis of SAA1 during long-standing course of RA inflammation.

B cell activation in the synovium has been considered to play an important role in the pathogenesis of rheumatoid arthritis (RA). Although previous studies revealed that type B synoviocytes have the capacity to support B cell differentiation, the role of type A synoviocytes in the B cell activation remains unclear. It has also been found that type A synoviocytes are derived from the bone marrow. The current studies therefore examined the capacity of bone marrow derived monocyte-lineage cells (BMMo) to activate human peripheral blood B cells. Bone marrow CD34⫹ cells from RA patients that had been stimulated with stem cell factor and GM-CSF for 3-4 weeks (⬎90% CD14⫹ HLA-DR⫹ cells, ⬍0.5% CD19⫹ B cells and ⬍0.5% CD3⫹ T cells) induced the production of IgG, but not that of IgM, by highly purified B cells from healthy donors in the presence of IL-2 and IL-10. The BMMo preferentially stimulated IgD- B cells, but not IgD⫹ B cells, to produce IgG. By contrast, soluble B cell activating factor belongiong to the TNF family (BAFF), but not BMMo, stimulated IgD⫹ B cells to produce IgM. The induction of IgG production by the BMMo was abrogated when they were separated from B cells by a membrane filter. Neither anti-CD154 mAb nor anti-CD70 mAb abrogated the IgG production induced by BMMo. These results indicate that upon stimulation with stem cell factor and GM-CSF, RA CD34⫹ progenitor cells differentiate into monocyte-lineage cells that preferentially activate IgD- B cells to produce IgG through direct cellular interactions that do not involve BAFF. The data therefore suggest that type A synoviocytes might also play a role in B cell activation in the RA synovium.

Disclosure:

Disclosure: Supported by the Promotion of fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Productive Review of Japan.

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p53R2, THE p53-DEPENDENT RIBONUCLEOTIDE REDUCTASE GENE, IS EXPRESSED IN RHEUMATOID SYNOVIUM. Kimio Masuda, Riako Masuda, Michel Neidhart, Christian A Seemayer, Beat R Simmen, Beat A Michel, Renate E Gay, Steffen Gay Zurich, Switzerland

INFLAMMATORY ACUTE PHASE REACTANT IN RHEUMATOID ARTHRITIS, ALPHA 1-ANTITRYPSIN AND C-REACTIVE PROTEIN, INHIBIT OSTEOCLASTOGENESIS. Shigeru Kotake, Nobuyuki Udagawa, Tasuku Okamoto, Yuki Nanke, Kang Jung Kim, Shigeki Momohara, Seiji Saito, Naoyuki Takahashi, Hideaki Oda, Taisuke Tomatsu, Naoyuki Kamatani Tokyo, Japan

Background: Although, it was reported that p53 was highly expressed in rheumatoid synovium, a low rate of apoptosis was also observed. Recently, p53R2 was identified as a p53-dependent ribonucleotide reductase gene associated with inhibiting apoptosis. The aim of this study was to investigate the expression of p53R2 in cultured rheumatoid arthritis synovial fibroblasts (RA-SF) and synovial tissues from patients with RA. Materials and Methods: Seven RA-SF, 2 osteoarthritis (OA)-SF, 1 SF from normal synovium, and 2 normal skin fibroblasts (passages 3-6) were used for RT-PCR. Total RNA was extracted from each cultured fibroblasts, reverse transcribed and amplified with the primers specific for p53R2. The products were analyzed on 1.0 % agarose gels in comparison with the expression of actin beta. The expression and distribution of p53R2 gene were also examined in paraffin sections of synovial tissues from 11 patients with RA and 3 normal individuals by in situ hybridization with digoxigenin-labeled RNA probes specific for p53R2. Results: In vitro, the expression of p53R2 gene was observed by RT-PCR in all RA- and OA-SF as well as in SF from normal synovium and normal skin fibroblasts. In situ hybridization showed that p53R2 mRNA was predominantly expressed in synovial tissues from patients with RA (9/11), especially in lining and sublining layers. Moreover, in some cases, the expression of p53R2 gene was also observed at sites of bone and cartilage destruction (2/4). In contrast, no expression was detected in normal synovial tissues (0/3). Conclusions: These data indicate that the induction of the p53R2 gene may present a novel mechanism contributing to the repair of damaged DNA and the inhibition of apoptotic pathways in the rheumatoid synovium.


MONOCYTE-LINEAGE CELLS INDUCED FROM BONE MARROW CD34⫹ PROGENITOR CELLS OF RHEUMATOID ARTHRITIS PATIENTS HAVE THE CAPACITY TO ACTIVATE IGD- B CELLS THROUGH DIRECT CELLULAR INTERACTIONS THAT DO NOT INVOLVE B CELL ACTIVATING FACTOR BELONGING TO THE TNF FAMILY (BAFF). Shunsei Hirohata, Tamiko Yanagida, Tetsuya Tomita, Takahiro Ochi Tokyo and Osaka

Objective:To identify a novel factor that inhibits osteoclastogenesis in the synovial tissue of patients with rheumatoid arthritis (RA). Materials and Methods:We purified a factor from RA synovial tissues by hydrophobic column chromatography followed by ion-exchange column chromatography, and chromatofocusing. A factor with the inhibitory effect of osteoclast formation in mouse co-culture system was further purified to apparent homogeneity by polyacrylamide gel electrophoresis. Results:The factor was finally identified as human alpha 1-antitrypsin by amino acid sequencing. Human alpha 1-antitrypsin (0.5 - 5␮g/ml) dose-dependently inhibited osteoclastogenesis in a co-culture of osteoblasts and bone marrow cells induced by 1,25-dihydroxyvitamine D3. Alpha 1-antitrypsin positive cells were immunohistologically detected in synovial tissue from patients with RA. Next, we investigated the effect of another inflammatory acute phase reactant, C-reactive protein (CRP), on osteoclastogenesis. CRP (1.0 - 3.0␮g/ml) dose-dependently inhibited osteoclastogenesis from human monocytes induced by soluble RANKL and M-CSF. Similarly, CRP strongly inhibited osteoclast differentiation in mouse co-culture system. Conclusions:These findings suggest that inflammatory acute phase reactants in RA, alpha 1-antitrypsin and CRP, inhibit osteoclastogenesis in human and mouse culture system. The production of these reactants is induced by proinflammatory cytokines such as IL-6, IL-1, or TNF alpha, which induce osteoclastogenesis. Thus, these acute phase reactants may play an important role in feedback inhibition in systemic and/or local osteoclastogenesis in inflammatory diseases such as RA.

Disclosure: K. Masuda is supported by Japan Rheumatism Foundation, R. Masuda by Uehara Memorial Foundation, C. A. Seemayer by Swiss Science Foundation, and all others by their respective institutions.

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THE CYTOKINE PROFILE OF THE RHEUMATOID SUBCUTANEOUS NODULE. John Highton, Paul Hessian, Annabel Kean, Cheuk Kwan Sun Dunedin, New Zealand

A POPULATION-BASED CASE-CONTROL STUDY OF RHEUMATOID ARTHRITIS AND SMOKING: RESULTS FROM THE BEHAVIORAL RISK FACTOR SURVEILLANCE SYSTEM. Eswar Krishnan Palo Alto, CA

Within the rheumatoid subcutaneous nodule, there is an inflammatory infiltrate in which monocytes/macrophages and T-lymphocytes are the predominant cells. The accumulated macrophages are in close opposition to resident fibroblasts, a situation similar to that occurring within the synovial membrane. Widespread production of the proinflammatory cytokines, interleukin-1␤ (IL-1␤) and tumor necrosis factor-␣ (TNF-␣) by macrophages is an additional feature common to both the nodule and synovial lesions. To further our understanding of the pathogenic mechanisms in operation within the rheumatoid nodule, we are examining the cytokine profile predominating within this lesion. Using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) methodology and mRNA extracted from excised nodules, our focus has been on cytokines that might reflect monocyte/macrophage and T-lymphocyte activation as well as polarisation towards a Th1 or Th2 immune response. The presence of transcripts for TNF-␣, IL-1␤, interferon-␥ (IFN-␥) and interleukins -2, -4, -5, -10, -12, -13, -15, -18 has been documented in separate nodules taken from 10 patients with rheumatoid arthriris. All nodules contained transcripts for TNF-␣, IL-1␤, and IL-10, -12, -15 and IL-18. In one nodule, transcript for IFN-␥ was not detected on repeated analysis. The remaining nodules were strongly positive for IFN-␥ transcript. In all 10 nodules examined, transcripts for IL-2, IL-4 and IL-5 were not detected while that for IL-13 was weakly positive in 5 of the 10 nodules. examined. The variability in IL-13 expression showed no correlation with the extent of expression of other cytokines examined. The cytokine profile of the rheumatoid subcutaneous nodule reflects significant monocyte/ macrophage activation (IL-10, IL-12, TNF-␣, IL-18). The presence of significant amounts of transcript for IFN-g, low levels of IL-13 and undetectable IL-4 and IL-5 is consistent with a Th1-type immune response driving the inflammatory process in the rheumatoid subcutaneous nodule.

Smoking has been reported to be associated with increased incidence and severity of Rheumatoid Arthritis (RA), Rheumatoid Factor (RF) and disability. Most of the case control studies reporting the association between smoking and RA have been hospital-based and thus potentially biased. This study is a population-based case- control study using the arthritis module data of the Behavioral Risk Factor Surveillance System (BRFSS)in 1999. BRFSS is a state-based annual telephone random-digit-dial sample survey of non-institutionalized adults. Cases: 563 self-reported cases of physician-diagnosed Rheumatoid Arthritis in seven states of the USA Controls: 1654 geographically matched individuals with no arthritis or rheumatic complaints were chosen as controls by computerized random sampling. Since the data was from a complex sample survey, a special analysis module in STATA was used to calculate means, proportions and to fit logistic regression models. Forward stepwise modeling was performed. Age, sex, income, education, insurance status, and race were retained in the model before introducing smoking variables. Cases were more likely to be women (68% vs. 57% p⬍0.001), older than 55 years (57% vs. 22% p⬍0.001) and have less than 12 years of education compared to controls (29% vs. 11% p⬍0.001). Cases and controls had similar racial composition. As expected, more cases than controls described their general health as poor (19% vs. 3%). Cases had activity limitation for longer time in the preceding 30 days than controls (15 versus 8 days). There was no statistically significant difference in the proportion of uninsured (15% vs. 17%). Cases were more likely to have ever smoked than controls (57% vs. 45 % p⬍0.01). On univariate analysis, ever-smokers had an odds ratio of 1.6 (95% Confidence Interval 1.2-2.1). In multivariate analysis after adjusting for other statistically significant covariates (age, sex, employment and poor general health) ever-smokers were at higher risk for Rheumatoid Arthritis (Odds ratio 1.5, 95% confidence interval 1.1-2.0) This study confirms the association between smoking and subsequent rheumatoid arthritis and provides additional data on illness impact. Ever smoking cigarettes confers a 50% higher risk of having RA compared to the never smoking.Because of the large size, population-based controls and special statistical analysis of this study, this observation is unlikely to be due to bias or random error.

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TENTATIVELY ASSIGNED 5 JAPANESE RA SUSCEPTIVILITY GENE AREAS, TIRA1-5, BY PRELIMINARY GENOME-WIDE SCREENING. Fujio Takeuchi, Maki Moritani, Hiromi Nabeta, Masaki Mori, Makoto Goto, Tadamasa Hanyu, Takashi Murayama, Sumiki Yamamoto, Nobuo Takubo, Norikazu Murata, Tsukasa Matsubara, Kunio Matsuta, Tadao Chihara, Akio Yamada, Hiroshi Sakuta, Mitsuo Itakura Tokyo, Tokyo, Tokushima, Tokushima, Niigata, Niigata, Kanazawa, Ishikawa, Matsuyama, Ehime, Osaka, Osaka, Kobe, Hyogo and Ichihara, Chiba, Japan

DIFFERENTIAL EXPRESSION OF THE ANGIOGENIC TIE RECEPTOR FAMILY IN ARTHRITIC AND NORMAL SYNOVIAL TISSUE. Shiva Shahrara, Michael V Volin, Mattew A Connors, G Kenneth Haines III, Alisa E Koch Chicago, IL

Objective: The susceptibility genes of rheumatoid arthritis (RA) have been studied by genomewide screening method to clarify the contribution of genetic factors of Japanese RA. Methods: Micro-satellites (MS) polymorphism (ABI-PRISM LMS-10 ver.1) were studied to investigate genetic linkages by affected sib-pair method. HLA-DR was also examined. As sample, 59 RA families with more than 2 affected sibs were used. Allele frequencies of MS of Japanese control have also been investigated. For analysis, MAPMAKER/SIB programs were used. Results: About 18% of MS showed heterozygosity less than 0.7. These data showed MS polymorphisms in Japanese were different from those in Caucasian. Preliminary data showed increases of Maximum LOD Score more than 1.5 in 5 lesions, tentatively identified as TIRA1-5. TIRA1-5 located near D3S1278, D11S904, D17S802, D18S452 and DXS1226, respectively, and showed LOD score of 1.8, 1.85, 2.1, 2.25 and 1.5, respectively.The frequency of the DR shared epitope was not significantly increased in the familial RA comparing to that in non-familial RA. Conclusion: Five RA susceptibility gene area, TIRA1-5, were tentatively shown by genome-wide screening using Japanese RA families. Disclosure:

Angiopoietins (Ang) are vascular endothelial cell-specific growth factors that play important roles principally during the later stages of angiogenesis. Angiogenesis is crucial for leukocyte ingress into the RA pannus. Using immunohistochemical analysis we examined the distribution of the receptor tyrosine kinase (Tie) and the angiopoietin ligands in human rheumatoid arthritis (RA) compared to osteoarthritis (OA) and normal (NL) synovial tissue (ST). Furthermore, Tie1 and 2 mRNA expression was quantified with real-time polymerase chain reaction in RA, OA, NL STs and RA synovial fibroblasts using GAPDH as an endogenous control. In RA, Ang-1 (agonist for Tie2) positive immunostaining on lining cells, macrophages and endothelial cells was significantly higher than on OA and NL ST. The Ang-2 (antagonist for Tie2) ST expression pattern was similar in RA and OA, whereas, Ang-2 expression was low in NL ST. The dominance of Ang-1 over Ang-2 protein expression in RA ST likely indicates a milieu favoring active neovascularization. Tie1 (an orphan receptor) and Tie2 (receptor for Ang-1 and -2) were more widely distributed in all disease groups compared to Ang-1 and Ang-2. RA and OA ST showed significant upregulation of Tie1 on lining cells, macrophages and endothelial cells compared to NL. Tie2 was significantly upregulated on RA and OA ST lining cells, macrophages and smooth muscle cells compared to NL ST. This may mean that the availability of Tie2 can be important for the initiation of angiogenesis. Tie1 and 2 mRNA levels were significantly higher in RA ST compared to OA (3 and 1.9 fold increase, respectively) and NL (9.5 and 4 fold increase, respectively). Also data obtained from OA ST demonstrated a significant increase in Tie1 and 2 mRNA expression compared to NL ST (3 and 2 fold increase, respectively). Interestingly, in correlation with protein data, we detected low levels of Tie1 and 2 mRNA in RA synovial fibroblasts. This finding indicates that Tie1 and Tie2 are not restricted to endothelium. In conclusion the distribution of Ang-1, Ang-2 and Tie2 in RA ST on macrophages and endothelial cells suggests an important role for these angiogenic molecules in the pathogenesis of RA. Hence, the upregulation of Ang-1 and Tie2 in RA ST is additional evidence for active angiogenesis in this disease. Disclosure:

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FIBRONECTIN GENE POLYMORPHISM IN SEVERE AND MILD RHEUMATOID ARTHRITIS (RA). Martina Fabris, Barbara Tolusso, Stefania Sacco, Grazia Favret, Gianfranco Ferraccioli Udine, Italy

ASSOCIATION OF RHEUMATOID ARTHRITIS (RA) TO CHROMOSOME 17Q22, HOMOLOGOUS TO A LOCUS LINKED TO ANIMAL MODELS OF INFLAMMATORY ARTHRITIS. Anne Barton, Johnny C Lorentzen, Lars Klareskog, William Ollier, Alan Silman, Jane Worthington Manchester, United Kingdom and Stockholm, Sweden

Background. The Extra-domain A containing Fibronectin (EDA⫹FN) is highly expressed in RA and it associates with activated or transformed states of synovium (Hino et al. Arth Rheum 1995; 38: 678-683). EDA⫹FN is generally expressed by proliferating and migrating cells and is more potent than FN lacking EDA (EDA-FN) in promoting cell adhesion and migration. We assessed the genotype segregation of the MspI FN gene polymorphism in RA comparing severe unresponsive and good MTX responder patients. Then we analyzed the role of this specific polymorphism on total FN and EDA⫹FN monocyte expression after LPS stimulation. Methods. MspI FN gene polymorphism genotyping was made according to Avila et al. (Am J Resp Cell Mol Biol 1999; 20: 106) on 61 controls and 79 RA patients split into two subgroups: 57 rheumatoids in stable partial remission (⬍3 swollen joints and a morning stiffness of ⬍20 min) since at least 6 months after MTX treatment (15 mg/w weekly, range 10-25 mg/w), thus called MTX-responders (MTX-R) and 22 rheumatoids (named MTX-NR) with still active disease (⬎6 swollen joints and a morning stiffness of at least 60 min) despite 6 months of combination therapy (MTX ⫹ Sulphasalazine ⫹ Hidroxychloroquine or MTX ⫹ Cyclosporine, plus prednisone 5 mg/day). p value was calculated by the Yates continuity corrected chi-square test. After 24 hours from isolation, monocytes from 5 RA patients and 5 healthy controls were incubated with LPS 100 ng/ml for 4, 8 and 24 hours and mRNA extracted using trizol reagent. FN expression and EDA⫹/EDA- FN ratio were evaluated by RT-PCR according to Tavian et al.(Int J Cancer 1994; 56: 820-5). Results are expressed as median value. Results. MTX-R had an overall frequency of DD homozygosity of 56.1% vs 52.5% in HDB, while in MTX-NR subgroup the DD genotype had a frequency of 81.8% (OR⫽4.1, CI⫽1.2-13, p⫽0.008 vs HDB). The comparison between MTX-NR and MTX-R was similarly striking (OR⫽3.5, CI⫽1.1-12, p⫽0.01). Monocytes stimulation did not disclosed any relevant difference between patients and controls, but after 4 hours of LPS treatment, we found a significant difference (p⫽0.03) in EDA⫹/EDA- ratio increase between DD (1,64 fold increase - CI: 1,2-8,19) and CD/CC subjects (1,25 fold increase; CI: 0,8-1,5). Conclusions. The DD genotype for MspI Fn polymorphism is prevalent among severe non-responder RA patients and seems to determine an important switch towards the expression of the more active EDA⫹FN isoform after LPS stimulation, probably contributing to a more aggressive phenotype in RA. Disclosure:

Background: RA is an oligogenic autoimmune disease. Although the major genetic susceptibility locus is known to map to the MHC region, this only accounts for 30-40% of the total genetic component to disease susceptibility. We have previously tested a number of regions homologous to loci identified in animal models of inflammatory arthritis for linkage in RA and have reported linkage to 17q22 (1,2). This region is syntenic to a locus linked to susceptibility in 2 animal models of inflammatory arthritis, oil-induced arthritis and collagen-induced arthritis. The linkage was replicated in independent cohorts of RA affected sibling pair (ASP) families. Demonstration of association to markers mapping to the region would narrow down the area thought to harbour the disease gene. Aim:To investigate association to 5 microsatellite markers mapping under the peak of linkage in RA ASP and single-case families. Patients and methods:Association was investigated in a test group of 300 RA ASP families and a replication cohort of 152 single-case families. Association was tested using a combined extended transmission disequilibrium test (ETDT) and sib-TDT to maximise the number of informative families available for analysis. Microsatellite markers were typed using fluorescence based automated genotyping. Results:Association to one of the markers mapping to the peak of linkage (D17S807) was demonstrated in the RA ASP families (p⫽0.02) with alleles 6 and 9 being preferentially transmitted. A haplotype of the same 2 alleles from this and an adjacent marker mapping just 0.3cM from it (D17S1821-D17S807) was associated with RA in nuclear families (␹22df 10.84, corrected p value ⫽ 0.014). Conclusion:Association to a marker mapping to the peak of linkage has been demonstrated in RA ASP families. This association has been replicated to a haplotype of alleles including this marker in an independent cohort of single-case RA families using extensions of the TDT. Following previous reports of linkage to a 4-5cM region of 17q, this association study has now refined the region likely to contain an RA disease susceptibility gene down to ⬃1cM, a distance which is amenable to physical mapping studies. (1) Barton et al. Arthritis Rheum 2000;43:Suppl 1. (2) Barton et al. Rheumatology 2001;40:Suppl 1. Disclosure:

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INCREASED SYSTEMIC ARTERIAL STIFFNESS, AS DETERMINED BY PULSE WAVE ANALYSIS, IN RHEUMATOID ARTHRITIS. Rainer Klocke, John R Cockcroft, Ian R Hall, Gordon J Taylor, David R Blake Bath and Cardiff, United Kingdom

MARKERS FOR BONE- AND CARTILAGE DAMAGE MEASURED DURING THE FIRST YEAR INDEPENDENTLY PREDICT RADIOLOGICAL PROGRESSION OVER 5 YEARS. R BM Landewe, P Geusens, D MFM van der Heijde, M Boers, Sj van der Linden The Netherlands

Background: Most epidemiological studies report increased cardiovascular (CV) mortality associated with rheumatoid arthritis (RA), for reasons that remain unclear. Chronic inflammation may impair endothelial function leading to increased arterial stiffness, which is an important determinant of CV risk. Non-invasive pulse wave analysis (PWA) (1) can measure systemic arterial stiffness as the augmentation index (AIx). AIx is the central systolic pressure augmentation, caused by peripheral pulse wave reflection, expressed as a percentage of the pulse pressure. AIx correlates with intra-arterial pressure recordings (2) and other measures of arterial stiffness (3). Method: 41 RA patients, aged ⱕ50 years, were screened and examined for the absence of clinical CV disease and risk factors, such as smoking, diabetes mellitus, hypercholesterolaemia and hypertension. 17 suitable subjects were further assessed regarding their RA activity, brachial blood pressure (BP) and AIx by radial PWA(SphygmoCor, PWV Medical, Sydney, Australia). Haemodynaemic measurements were in duplicates. Comparison was made with PWA data from healthy controls, matched closely for gender, age, peripheral BP, heart rate and height. Appropriate matches could be identified for 14 subjects with RA: 11 females; mean (SD) age 42.4 (5.8); mean RA duration 11.1 (6.3) years; mean C-reactive protein 19 (15) mg/l; median (IQR) 28-Tender-/Swollen Joint Score 6.0 (8.0) / 5.5 (7.5) resp. No subjects had clinical systemic rheumatoid vasculitis or were on prednisolone ⬎7.5 mg/day. Results: The mean AIx in the 14 RA subjects was higher compared to the healthy controls: 26.2⫾6.7(SD) vs. 18.9⫾10.8 %, p⬍0.03 by 2-tailed paired t-test. This difference is of similar magnitude, reported previously for type-1 diabetes mellitus subjects (mean age 30 years) without overt cardiovascular disease (4). Conclusion: RA patients exhibit evidence of increased systemic arterial stiffness compared to matched healthy controls, as assessed by PWA. Since they had no evidence of clinical cardiovascular disease or risk factors, it is suggested that the inflammatory process associated with RA is responsible for the increased arterial stiffness. References:(1)Kelly R,et al.Circulation 80,1652-1659,1989. (2)Chen CH,et al.Circulation 95,1827-1836,1997. (3)Liang YL, et al.Clin Sci (Colch) 95,669-679,1998. (4)Wilkinson IB,et al.QJM 93,441-448,2000.

Objectives: to investigate whether measures of bone/cartilage degradation can predict radiological progression over time independent of measures of disease activity/inflammation Patients & methods: Data of the COBRA-treatment cohort were used. Linear regression analysis with stepwise selction of variables was applied with “radiological progression rate” as dependent-, and urine deoxypyridinoline (DPD) excretion, DAS28, ESR, demographic data, Sharp-score, RF and HLA-DR4 as explanatory variables. Results: DPD correlated moderately well with DAS28 and ESR, but not with Sharp-score. DPD was a weak independent predictor of radiological progression in the “baseline model” (only baseline values) but a strong (stronger than DAS28 or ESR) independent predictor of radiological progression in the “time-averaged” model (first-year time-averaged estimates for DPD, DAS28, ESR). Time-averaged model

Change R2

P-value

Independent predictor

Change R2

P-value

1. ESR 2. Sharp-score 3. RF-status 4. Treatment (COBRA/SSZ) 5. DPD Adjusted model R2

0.19 0.11 0.07 0.04 0.03 0.41

0.005 0.0001 0.001 0.013 0.038

1. 2. 3. 4.

0.39 0.10 0.04 0.02

0.0001 0.001 0.003 0.02

Sharp-score time-averaged DPD RF-status time averaged DAS28

Adjusted model R2

0.56

Compared to DAS28 and ESR, DPD showed little tendency to change during the first year. Conclusions: DPD measured during the first year is associated with radiological progression later on. Although DPD partially follows fluctuations in DAS28/ESR, these results indicate that DPD also reflects separate pathophysiological processes not measured with DAS28 and/or ESR. Treatment modalities that specifically interfere with bone-and cartilage turnover should be sought to better inhibit radiological progression. Disclosure:

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OSTEOPROTEGERIN (OPG) IS A NATURAL INHIBITOR FOR TUMOR NECROSIS FACTORRELATED APOPTOSIS INDUCING LIGAND (TRAIL)-INDUCED APOPTOSIS IN SYNOVIAL CELLS. Taiichirou Miyashita, Atsushi Kawakami, Satoshi Yamasaki, Kiyoshi Migita, Katsumi Eguchi Nagasaki, Japan

ANGIOPOIETIN-1 IS EXPRESSED IN RA SYNOVIUM AND IS INDUCED BY TNF-␣ IN CULTURED SYNOVIAL FIBROBLASTS. Allison R Pettit, Ellen M Gravallese, Cathy Manning, Alfie Tsay, Rebecca Madore, John Gaspar, Steven R Goldring, Peter Oettgen Boston, MA

TRAIL is a dominant death-inducing ligand, and OPG is known as a soluble receptor for TRAIL. Inhibitory action of OPG for osteoclastgenesis, which is mediated through binding with receptor activator of NF-k B ligand (RANKL), has well been recognized, however, the role of OPG for TRAIL-eliciting signals remains to be obscure. Here we showed that OPG is a endogenous inhibitor for TRAIL-induced apoptosis in cultured synovial cells, which may involve in rheumatoid synovial cell hyperplasia. [MATERIALS AND METHODS] Protein concentration of OPG and IL-1 b in synovial fluids (SF) from RA patients was examined by ELISA. Fibroblast-like synovial cells (FLS) were isolated from the synovial tissue from patients with arthritis (RA), and functional significance of OPG for TRAILinduced apoptosis in FLS was investigated in vitro. [RESULTS] Protein concentration of OPG in SF from RA patients was positively correlated with that of IL-1 b. FLS produced OPG in the culture supernatants, and its production was significantly augmented by IL-1 b. Death receptor 5 (DR5), a death-inducing receptor for TRAIL, was expressed on FLS, and the addition of soluble TRAIL clearly induced apoptosis in FLS. Functional role of OPG for TRAIL-induced apoptosis in FLS was demonstrated, which showed that most of cytotoxic activity of soluble TRAIL is abolished by adding OPG fusion protein. [CONCLUSIONS] Our present study suggests that TRAIL is an important regulator for apoptotic cell death of synovial cells, and OPG appears to be an autocrine inhibitor for TRAIL-induced synovial cell apoptosis. Furthermore, the known role of IL-1 b for the progression of synovial cell hyperplasia in RA patients, may, in part, to be attributed with augmentation of OPG synthesis from synovial cells.

Angiogenesis is a critical component of the inflammation associated with rheumatoid arthritis (RA). Several angiogenic growth factors that promote the early stages of angiogenesis, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), are expressed in rheumatoid synovium. The angiopoietins are a newly identified family of angiogenic factors. Angiopoietin-1 (Ang-1) and its endothelial-specific receptor Tie2 are critical mediators of the later stages of blood vessel development and maturation. Ang-2 is a competitive blocker of Ang-1. The role of the angiopoietins in inflammatory disorders has not been studied. To evaluate the potential role of these proteins in RA we examined Ang-1 and Ang-2 expression in RA synovial tissue samples. In situ hybridization (ISH) and immunohistochemistry demonstrated that both Ang-1 and Ang-2 are expressed in RA synovium. Ang-1 was observed predominantly in cells in perivascular sites, and in the synovial lining layer in 6 of 8 samples analyzed. Some heterogeneity of Ang-1 expression was noted among samples, which may reflect differences in the disease stage in these patients. To define the factors involved in regulation of Ang-1 and Ang-2 in RA synovium, RNA was prepared from cultured synovial fibroblasts before and after stimulation with tumor necrosis factor alpha (TNF-␣). Northern blot analysis revealed induction of Ang-1 mRNA with TNF-␣. Furthermore, significant induction by TNF-␣ of Ang-1 protein was demonstrated by ELISA analysis of culture supernatants and cell lysates. Focal expression of Ang-2 mRNA was detected by ISH within blood vessels in RA synovial tissues. Ang-2 mRNA was weakly expressed in cultured synovial fibroblasts and could not be induced in response to TNF-␣. These results suggest that Ang-1 may be an important regulator of angiogenesis in RA, and that TNF-␣ may act to promote blood vessel maturation in this disease through induction of Ang-1 in synovial fibroblasts. Disclosure:


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PRO-INFLAMMATORY ROLE OF PROTEIN I/II, A MODULIN FROM ORAL STREPTOCOCCI IN SYNOVIOCYTES ISOLATED EITHER FROM RHEUMATOID ARTHRITIS OR OSTEOARTHRITIS. Laurence Neff, Mirjam Zeisel, Marie Scholler, Francois-Xavier Limbach, Jean-Paul Klein, Dominique Wachsmann, Jean Sibilia Strasbourg, France

TNF-␣ PROTECTS HUMAN SYNOVIOCYTES FROM APOPTOSIS INDUCED BY NITRIC OXIDE. Clio Ribbens, Biserka Relic, Nathalie Franchimont, Alfredo Rodriguez, Nicole Schaaf-Lafontaine, PierreAndre Guerne, Michel G Malaise Liege, Belgium and Geneva, Switzerland

Synoviocyte fibroblast-like (FLSs) are key cells involved in joint inflammation and destruction in rheumatoid arthritis (RA) and recent studies have shown that they are permanently modified by the rheumatoid environment. Objective-Methods : we examined potential mechanisms of pro-inflammatory cytokines release in RA, in response to bacterial components, by studying IL-6, IL-8 and IL-18 gene expression and signal trasnduction in FLSs isolated either from RA or osteoarthritis (OA) patients used as control. Results : 1.In this study using ELISA and RT-PCR, we showed that protein I/II, a modulin from oral streptococci, bacteria which rRNA could be present in the joint cavity of RA patients (Kempsell et al. Infect Immun 2000;68:6012), increases the synthesis of IL-6, IL-8 and IL-18 through transcriptional up regulation of the corresponding genes. 2.We found that RA FLSs release significantly more cytokines than OA FLSs after protein I/II stimulation. 3.We further investigated the signaling pathways leading to protein I/II-induced IL-6 and IL-8 release. The three main MAPKinases ( p38, ERK1/2 and JNK) were readily phosphorylated in protein I/II-stimulated RA and OA FLSs We further showed with SB 203580 and PD 98059, that protein I/II-induced IL-6 and IL-8 release is mediated by ERK 1/2 and JNK but not by p38 in both RA and OA synoviocytes. Conclusions -Some bacterial antigens (streptococcus or others) can modulate the inflammatory pathways of synoviocytes (FLSs) from RA or OA patients. -Streptococcal proteins (protein I/II) induced IL-6 and IL-8 release mediated by ERK 1/2 and JNK but not by P38 in RA and OA synoviocytes.

Introduction. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by hyperplasia of the synovium due, at least in part, to inadequate apoptosis. Nitric oxide (NO) is spontaneously produced by primary synovial cultures from RA and osteoarthritis (OA) patients and is able to induce apoptosis of human synoviocytes in vitro. The RA synovium is also characterized by an overproduction of the pro-inflammatory cytokine tumor necrosis factor-␣ (TNF-␣) which is targeted by biological agents used for the treatment of RA patients. Objective. The aim of our study was to evaluate the effect of TNF-␣ on synoviocyte apoptosis induced by the NO donor sodium-nitro-prusside (SNP). Methods. Synoviocytes were obtained from RA and OA patients undergoing arthroscopy or joint replacement surgery. The cells were pretreated with TNF-␣ in DMEM ⫹ 1% FCS for various time periods then treated with SNP for an additional 24h. In some experiments, cells were pretreated with anti-TNF␣ antibodies or the phosphatidylinositol-3 kinase (PI-3-K) inhibitor LY294002 30 min before TNF-␣ treatment. Cell survival was measured by the MTS colorimetric test. Apoptosis was confirmed by annexin V and propidium iodide FACS analysis. Results. TNF-␣ induced the proliferation of synoviocytes after 48 h in the absence of SNP (MTS test). SNP at 1 or 2 mM resulted in a 70 - 100% cytotoxic activity against synoviocytes. Pretreatment with TNF-␣ completely protected OA and RA synoviocytes from SNP-induced cell death at doses ranging from 0.1 to 30 ng/ml (MTS test). FACS analysis confirmed that SNP induced synoviocyte apoptosis as shown by the induction of annexin V-positive propidium iodide-negative cells (respectively 52% and 87% of cells at 1 and 2 mM SNP versus 5% of cells in control conditions). The protective effect of TNF-␣ was inhibited by anti-TNF-␣ antibodies. Pre-incubation of cells with the PI-3-K inhibitor Ly294002 at 10 mM decreased the anti-apoptotic effect of TNF-␣ by 20 to 50%. Conclusions. TNF-␣ inhibits synoviocyte apoptosis induced by NO and may therefore contribute to synovial hyperplasia. The antiapoptotic effect of TNF-␣ is decreased by a PI-3-K inhibitor, suggesting that it is mediated at least in part through the insulin receptor substrate pathway. This antiapoptotic effect of TNF-␣ may be another favorable feature of anti-TNF therapies used in RA.

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FIBROBLAST-LIKE SYNOVIOCYTES (FLS) FROM PATIENTS WITH RHEUMATOID ARTHRITIS SHOW AUGMENTED PROLIFERATIVE RESPONSES THAT MAY BE RELATED TO DEFECTIVE INDUCTION OF MITOGEN ACTIVATED PROTEIN KINASE (MAPK) ACTIVITY AFTER LIGATION OF THE FIBROFLAST GROWTH RECEPTOR (FGF-R). Fabiola A Placeres, Consuelo Lopez-Benitez, Evelyn J Alonzo, Ana M Blasini, Martin A Rodriguez Caracas, Venezuela

DIFFERENT REGULATION OF COLLAGENASE 3 AND INTERSTITIAL COLLAGENASE IN SYNOVIAL FIBROBLASTS WHEN MAINTAINED WITHIN THREE-DIMENSIONAL COLLAGEN GELS OR COIMPLANTED WITH NORMAL CARTILAGE IN NOD/SCID MICE. Dirk Wernicke, Claudia SchulzeWesthoff, Peter Petrow, Rolf Brauer, Wolfgang A Schmidt, Josef Zacher, Steffen Gay, Erika Gromnica-Ihle Berlin-Buch and Jena, Germany and Zurich, Switzerland

FLS from patients with rheumatoid arthritis (RA) exhibit a transformed phenotype that may be related to abnormal post-receptor signaling. Highly purified FLS (100 % vimentin-expressing, CD3-, CD14-, CD68- cells) were obtained from synovium during total joint prosthesis or synovectomy in patients with RA (n⫽5) and osteoarthritis(OA) (n⫽7). Cells were isolated by collagenase digestion, grew on DMEM-F12 medium,and studied in a period between the third to sixth passage. Unstimulated FLS from RA patients showed increased uptake of [ 3H]-thymidine, as compared to FLS from OA patients: 810⫾101 vs. 513⫾36 (m ⫾ SEM cpm, p⫽ 0.0001). Stimulation in vitro with increasing concentrations of human basic fibroblast growth factor (bFGF) led to an increment of [3H]-thymidine uptake in FLS from both groups. However, at all concentrations tested, RA FLS showed higher proliferative rates than FLS from OA patients (p⫽ 0.01). This pattern was consistent at 24, 48 and 72 hours time points of stimulation. Growth rates of various cell types can be influenced by the Ras/Raf/MAPK signaling pathway. We examined this cascade using a Western blot with a specific MAb recognizing the double phosphorylated phenotype of activated forms of ERK-1 and ERK-2, two relevant MAPKs in fibroblasts. As compared to FLS from OA patients, FLS from RA patients showed a significant decrease in the double phosphorylation of 44-kDa (ERK-1, p⫽0.0007) and 42-kDa (ERK-2, p⫽0.02), both in unstimulated cells and in cultures activated with increasing concentrations of bFGF. There was a negative correlation between ERK-1 and ERK-2 activity and proliferative responses in FLS: ERK-1: r⫽ -0.532, (p⫽0.0002), ERK-2: r⫽ -0.319, (p⫽0.03). Abnormal signaling in response to bFGF may interfere with activation of the MAPK cascade and contribute to unchecked growth of FLS in rheumatoid synovium. CONICIT No. 97000808.

In rheumatoid arthritis (RA) the matrix-metalloproteinases (MMPs) collagenase 3 and interstitial collagenase are expressed in synovial fibroblasts at sites of synovial invasion into cartilage. In order to compare the role of cell-matrix interactions for the induction of collagenase 3 and interstitial collagenase in synovial fibroblasts, the expression of both MMPs was investigated in primary synovial fibroblasts maintained within three-dimensional collagen gels or coimplanted with normal cartilage in immunodeficient NOD/SCID mice. Synovial fibroblasts coimplanted with normal cartilage in NOD/SCID mice invaded deeply into adjacent cartilage. In this in vivo system of cartilage destruction, collagenase 3 mRNA was induced in synovial fibroblasts at sites of cartilage erosion, whereas the expression of interstitial collagenase mRNA could not be detected. Culture of synovial fibroblasts within three-dimensional collagen gels was associated with a marked increase in collagenase 3 mRNA expression and proenzyme production. This stimulatory effect was one order of magnitude higher compared with the stimulation by IL-1␤ or TNF-␣. In contrast, mRNA expression and proenzyme production of interstitial collagenase were increased strongly and to a similar extent either by contact with three-dimensional collagen or by proinflammatory cytokines. Membrane-type 1 MMP and gelatinase A known to activate procollagenase 3 by a cell-surface associated proteolytic cascade are coordinately expressed with collagenase 3 in synovial fibroblasts at sites of cartilage erosion in cell-cartilage implants in NOD/SCID mice. The results suggest that the induction of collagenase 3 by cell-matrix interactions and its membrane-associated activation represent a pivotal mechanism contributing to the invasive phenotype of synovial fibroblasts at sites of synovial invasion into cartilage in RA. Disclosure:

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SYNOVIOCYTES FROM RHEUMATOID ARTHRITIS (RA) PRESENT FIBRINOLYTIC PATTERN OF THE INVASIVE CELLS. Marina Cinelli, Serena Guiducci, Angela Del Rosso, Marco Matucci Cerinic, Gabriella Fibbi, Mario Del Rosso Florence, Italy

EXPRESSION OF MAGE (MELANOMA ANTIGEN), A NOVEL AUTOANTIGEN, IN JUVENILE RHEUMATOID ARTHRITIS (JRA). Deborah McCurdy, LeiQian Tai, Karen L Imfeld, Frank Zaldivar, Monique Berman Orange, CA

Background: in RA, the fibrinolytic cascade is involved both in the generation of cartilage damage and in the genesis and maintenance of synovial inflammation. Aim: to evaluate on synoviocytes (SY) from RA patients and healthy donors (H): 1) the fibrinolytic pattern 2) the proliferative potential of urokinase-Plasminogen Activator (u-PA), in order to establish the role of each component of the fibrinolytic cascade in the genesis and progression of the rheumatoid synovitis. Materials and methods: the levels of plasminogen activators and inhibitors, and the u-PA dependent proliferation were studied in vitro on SY from 4 H and 4 RA subjects undergoing joint surgery. SY monolayers were used within the 7th passage in culture. Cells were seeded on to multiwell plates with RPMI 1640 supplemented with 10% fetal calf serum (FCS) and incubated for 48 h. Then, FCS concentration was reduced to 0.1% for additional 48 h. At the end of the incubation, the supernatants were removed, centrifugated and stocked at -20C° until the determination of u-PA and Plasminogen Activator Inhibitor-1 (PAI-1). Cells were detached, counted and lysed. The lysates were stocked at -20°C until the determination of urokinase Plasminogen Activator Receptor (u-PAR). The levels of u-PA, u-PAR and PAI-1 were assayed by specific ELISA. To evaluate the SY proliferative potential, cells were seeded on to multiwell plates with RPMI 1640 supplemented with 10% FCS and incubated for 48 h. Then, FCS concentration was reduced to 0.1% for additional 48 h. Then, SY were treated with u-PA 500 ng/ml and/or antibodies anti u-PA (5B4) and anti-u-PAR (R3) for 48 h and counted. Results: SY from RA, in respect to SY from H, showed significantly higher levels of PAI-1 [(6.34 mg/million cells ⫾ 1.13 standard deviation (SD) vs 2.9 mg/million cells ⫾ 0.7 SD, p⫽ 0.01], minor levels of u-PA (2.58 ng/million cells ⫾ 1.4 SD vs 10.94 ng/million cells ⫾ 2.1 SD, p⫽0.01), and more u-PAR on their surface (28.5 ng/million cells ⫾ 4.8 SD vs 13 ng/million cells ⫾ 3.0 SD, p⬍0.05). Treatment of SY from RA and H with u-PA provides a proliferative effect similar to 10% FCS (p⬍0.05), blocked by 5B4 and R3 and not different in RA in respect to H SY (p⬍0.05). Conclusions: These data show that RA SY present the typical fibrinolytic pattern of the invasive cells and that SY proliferation is stimulated by u-PA. Fibrinolytic system provide an extracellular proteolysis required for the invasion of articular tissues from SY and for the first steps of synovitis. The components of this system may be a future target for new RA therapies.

The Melanoma Antigen gene family (MAGE) is one of the important tumor antigen families recognized by cytotoxic T cells and is being studied as a vaccine in cancer therapies. MAGE A1-12 (located on chromosome Xq28) and MAGE B1-6 (Xp21.3-Xp22), and MAGE C1-3 (Xq26-27) code for proteins that are recognized as tumor antigens and may play a role in immune surveillance. MAGE family genes are important in development and are expressed during embryogenesis. MAGE proteins are not expressed in normal cells, except in spermatogonia. Using sera from two patients with systemic lupus erythematosus (SLE), we previously identified a member of the MAGE B family, MAGE Xp-2, from a cDNA expression library from a HEp-2 laryngeal carcinoma cell line. This suggested that this gene is not only expressed in certain tumors, but also in autoimmune disease. In this study we demonstrate the expression of MAGE A1 in cells from synovial fluid aspirates from patients with juvenile rheumatoid arthritis (JRA) using RT-PCR. Monoclonal antibody to MAGE A1 (Ab-4, Neomarkers, Fremont, CA) stains small and large aggregates of cells from synovial fluid. Flow cytometry shows expression of MAGE-1 in both the cytoplasm and on the cell surface. We hypothesize that viral infections or other environmental triggers induce the expression of developmental genes, perhaps as a mechanism of repair and replacement of damaged tissue. Expression and presentation of MAGE proteins, normally only expressed early in development, may not be tolerated as self-antigens and may elicit an immune response that initiates and perpetuates an autoimmune process. Disclosure:

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OVEREXPRESSION OF THE TRANSFORMING GROWTH FACTOR- ␤ - INDUCIBLE GENE H3, bIG-H3 IN RHEUMATOID ARTHRITIS. Chae-Gi Kim, Wern-Chan Yoon, Yong-Ho Song, Jung-Yoon Choe, Sang-Gyung Kim, Jong-Sup Bae, In-San Kim Daegu, Republic of Korea

DIVERSIFICATION OF THE EXPRESSED Ig HEAVY CHAIN VARIABLE REGION GENE REPERTOIRE IN SYNOVIAL TISSUE B LYMPHOCYTES BY NUCLEOTIDE INSERTION AND DELETION AND BY VH GENE REPLACEMENT. Yasushi Miura, Richard A Furie, Eugene S Krauss, David M Dines, Nicholas Chiorazzi Manhasset, NY

Background : In rheumatoid arthritis (RA), TGF-␤ has been implicated as an important modulator of synovial fibroblast hyperplasia and pathology, though the precise mechanism was not established yet. TGF-␤-inducible gene h3 (␤ig-h3) is a novel protein that can be induced by treatment of TGF-␤ in some kinds of cells and it has been detected in several human tissues. Recently, it was suggested that the ␤ig-h3 might play a potential role in the extracellular matrix formation and tissue fibrosis in several corneal and skin diseases. But, its expression in RA has not been studied. Purposes: The purposes of this study were 1) to investigate the ␤ig-h3 expression in RA and compare it with those in osteoarthritis (OA), and 2) to evaluate the interaction between ␤ig-h3 and cultured synoviocytes of RA. Methods: ␤ig -h3, TGF-␤1, and TGF- ␤2 levels were analyzed quantitatively in peripheral blood (PB) and synovial fluid (SF) of 20 patients with active RA and 20 patients with knee OA using ELISA. Expression of ␤ig-h3 in cultured synoviocytes was measured before and after treatment of TGF- ␤1, using Western blot analysis. Cell adhesion and spreading activities of ␤ig -h3 were assayed in synoviocytes from RA and OA patients after culturing the cells on each dish coated with bovine serum albumin, plasma fibronectin, and recombinant ␤ig-h3, respectively. In addition, clinical and laboratory parameters of disease activity of RA were measured at the time of sampling. Results: 1) Significantly more expression of ␤ig-h3 was detected in synovial fluid of RA patients (201.8 ⫾ 211.4 ng/ml) than in those of OA patients (62.6 ⫾ 45.1 ng/ml) (p⫽ 0.004), while there was no difference in the level of PB ␤ig-h3 between RA and OA patients. 2) A positive correlation between TGF- ␤1 and ␤ig-h3 was apparent in all 4 kinds of samples ; PB and SF of each RA and OA patients. 3) While ␤ig-h3 protein was detected in the cultured synoviocytes of both RA and OA, more enhanced expression of ␤ig-h3 was observed in RA. Treatment of synoviocytes with TGF- ␤1 led to an increase in ␤ig-h3 protein. 4) Adhesion and spreading of synoviocytes were promoted prominently on surfaces coated with ␤ig-h3, and the promoting activity of ␤ig-h3 was similar to that of fibronectin. 5) There was no correlation between ␤ig-h3 and the parameters of disease activity of RA. Conclusions: In rheumatoid arthritis, significantly elevated ␤ig-h3 might be involved in mediating some of the varying effects of TGF-␤, such as the hyperplasia of synovium. The precise functions of this protein in synovial pathology of RA need to be further investigated.

B lymphocyte clonal expansion and differentiation are usually accompanied by changes in the structure of the Ig produced by the individual B cell. These changes involve both the variable (V) and constant (C) portions of the Ig. Although changes in the B cell’s V region occur most frequently as a consequence of somatic point mutations, other mechanisms such as nucleotide insertion or deletion and VH gene replacement can also alter V gene structure. Changes in the structure of the CH region of Ig molecule occur during isotype switching. While analyzing the cDNA sequences of the B lymphocytes that expand in the synovial tissues of patients with rheumatoid arthritis (RA), we identified examples of nucleotide insertion or deletion and VH gene replacement that occurred at a frequency of ⬃1% of the transcripts analyzed. Since the nucleotide insertions occurred in multiples of 3 (3-12 bp) and were usually found in the CDR, they maintained an appropriate reading frame and probably coded for an intact receptor. In addition, since the sequences of the inserted nucleotides were very similar to the adjacent nucleotide sequence, they probably arose by DNA duplication due to DNA polymerase slippage. The nucleotide deletions detected in these cDNAs varied considerably in length. Whereas the shorter deletions (3-18 bp in multiple of 3) usually occurred in the CDR and probably coded for acceptable Ig molecules, the longer deletions (⬎54 bp) spanned several FR and CDR and likely coded inadequate Igs. Furthermore, four examples of VH gene replacement were identified among VH1- and VH2-expressing transcripts. Since these three types of diversification were detected more frequently in IgG⫹ and IgA⫹ transcripts that exhibited more somatic point mutations than the IgM transcripts, these mechanisms may be related to Ig V gene hypermutation process. Finally, several clonally-related transcripts with identical HCDR3 were found indicating clearly that isotype class switching occurs in these cells. These clonal diversification events, along with typical somatic point mutations, may contribute to or be attempts to rescue B cells from the autoreactivity characteristic of RA.

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PREDICTIVE MARKERS OF DISEASE OUTCOME IN SYNOVIAL TISSUE OF PATIENTS WITH RECENT ONSET RA. Maarten C Kraan, Ella C Barg, Jasper J Haringman, Malcolm D Smith, Ferdinand C Breedveld, Michael J Ahern, Paul P Tak Amsterdam and Leiden, Netherlands and Adelaide, Australia

ANGIOGENESIS AND STROMAL CELL DERIVED FACTOR-1 (SDF-1) EXPRESSION IN RHEUMATOID SYNOVIUM. Jose L Pablos, Begona Santiago, Carmen Torres, Maria T Brehmer, Maria Galindo, Francisco J Blanco Madrid and La Coruna, Spain SDF-1 is a CXC chemokine constitutively expressed by bone marrow stromal cells. SDF-1 and CXCR4 are a monogamous receptor/ligand pair that is essential during prenatal myelo- and lymphopoiesis and vascular development. SDF-1 is a potent chemoattractant for mononuclear and endothelial cells and induces angiogenesis in animal models. In rheumatoid arthritis (RA) synovium, we have previously observed SDF-1 expression in synoviocytes, stromal fibroblasts and endothelium by immunohistochemistry. In vitro, fibroblast like synoviocytes but not endothelial cells expressed SDF-1 mRNA, suggesting that SDF-1 protein could be present in endothelial cells but synthesized by other cell types. To analyze whether endothelial SDF-1 protein is intracellular or bound to surface heparan sulfate proteoglycans, we performed SDF-1 immunostaining of synovial tissue cryosections after heparitinase I (10 mU/ml) pretreatment for 1 hour at 37° C. Synovial tissues were obtained from RA (n⫽ 7) and osteoarthritis (OA) (n⫽7) patients during arthroplasty. Heparitinase I pretreatment abrogated endothelial cell SDF-1 immunostaining, whereas synoviocytes and stromal fibroblasts remained immunostained. To better understand the potential relevance of SDF-1 in RA angiogenesis, we also performed triple labeling with anti-SDF-1 mAb (clone K15C), the angiogenesis marker anti-CD51/61 (integrin ␣v␤3) and the endothelial cell marker Ullex europaeus aglutinin I, in cryosections from RA and OA synovium. We found an increased number of integrin ␣v␤3 expressing vessels in RA compared to OA synovial sections, although some integrin ␣v␤3 positive vessels were also observed in OA. SDF-1 was immunodetected in the endothelium of some synovial vessels, with adjacent vessels negative. Integrin ␣v␤3 immunoreactive endothelial cells colocalized with SDF-1 immunoreactivity. Synoviocytes and stromal fibroblasts seem the main source of SDF-1 in RA synovium. Endothelial cells SDF-1 seems bound to surface heparan sulfate proteoglycans and its presence correlates with that of integrin ␣v␤3 expression, suggesting its potential participation in angiogenesis. Disclosure: Supported by grant SAF 2000/0062

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Objective. To determine the relationship between the characteristics of synovial tissue in early rheumatoid arthritis (RA) and disease outcome. Methods. Synovial tissue samples were obtained from an inflamed knee joint by blind needle biopsy in 18 patients with RA of ⬍ 1-year disease duration. In addition to clinical and laboratory assessment, immunohistologic analysis was performed to detect T cells, plasma cells, macrophages, B-cells and granzyme B positive cytotoxic cells on frozen tissue sections. Sections were evaluated by digital image analysis. Clinical, laboratory and radiological assessment was performed at the time of the biopsy and after follow up for 6 years. Results. Both rheumatoid factor (P ⬍ 0.05) and female sex (P ⫽ 0.001) were associated with higher erosion scores at follow up. There was a significant positive correlation between the DAS at entry of the study and radiological deterioration over 6 years (r ⫽ 0.514, P ⬍ 0.05). Patients with severe radiological progression (ⱖ 10 points increase in Larsen score in 6 years) had significantly higher numbers of plasma cells (556 ⫾ 114 (mean ⫾ SEM)) and granzyme B positive cells (25 ⫾ 10) in early RA synovial tissue than patients with less severe radiological progression (⬍ 10 points increase in Larsen score in 6 years) (244 ⫾ 157; P ⬍ 0.05 for plasma cells and 0 ⫾ 0; P ⬍ 0.005 for granzyme B positive cytotoxic cells). In addition, granzyme B positive cells were significantly higher in synovium of patients with persistent disease activity, evaluated by DAS (responders 6 ⫾ 8, non-responders 34 ⫾ 17, P ⬍ 0.05). Intimal lining macrophages, B-cells and T-cells were higher in patients with severe radiological progression but these differences did not reach statistical significance. Conclusion. As demonstrated in previous studies both plasma-cells and granzyme B in synovial tissue of early RA patients are related with the severity of joint damage after 6 years and, therefore, may be useful prognostic markers in recent onset RA. Also these results suggest that plasma-cells and granzyme B might have an important role in the pathogenesis and joint erosion of RA.

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LOCAL PRODUCTION OF B LYMPHOCYTE STIMULATOR (BLySTM) PROTEIN IN HUMAN ARTHRITIC JOINTS.

SHARED EPITOPE HYPOTHESIS VERSION 2.0 IN RHEUMATOID ARTHRITIS: DEFINING THE “SHARED EPITOPE HYPOTHESIS” BY AMINO ACID POSITION, NOT BY AMINO ACID SEQUENCE. Niek De Vries, Henk Tijssen, Piet LCM Van Riel, Leo BA Van de Putte Utrecht and Nijmegen, The Netherlands

Soon-Min Tan, Dong Xu, Viktor Roschke, Kevin P Baker, David M Hilbert, William Stohl Los Angeles, CA and Rockville, MD BLyS (also known as BAFF, zTNF4, TALL-1, and THANK) is a 285-amino acid protein member of the tumor necrosis factor ligand superfamily which is expressed predominantly (solely) by myeloid lineage cells (e.g., monocytes) and the biologic effects of which are predominantly (solely) targeted at B cells. Constitutive overexpression of BLyS in mice leads to systemic illness characterized by polyclonal hypergammaglobulinemia, circulating immune complexes, elevated titers of ANA, RF, and anti-dsDNA autoantibodies, and renal Ig deposits. In vivo treatment of lupus mice with BlyS antagonists ameliorates disease progression and improves survival. In humans, a substantial percentage of RA and SLE patients harbor elevated serum BlyS levels, and titers of RF and anti-dsDNA autoantibodies in RA and SLE patients respectively correlate positively with serum BlyS levels. To assess whether local production of BlyS in the joint might play a role in inflammatory arthritis, we measured simultaneous serum and synovial fluid (SF) BlyS levels in 24 patients with inflammatory arthritis (Inflam) and in 8 patients with OA or traumatic arthritis (OA/Tr). SF and serum BLyS levels in Inflam were each significantly greater than those in OA/Tr (p ⬍ 0.001 and p ⫽ 0.043 respectively). In each cohort, BLyS levels were greater in SF than in serum, with geometric mean SF and serum BLyS levels of 12.4 and 5.1 ng/ml respectively in Inflam (p ⬍ 0.001) and 6.4 and 3.2 ng/ml respectively in OA/Tr (p ⬍ 0.001). Double-staining for surface CD14 (monocyte marker) and BLyS demonstrated increased surface expression of BLyS on SF mononuclear, especially CD14⫹, cells compared to that on corresponding blood mononuclear cells. SF BLyS levels tended to correlate with SF leukocyte number (r ⫽ 0.310, p ⫽ 0.074) and did correlate with SF monocyte/histiocyte number (r ⫽ 0.363, p ⫽ 0.035). In contrast, no correlation was detected between serum BLyS levels and WBC number (r ⫽ 0.197, p ⫽ 0.280) or blood monocyte number (r ⫽ 0.024, p ⫽ 0.895). Quantitative RT-PCR failed to demonstrate consistently increased BLyS mRNA levels per monocyte in SF, suggesting important post-transcriptional events in the upregulation of BLyS protein expression in SF monocytes. Taken together, these findings strongly point to local production of BLyS in the arthritic joint. Elevated local BLyS levels in diseases such as RA could promote local B cell survival, expansion, and differentiation and facilitate development of pathogenic immune complexes.

Objective & Methods: To further analyze the association of HLA-DRB1 alleles with disease susceptibility in recent onset rheumatoid arthritis (RA) 167 caucasian RA patients and 166 healthy controls were typed for HLA-DRB1. Results: The association of susceptibility to RA with the group of alleles encoding the shared epitope susceptibility sequences was confirmed in recent onset RA. Among non-susceptibility alleles DRB1*07, *1201, *1301 and *1501 showed significant protective effects. Even after correction for the influence of susceptibility alleles, sigificant independent protective effects of DRB1 alleles were observed. Protective alleles shared a third hypervariable region motif. Independent homozygosity effects were observed for susceptibility and protective alleles. Conclusion: Non-susceptibility alleles differ significantly regarding RA risk. Protective alleles show clear homology at positions 67-74, often encoding Isoleucine at position 67 or Aspartic acid at position 70. Both susceptibility and protective alleles show homozygosity effects. These results and literature data indicate that the shared epitope hypothesis should be rephrased to account for differential risks among non-susceptibility alleles, thus defining the “shared epitope hypothesis” by amino acid position, not by amino acid sequence. Disclosure:

Disclosure: William Stohl is a consultant to Human Genome Sciences. Viktor Roschke, Kevin P Baker, and David M Hilbert are employees of Human Genome Sciences. BLyS is a trademark of Human Genome Sciences.

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EXPRESSION OF G0S24 (TRISTETRAPROLINE) GENE IS INCREASED IN SYNOVIAL TISSUE FROM PATIENTS WITH RHEUMATOID ARTHRITIS. Akito Tsutsumi, Yoshihiro Adachi, Satoshi Kojo, Kazuko Shibuya, Hideyuki Murata, Hiroshi Nakamura, Takayuki Sumida Kawasaki, Kanagawa and Tsukuba, Ibaraki, Japan

DEXAMETHASONE SUPRESSES OSTEOCLAST FORMATION FROM CULTURED SYNOVIAL CELLS VIA DOWNREGULATION OF RANKL(RECEOPTOR ACTIVATOR OF NF-␬B ligand)/ODF IN SYNOVIAL FIBROBLASTS. Yeong-Shil Joo, Jae-Il Park, Kyoung-Ho Suk, Min-Ju Kang, Byoung-Joon Kim, Kyu-Jeung Ahn, Duke-Whan Chung, Yeong-Seol Kim, Young-Kil Choi Seoul, Republic of Korea

Objective: In order to understand the molecular mechanisims involved in the pathogenesis of rheumatoid arthritis (RA), and to search for a possible target of RA specific therapies, we wished to identify genes specifically expressed in synovial tissues of patients with RA. Methods: Samples from a patient who fulfilled the ACR criteria for classification of RA were used. RNA was extracted from synovial tissue from an operated knee joint and peripheral blood mononuclear cells (PBMC). Gene expression between synovial and PBMC samples was compared by the differential display-PCR (DD-PCR) method. Genes specifically expressed by synovial tissue samples were cloned and sequenced. Genes considered of interest were selected, and their mRNA expression was compared between synovial tissues from 10 RA and 10 osteoarthritis (OA) patients by TaqMan realtime semiquantification PCR method. Mann-Whitney’s U test was used for statistical analyses. Results: 1) A large number of genes highly expressed in synovial tissues of RA patients compared to their PBMC were identified. 2) Identified genes included procollagen type 1 alpha-2 chain, YKL-39, CD63, Ferritin L chain, complement C1r, NADH dehydrogenase subunit 2, G0S24, Immunoglobulin binding protein, mortality factor 4, and many genes not present in accessible data bases. 3) In particular, the expression of G0S24 gene, a gene encoding a protein that reduces the stability of TNF␣ mRNA, was significantly increased in synovial tissues from RA patients than those from OA patients. 4) The expression of CD63, Immunoguloblin binding protein, mortality factor 4 and Ki nuclear autoantigen did not significantly differ between synovium from RA or OA patients. Conclusion: DD-PCR method may be used to identify genes specifically present in RA synovial tissues. Since previous studies from other laboratories have shown that mice lacking the G0S24 gene develop severe arthritis similar to that seen in human RA, we speculate that the G0S24 gene product may affect the course of RA by reducing the production of TNF␣ in the synovium.

A number of soluble factors which play important role in the pathophysiology of rheumatoid synovitis are also known to be involved in osteoclast differentiation and activation through RANKL/ODF. Investigations into these factors in relation to their osteotropic effect can provide better understanding of the pathophysiology of bone erosion and therapeutic implication. We have previously shown that dexamethasone downregulates RANKL in primary culture synoviocytes which could be a putative mechanism explaining its possible bone-protecting effect in RA. In order to confirm the inhibitory effect of dexamethasone on osteoclastogenesis in rheumatoid synovium, we examined the expression of RANKL and formation of TRAP(⫹) MNCs from synoviocytes under the influence of dexamethasone. Primary culture synoviocytes or synovial fibroblasts isolated from synovial tissues of 8 RA patients were treated with IL-1␤(10ng/ml) or dexamethasone(10-9M) and the expression of RANKL was observed using semiquantitative RT-PCR. TRAP(⫹)MNC formation was induced from primary culture synoviocytes or in coculture system of synovial fibroblasts with PBMCs in the presence of M-CSF and 1,25(OH)2VD3. Primary culture synoviocytes and synovial fibroblasts express RANKL and OPG mRNA with decreasing intensity when they are passaged. The intensity of expression was different from patient to patient. Expression of RANKL mRNA was significantly increased by IL-1␤ and decreased by dexamethasone, in a dose- and time-dependant manner. The upregulated RANKL by IL-1␤ was also blocked by dexamethasone. TRAP(⫹) MNCs are formed from primary culture synoviocytes or in coculture system of synovial fibroblasts and PBMC in the presence of M-CSF and 1,25(OH)2VD3. Dexamethasone clearly inhibited TRAP(⫹) MNCs formation from synovial cells(401⫾9.2/well vs. 17⫾2.2/well). These data could provide in vitro evidence for the putative mechanism for protecting bone erosion by long-term low dose glucocorticoid in RA. Further investigations dissecting the role of various soluble inflammatory mediators and antirheumatic agents in osteoclastogenesis in rheumatoid synovium are needed. Disclosure:


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THE DISINTEGRIN METALLOPROTEINASE MDC15 IS HIGHLY UPREGULATED IN RHEUMATOID SYNOVIAL MEMBRANES. Beate B Boehm, Thomas Aigner, Carl P Blobel, Joachim R Kalden, Harald Burkhardt Erlangen and Erlangen, Germany and New York, NY

MARKED OVEREXPRESSION OF SOLUBLE INDUCIBLE HEAT SHOCK PROTEIN 70 IN RHEUMATOID ARTHRITIS (RA) SYNOVIAL FLUID (SF). Robert Kowalewski, Steven E Carsons, Frances Santiago-Schwarz Mineola and Stony Brook, NY

Introduction. MDC15 (ADAM15) belongs to the family of membrane-bound disintegrin metalloproteinases (ADAM or MDC). Some MDCs are involved in (patho-) physiological relevant processes such as remodeling of the extracellular matrix as well as shedding of membrane-bound cytokines (e.g. TNF-alpha) (1, 2). Furthermore, the markedly strong up-regulated expression of his proteinase in arthritic cartilage implicates an important role in tissue destruction (3). Objectives. In the current study the expression of MDC15 in synovial membranes of patients with Rheumatoid Arthritis (RA) in comparison to osteoarthritic (OA) and normal specimens was analyzed. Methods. Using in situ hybridization the expression of MDC15 mRNA was investigated in 25 RA synovial tissues, in 55 OA tissues and 8 normal synovial tissues. The detection of MDC15 on protein level was performed by immunohistochemistry. A specific polyclonal antibody was generated by immunization with a synthetic cyclized peptide from the disintegrin domain and specificity was confirmed by immunoblot analysis on COS-7 cell lysates transfected with the MDC15 cDNA. Results. By in situ hybridization as well as by immunohistochemistry highest expression of MDC15 could be detected in the synovial lining cells in all RA synovial tissues analyzed. In comparison to the RA specimens, far lower levels of expression were observed in the synoviocytes of OA specimens that still exceeded the very low expression found in normal synovial tissues. Double labeling experiments using anti MDC15 and CD68 antibodies revealed that both CD68⫹ macrophage-like A-type synoviocytes and CD68- fibroblast-like cells produce MDC15. Moreover, very strong expression of MDC15 could be detected in CD138⫹ plasma cells, whereas CD20⫹ B-cells and CD4⫹ and CD8⫹ T-cells exhibited no expression in situ in the tissue as well as in lymphocytes isolated from peripheral blood mononuclear cells. Conclusion. The enhanced expression of MDC15 in the rheumatoid synovium suggests a potential role in the pathophysiology of cartilage destruction, since it might be involved in the direct proteolytic degradation of extracellular matrix molecules. References. 1. Chubinskaya S et al. J Histochem Cytochem 1998; 46:723-730; 2. Patel IR et al. J Immunol 1998; 160:4570-79; 3. Bohm BB et al. Arthritis Rheum 1999; 42: 1946-50

Heat shock proteins (hsp) are stress-related molecules that promote protein stability and transport. Emerging concepts are that soluble hsp chaperone antigens into antigen presenting cells such as dendritic cells and thus may contribute to cross-priming and the induction of autoimmunity. In RA, strong genetic associations between hsp70 and major histocompatibility complex (MHC) class II shared epitopes have been reported. While abnormally elevated hsp70 mRNA and inducible (i) hsp70 protein have been described in RA synovial cells, the distribution of soluble ihsp70 in RA synovial fluid (which may be a source of immunogen) is unknown. We compared soluble ihsp70 levels in RA SF (N⫽20), RA sera, (N⫽10) normal sera (N⫽10) and non autoimmune arthritic SF (osteoarthritis, OA, N⫽10; gout, N⫽5) using a sandwich ELISA. The antibodies used were specific for ihsp70 and did not cross react with constitutive hsp70. Measurement of ihsp70 was not influenced by rheumatoid factor, as revealed in spike recovery and isotype antibody control studies. Inducible hsp70 was increased ⬃90 fold in RA SF compared to normal sera (348 ng/ml vs. 4 ng/ml, P⬍.0001). Compared to RA sera, the levels of ihsp70 in RA SF were increased ⬃30 fold (P⬍.0001). While trends toward increases (3 fold) in ihsp70 were noted in RA sera vs. normal sera, results were not significant (P⫽.064). SF obtained from OA and gout contained higher ihsp70 levels than normal sera (24 ng/ml and 59 ng/ml vs. 4 ng/ml, respectively, Pⱕ.02). However, relative to RA SF, ihsp70 levels were significantly reduced in OA and gout SF (Pⱕ.0025). These results demonstrate that soluble ihsp70 is particularly over-expressed in RA SF. Our data substantiate that alterations in the distribution of hsp70 occur in RA and support the evolving idea that increased hsp may facilitate cross-priming events in autoimmune diseases. Disclosure:

Disclosure:

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EXPRESSION OF GALECTIN-1, GALECTIN-3 AND GALECTIN-3 BINDING PROTEIN IN RHEUMATOID ARTHRITIS SYNOVIUM. Stefan Kuchen, Christian A Seemayer, Peter Kuenzler, Renate E Gay, Beat A Michel, Steffen Gay, Michel Neidhart Zurich, Switzerland Objective: To investigate the expression of galectin-1 (Gal-1) and galectin-3 (Gal-3), two human ␤-galactoside specific lectins implicated in inflammation, cell growth, differentiation and transformation, as well as of the galectin-3 binding protein (G3BP) in rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissues (NS). Material and Methods: The expression of Gal-1, Gal-3 and G3BP mRNA was investigated on paraffin-sections of 7 RA, 3 OA and 2 normal synovial specimens by non radioactive in situ hybridization (ISH). On parallel sections immunohistochemistry (IHC) for the macrophage marker CD68 was performed. In addition, the expression of Gal-3 and G3BP was confirmed by IHC on paraffin and snap frozen sections using specific antibodies. Results: Gal-1, Gal-3, and G3BP mRNA were abundantly expressed in all RA synovial tissues. Gal-3 and G3BP mRNA and protein were predominantly expressed in the sublining and to a lesser extent in the lining layer and at sites of invasion. In contrast, the expression of Gal-1 was not uniform in different RA specimens and either expressed in the lining or in the sublining layer. In general, blood vessels were negative for Gal-1, Gal-3 and G3BP. Neither the expression of Gal-1, Gal-3 nor G3BP corresponded with the expression of CD68. In OA synovial specimens only a limited number of cells were positive for Gal-1, Gal-3 and G3BP. In NS Gal-1, Gal-3, and G3BP were not detectable. Conclusion: This data indicate that Gal-1, Gal-3 and G3BP appear not only involved in the inflammatory process but may contribute also to the activation of CD68 negative synovial fibroblast-like cells in the RA synovium. Disclosure: S. Kuchen is supported by the EMDO Foundation, C.A. Seemayer by the Swiss-National Science Foundation, P. Kuenzler by Novartis (Basel, Switzerland), and all others by their respective institutions

INVESTIGATION OF THE ROLE OF P16INK4a IN RHEUMATOID ARTHRITIS (RA). Peter Kuenzler, Christian A Seemayer, Stefan Kuchen, Michel Neidhart, Martin Pruschy, Beat A Michel, Renate E Gay, Steffen Gay Zurich, Switzerland To investigate the expression of the tumor suppressor/cdk inhibitor p16INK4a in vivo, and to test its presence and inducibility in RA synovial fibroblasts (SF) in vitro. Tissue sections from RA, osteoarthritis (OA) and control (normal synovia) as well as cultured RA-SF and controls [normal SF (N-SF) and foreskin fibroblasts (FSFB)] were analyzed for the presence of p16 using immunohistochemistry (IHC) and -fluorescence (IF) techniques, respectively. The in vitro inducibility of p16 in RA-SF and control FSFB was analyzed after irradiation with X-rays (10 Gy) or serum starvation by IF staining after 25 hours or 13 days. The levels of p16 mRNA in RA-SF, OA-SF and FSFB were determined by real time PCR (TaqMan) with and without serum starvation. Cells from 1 RA-SF and 1 N-SF were coimplanted with normal human cartilage under the renal capsule of SCID mice and kept there for 60 days. Paraffin embedded SCID mice sections were stained with p16 specific antibodies. In 10/13 RA specimen, 1-6% of the cells stained positive for p16, whereas on control tissue sections less than 1% were positive. In 3 RA samples, p16 was present in 14-23% of the cells, which were predominantly located in the sublining and in one case also in the lining layer. Cultured RA-SF and controls expressed p16 neither constitutively nor after serum starvation, but 13 days after X-ray irradiation, 30-50% of the cells stained positive for p16. Real time PCR data show a very low level of p16 mRNA even after serum starvation. In the SCID mouse coimplantation model, most remarkable, the cartilage invading RA-SF expressed p16 but neither the RA-SF adjacent to the cartilage nor N-SF. These results indicate a role of p16 in RA in a subset of patients and a correlation between p16 expression and the invasive process. Since p16 is associated with the arrest of the cell cycle, the data also suggest a dissociation of proliferation and invasion. Disclosure: P. Kuenzler was supported by Novartis (Basel, Switzerland), C.A. Seemayer by the Swiss National Science Foundation, S. Kuchen by the EMDO Foundation, and all others by their institutions.

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FIBROBLAST-LIKE SYNOVIOCYTES (FLS) FROM PATIENTS WITH RHEUMATOID ARTHRITIS SHOW AUGMENTED BASAL AND HUMAN BASIC FIBROBLAST GROWTH FACTOR-INDUCED TYROSINE PHOSPHORYLATION. Consuelo Lopez-Benitez, Fabiola A Placeres, Evelyn J Alonzo, Ana M Blasini, Martin A Rodriguez Caracas, Venezuela

HIGH DIAGNOSTIC VALUE OF ANTI-FILAGGRIN AUTOANTIBODIES (AFA) DETECTED BY A NEW ELISA USING CITRULLINATED AND NON-CITRULLINATED RECOMBINANT PROTEINS AS ANTIGENS IN A COHORT OF 152 COMMUNITY CASES OF VERY EARLY ARTHRITIS. Olivier Vittecoq, Brigitte Incaurgarat, Vincent Saulot, Jocelyne Legoedec, Fabienne Jouen-Beades, Alain Gayet, Patrice Fardellone, Patrick Boumier, Anne Phan Van, Jean-Marie Bertho, Charles Zarnitsky, Othmane Mejjad, Alain Daragon, Michel Jolivet, Francois Tron, Xavier Le Loet Rouen, Marcy l’Etoile, Rouen, Amiens and Tours, France

Abnormal post-receptor signaling may contribute to the transformed phenotype of FLS in patients with rheumatoid arthritis (RA). Highly purified synovial FLS (100 % vimentin-expressing, CD3-, CD14-, CD68- cells) were obtained during total joint prosthesis or synovectomy, in patients with RA (n⫽5) and osteoarthritis (OA) (n⫽7). Cells were isolated by collagenase digestion and grew on DMEM-F12 medium, then lysed and studied by Western blot in a period between the third to sixth passage. Unstimulated FLS from RA patients showed enhanced tyrosine phosphorylation of various substrates, ranging from 45- to 146-kDa, as compared to FLS from OA patients: 71.3⫾11.7 vs. 42.2⫾4.8 (m ⫾ SEM, total O.D, p⫽0.02). Further, in comparison to OA FLS, FLS from RA patients showed increased tyrosine phosphorylation of all these substrates after stimulation in vitro with human basic fibroblast growth factor (bFGF) (10-400 ng/ml), at all FGF concentrations tested. In addition, RA FLS showed tyrosine phosphorylation of an unidentified 64-kDa protein in 5/5 RA patients as compared to 1/7 OA patients (p⫽0.01). FLS from RA and OA patients responded similarly to inhibition of tyrosine phosphorylation, in cultures preincubated with genistein (160mg/ml), a specific tyrosine kinase inhibitor. On the contrary, preincubation with sodium orthovanadate (100 mM), a specific inhibitor of tyrosine phosphatases, induced a striking increment in baseline tyrosine phosphorylation of FLS from OA, but not RA patients: 336⫾28 vs. 136⫾21 (m ⫾ SEM % enhancement, p⫽0.0004). Similar differences were observed in FLS cultures stimulated with bFGF, and pretreated with sodium orthovanadate, at all bFGF concentrations tested. FLS from RA patients exhibit an abnormal pattern of tyrosine phosphorylation that may be related to a potential defect of down-regulatory tyrosine phosphatases. CONICIT No. 97000808.

Objective - To determine the diagnostic value of autoantibodies (Ab) recognizing citrullinated recombinant rat filaggrin (ACRF) for very early RA. Methods - Sera from patients with different classified rheumatic diseases and healthy subjects (group 1, n:415) and 152 community cases of very early arthritis [swelling of at least two joints persisting more than 4 weeks ; median disease duration : 4 months] (group 2) that were classified as RA (n:88) or as other rheumatic diseases after 1 year of follow up, were studied. ACRF were measured using a new ELISA, with results expressed as the difference between the OD value obtained on citrullinated minus that on non-citrullinated rat filaggrin (differential ACRF [dACRF]). For group 2, rheumatoid factors (RF), anti-keratin Ab (AKA) and anti-perinuclear factor (APF) were also tested. Results - Different reactivity patterns against citrullinated and non-citrullinated filaggrin were observed. Almost all sera reacting with citrullinated but not non-citrullinated filaggrin were from RA patients. Among RA and non-RA sera that recognized both forms of filaggrin, a positive result was obtained only with RA sera. For groups 1 and 2, dACRF specificity for RA was 99.5% and 98.4% respectively. In group 2, dACRF specificity for RA was better than that of RF (92.1%), APF (92.1%) and AKA (95.3%). dACRF positive predictive value was high (96.4%) with higher sensitivity (30.7%) than AKA (23.8%). Despite a high positive correlation among AKA, APF and dACRF test results, they were complementary since some sera were positive for only one test. Conclusion - In a community setting, anti- citrullinated rat filaggrin reactivity detected by a new ELISA, whose originality is based on the difference between serum’s reactivities on the citrullinated and native forms of filaggrin, had a higher diagnostic value for RA than AFA identified by previous tests. Disclosure: Study granted by the Fondation de la Recherche Me´dicale (FRM), the Association de Recherche sur la Polyarthrite (ARP) and for clinical assessment Pharmacia, France.

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EXPRESSION OF ANTIZYME INHIBITOR IN RHEUMATOID SYNOVIUM. Kimio Masuda, Riako Masuda, Shiro Ohshima, Beat R Simmen, Beat A Michel, Renate E Gay, Steffen Gay Zurich, Switzerland

ANTI-FILAGGRIN AUTOANTIBODIES DETECTED BY A NEW ELISA USING CITRULLINATED AND NON-CITRULLINATED RECOMBINANT PROTEINS AS ANTIGENS ARE NOT PREDICTIVE OF RADIOLOGICAL PROGRESSION IN RHEUMATOID ARTHRITIS (RA) - RESULTS OF A 3-YEAR LONGITUDINAL PROSPECTIVE STUDY FROM A POPULATION BASED-RECRUITMENT OF 127 RA. Olivier Vittecoq, Brigitte Incaurgarat, Vincent Saulot, Sophie Pouplin, Jocelyne Legoedec, Odile Letourneur, Dominique Rolland, Gaspard Gervasi, Kataryna Krzanowska, Jean-Francois Menard, Alain Gayet, Michel Jolivet, Francois Tron, Xavier Le Loet Rouen and Marcy l’Etoile, France

Disclosure: K. Masuda and S. Ohshima are supported by Japan Rheumatism Foundation, R. Masuda by Uehara Memorial Foundation, S. Ohshima by Toyobo Biotechnology Foundation, and all others by their respective institutions.

Objective -To evaluate the predictive value of autoantibodies (Ab) recognizing citrullinated recombinant rat filaggrin (ACRF) detected by a new ELISA for prognosis in a cohort of community cases of RA. Methods - 127 patients (mean age :50.5 yr, F/M :2.9) with early RA (median disease duration :2 yr) were enrolled prospectively in 1996 (T1), primarily from a population based-recruitment (80%), and were followed until 1999 (T2). At T1 were tested rheumatoid factors determined by the latex fixation test (LFT) and anti-filaggrin Abs identified by different assays, i.e. anti-keratin Ab (AKA) and anti-perinuclear factor (APF) by indirect immunofluorescence, ACRF by an ELISA test whose results are expressed as the difference of OD values given by sera tested against the citrullinated and non-citrullinated forms of filaggrin. At T1 and T2 : radiographs of the hands and wrists. Prognosis was defined by (1) radiological damage (van der Heijde modified Sharp’s method) at T2 ; (2) progression of radiological damage between T1 and T2. Results - At T1 :RA had mild activity (Ritchie articular index :11/78, mean CRP :15 mg/l), mild functional disability (HAQ :0.8/3), mild X-ray destruction (Sharp :9.2/280) ; 96% of patients were treated (DMARDs :95%; prednisone :72%); frequency of LFT, AKA, APF and ACRF were respectively : 50%, 33%, 45% and 45%. At T2 : total Sharp score :22.8/280. The only parameter at T1 associated with a higher Sharp’s score at T2 (p ⫽ 0.03) and particularly with a more important radiological progression (p ⫽ 0.03) was the LFT. Conclusion - Whatever the method used, indirect immunofluorescence or ELISA, anti-filaggrin Ab do not allow to predict radiological damage in community cases of RA of limited duration. Disclosure:

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Background: Antizyme inhibitor (AZI) is an “anti-enzyme” for ornithine decarboxylase (ODC) and induces polyamine synthesis. Polyamines are essential for cell growth, and their synthesis and catabolism are strictly regulated in mammalian cells. Most recently, it was reported that AZI was differentially expressed in tumor tissues versus in normal tissues. Moreover, we identified that AZI was predominantly expressed in proliferating, rather than quiescent, RA-SF by suppression subtractive hybridization. The aim of this study was to investigate the expression of AZI in cultured rheumatoid arthritis synovial fibroblasts (RA-SF) and synovial tissues from patients with RA. Materials and Methods: Seven RA-SF, 2 osteoarthritis (OA)-SF, 1 SF from normal synovium, and 2 normal skin fibroblasts (passages 3-6) were used for RT-PCR. Total RNA was extracted from each cultured fibroblasts, reverse transcribed and amplified with the primers specific for AZI. The products were analyzed on 1.0 % agarose gels in comparison with the expression of actin beta. The expression and distribution of AZI gene were also examined in paraffin sections of synovial tissues from 7 patients with RA and 3 normal individuals by in situ hybridization with digoxigenin-labeled RNA probes specific for AZI. Results: In vitro, AZI gene was highly expressed in RA- and OA-SF, although basal expression was also seen in SF from normal synovium and normal skin fibroblasts by RT-PCR. Interestingly, in situ hybridization showed that AZI mRNA were specifically expressed in synovial tissues from patients with RA (5/7), especially in lining and sublining layers, while no expression was observed in synovial tissues from normal individuals (0/3). Conclusions: Since AZI could be detected by subtractive hybridization of proliferating RA-SF from quiescent non-proliferating RA-SF, high expression of AZI in RA-SF and rheumatoid synovium suggests that AZI may play a role in the activation of synovial fibroblasts in RA.

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THE PROGNOSTIC VALUE OF ANTI-CYCLIC CITRULLINATED PEPTIDE (CCP) ANTIBODIES IN EARLY POLYARTHRITIS. Annemarie LMA Jansen, Dirkjan van Schaardenburg, Irene E van der Horst-Bruinsma, Rob J van de Stadt, Margret HMT de Koning, Ben AC Dijkmans Amsterdam, The Netherlands

PROGNOSTIC VALUE OF SERUM BONE MARKERS AND HAND BONE DENSITOMETRY IN VERY EARLY RHEUMATOID ARTHRITIS / PRELIMINARY RESULTS OF THE VERA STUDY. Alain Daragon, Olivier Vittecoq, Michel Brazier, Othmane Mejjad, Patrice Fardellone, Katarzyna Krzanowska, Patrick Boumier, Charles Zarnitsky, Anne Phan Van, Alain Gayet, Jean Francois Menard, Xavier Le Loet Rouen, Amiens, Le Havre and Tours, France

OBJECTIVE: to assess the prognostic value of anti-Cyclic Citrullinated Peptide (anti-CCP) antibodies for progressive erosive or disabling disease in a cohort of patients with early inflammatory polyarthritis. METHODS: consecutive new patients with peripheral arthritis of ⱖ 2 joints and ⱕ 2 years of symptom duration were studied. Excluded were patients with bacterial, psoriatic, crystal-induced arthritis or spondylarthropathy. Optimal cut-off values for serum IgM-RF, IgA-RF and anti-CCP were deduced from Receiver Operating Characteristics curves. At 2 year follow-up, progressive erosive disease was defined as: radiographic damage ⱖ 10 or radiographic progression ⱖ 4 (Sharp-van der Heijde units), and low functional capacity as a HAQ-score ⱖ 1. For the statistical analysis, a logistic regression model was used. RESULTS: 362 patients (68% female, median age 57 (17-85)) were included. Thirty-two percent of the patients was positive for anti-CCP at baseline. Anti-CCP was correlated significantly with radiographic progression (p ⬍.001) and low functional capacity (p ⬍.01). The combination of anti-CCP⫹, IgARF⫹ and radiographic damage at baseline contributed significantly (accuracy of 80%) to the prediction of radiological progression after 2 years, whereas IgMRF⫹, disease activity (DAS28) and demographic variables did not. In the multivariate model for functional capacity, anti-CCP did not significantly contribute. In a subgroup of IgM RF- patients, 24 of 243 (10%) patients were anti-CCP⫹. Multivariate analysis of this subgroup also showed a significant association of anti-CCP with radiological progression. CONCLUSION: Radiographic progression is independently associated with anti-CCP positivity in the whole group of early arthritis patients as well as in the subgroup of IgM RF negative patients. Disclosure:

Disclosure: Study granted by La Fondation pour la recherche Me´dicale and by Pharmacia, France.

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IS IT FEASIBLE TO ADOPT A COMMERCIALIZED APF KIT (IT-APF™ ) AS A ROUTINE DIAGNOSTIC TOOL FOR RHEUMATOID ARTHRITIS ? Sang-Gyung Kim, Kyung-Yun Jung, Hun-Suk Suh, JungYoon Choe, Seung-hie Chung, Chae-Gi Kim, Yong-Ho Song Daegu, Republic of Korea

EARLY AND FREQUENT INCIDENCE OF LOW TESTOSTERONE IN MEN WITH RHEUMATOID ARTHRITIS. John C Davis, Jr. San Francisco, CA

Objectives: Diagnostic value of antiperinuclear factor (APF) for rheumatoid arthritis (RA) is widely accepted, however this marker was only used in a limited number of laboratories because of technical difficulties of their detection and lack of standardization. Recently, a diagnostic kit using epithelial cells from human buccal mucosa became commercially available. Therefore, the objective of this study was to evaluate feasibility of the kit as a routine diagnostic tool for RA. Methods: The ready-made slides and indirect immunofluorescence (IIF) procedure were evaluated by two trained investigators. A total of 1147 APF tests were analyzed. IIF procedure followed the kit‘s instruction. Patient‘s sera were diluted 1:20 in PBS and incubated for 60 minutes for primary reaction. After washing step, secondary antibody was applied for 30 minutes which was mixed with ethidium bromide for both secondary reaction and counterstaining of nuclei at once. Results: 1) Total epithelial cell count was 482 ⫾ 9 per well. 2) There were only 18 ⫾ 4 epithelial cells (3.7 %) in a well which had normal oral cavity flora. 3) Percentage of positive cells out of total cells was 38 % (183 ⫾ 2 cells) in positive control well. 4) We found 280 positive cases out of 1147 tests (24 %). Positive cases consisted of 249 RA (88 %) including 42 seronegative RA (17 %), osteoarthritis (7 %), and 7 palindromic rheumatism (2.5 %), following ACR criteria and a couple of other arthritis. 5) Fluoroscence intensity grade of the positive cases were 1⫹ 40 %, 2⫹ 16 %, 3⫹ 9 %, and 4⫹ 35 %. 6) 1:20 dilution was found appropriate, which was in contrary to the dilution of 1:5 used in conventional IIF method. 7) Sixty minutes of primary reaction time was appropriate for detecting APF, instead of 90 minutes required in usual IIF method. 8) The secondary antibody mixed with ethidium bromide shortened total turnaround time (TAT). Thus, 2 hours of TAT was found enough. Conclusions: The commercialized APF kit was found appropriate for the future use because of not only the simplicity of procedure, relatively short TAT, easy scoring system according to fluorescene intensity and small number of normal oral cavity flora laden cells but also especially helpfulness in diagnosing seronegative RA cases.


The aim of the study was to determine the prognostic value of serum bone markers and hand bone densitometry in very early rheumatoid arthritis (RA). From a population-based recruitment, 66 patients with very early (median duration: 4.3 months [1-6 mo]) peripheral arthritis (swelling ⱖ2 joints for ⬎4weeks) were studied. Patients were steroid and DMARD naive. After a year, patients were classified as RA or non RA. Assessments were performed at entry (T1), after 9 (T2) and 12 (T3) months. Serum bone markers were C telopeptide (sCTX) by ELISA test, free pyridinoline (Pyr) and free deoxypyridinoline (Dpyr) by HPLC. Other investigation : hand bone densitometry (DEXA), hands and feet X Rays. Prognosis was defined as the progression of radiological damage from T1 to T3 (van der Heijde modified Sharp’s method). At T3, 26 patients had radiological progressive disease, and 40 no progressive disease. 50/66 patients fulfilled ACR criteria for RA (75% in non progressive group, 77% in progressive group, NS). At T1, no difference was found between progressive group and non progressive group, excepted for rheumatoid factors more frequent in progressive group (p⬍0,03). Bone mineral density (BMD) of both hands decreased significantly from T1 to T3 in the progressive group (p⬍0,03), whereas it remains unchanged in the non progressive group. Furthermore, sCTX, Pyr and Dpyr levels at T1 were negatively correlated to the evolution of hand BMD from T1 to T3 (p⬍0,02, and p⬍0,01 respectively). In patients with early RA, a structural bone damage progression after 12 months duration is associated with a decrease of hand BMD. Moreover, this decrease was negatively correlated with levels of serum bone markers at entry. These preliminary results of the VERA study need to be confirmed on a larger population.

[Background.] Several lines of evidence suggest that testosterone (T) deficiency might contribute to increased disease morbidity in men with rheumatoid arthritis (RA). Although the incidence of RA in younger men is low, the incidence increases significantly in older age groups coinciding with decreasing levels of sex hormones. Compared with age-matched controls or those with osteoarthritis or ankylosing spondylitis, male RA patients have lower levels of T, a hormone shown to increase nitrogen retention, lean body mass, strength, body weight, and stimulate the proliferation and differentiation of osteoblasts. [Methods.] We measured resting venous levels of multiple sex hormones between 8 and 10 a.m. in 33 men ⬎ 30 with RA in order to determine the frequency of hypogonadism by age group and associated predictive surrogate markers of disease activity. [Results.] Hypogonadism (total T ⬍300 ng/l) was identified in 30% of patients. Hypogonadism was found to be equally common in men under (31%) and over (30%) the age of 50. T levels did not correlate with markers of inflammation (ESR & CRP), nor with disease duration and prednisone dose. Sex binding globulin (SBG) was found to increase with age, but not prednisone dose. Follicle stimulating hormone (FSH) and leutinizing hormone (LH) were not appropriately elevated in men with low T levels. [Conclusions.] Hypogonadism is relatively common in men with RA and occurs at an earlier age than in reported age-matched healthy populations. Furthermore, age, prednisone dose, disease duration, and markers of inflammation do no adequately predict low androgen levels. These findings suggest that many men with RA would benefit from increased T screening and replacement therapy. Mean Age

56.8 yrs

Disease Duration

10.3 yrs

Prednisone Dose LH Total T % Free T SBG Incidence of Low T ⬍50 yrs Incidence Age 30-39 Incidence Age 50-59 Incidence 70-79

4 mg/day 5.51 IU/L 479 ng/dL 1.77 1.28 ug/dL 4/13 (31%) 1/2 (50%) 7/11 (64%) 0/5 (0%)

ESR FSH Free T T Index (T/SBG) Overall Incidence of Low T Incidence of Low T ⱖ50 yrs Incidence Age 40-49 Incidnece Age 60-69 Incidence ⱖ80

21 mm/hr 8.21 IU/L 80 ng/L 425 10/33 (30%) 6/20 (30%) 1/9 (11%) 1/5 (20%) 0/1 (0%)

Disclosure:

Disclosure: Supported by NIH 5K23 AR02113-03 and UCSF GCRC 37-17

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MEASUREMENTS CONTRIBUTING TO AN EARLY DIAGNOSIS OF RHEUMATOID ARTHRITIS (RA) IN PATIENTS WHO PRESENT WITH UNDIFFERENTIATED SYNOVITIS. Gaye Cunnane, Oliver FitzGerald, Tim E Cawston, Barry Bresnihan Dublin, Ireland; San Francisco, CA and Newcastle, United Kingdom

AUTONOMIC NEUROPATHY (AN) IN RHEUMATOID ARTHRITIS (RA). Fabiola Alcaraz-Lopez, Maria G Jimenez-Godoy, Francisco Fuentes-Ramirez, Ana V Perla-Navarro, Cesar Ramos-Remus, Federico Galvan-Villegas Guadalajara, Jal., Mexico

Background : The early diagnosis of RA in patients who present with undifferentiated synovitis remains challenging in the absence of specific markers for RA. Objectives : To test the ability of clinical and laboratory parameters to identify RA patients presenting with early undifferentiated arthritis (UA). Methods : Patients with UA (n ⫽ 72) and disease duration ⬍ 1 year were assessed at 6 monthly intervals for the first 18 months after presentation. Tender and swollen joint counts were recorded and blood was examined for erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), serum amyloid A (SAA), matrix metalloproteinase (MMP)-1, MMP-3 and tissue inhibitor of metalloproteinases (TIMP)-1 at these time intervals. Rheumatoid factor (RF) was determined at presentation. Joint erosions were counted in hand and feet radiographs taken at presentation and 18 months later. Non-parametric statistics were used to compare groups and the longitudinal data was calculated by the ’area-underthe-curve’ (AUC) analysis. Results : Thirty-eight patients remained undifferentiated even with follow-up, while 18 were ultimately determined to have RA; 3 evolved into a seronegative spondyloarthropathy and 13 had self-limiting disease. At presentation, only RF (p ⫽ 0.0004; sensitivity 0.83, specificity 0.75) and MMP-1 (p ⫽ 0.0002; sensitivity 0.67, specificity 0.79) could differentiate patients who evolved into RA from those who did not. When all UA patients were followed over the next 18 months, only sustained elevation (AUC values) of MMP-1 (p ⫽ 0.0005) and swollen joint count (p ⫽ 0.017) identified those patients with an ultimate diagnosis of RA. Although persistent disease at 18 months also distinguished RA from non-RA (swollen joint count: p ⫽ 0.0005), many RA patients had also developed significantly more joint erosions over this time period (p ⫽ 0.007). Conclusions : In patients with undifferentiated synovitis, certain clinical and laboratory features at presentation help identify patients who will evolve into RA. High serum levels of MMP-1 are of particular value in recognizing patients at risk of persistent synovitis before the development of early erosive disease.

AN is a well described condition in RA. Yet the relationship between RA duration and the development of AN has not been assessed. Mortality in RA is mainly due to cardiovascular causes, and AN may produce cardiovascular-related death, at least in diabetic patients. Objectives: To assess the prevalence of AN in RA using a pseudocohort design of RA patients assembled by disease duration. Methods: Consecutive patients with RA (ACR) attending one tertiary-care and one secondary-care rheumatology clinics were included and stratified according to disease duration (early RA 6 mo. to 3 yrs., established RA 3.1 to 10 yrs., late RA ⬎10 yrs.). 102 healthy women served as controls. Patients with comorbid conditions and/or on drugs affecting cardiovascular system were excluded. Demographic and clinical variables, including m-HAQ, VAS, joint counts and Larsen index, were prospectively assessed following an structured questionnaire. Patients and controls underwent autonomic function assessment using 5 non-invasive tests (3 based on heart-rate responses and 2 on blood pressure responses), as suggested by Ewing. Results: 57 RA patients were included; the mean age was 44.5 ⫾ 10; 28% had early RA, 37% had established RA and 33% had late RA. All five tests were significantly different in RA patients when compared with controls: abnormal hand-grip test in RA 42% vs 11%, p⬍0.001; abnormal Valsalva test in RA 53% vs 31%, p⫽0.008; abnormal minimum-maximum heart rate test in RA 26% vs 3%, p⬍0.001; abnormal 30:15 ratio in RA 16% vs 4%, p⫽0.01; orthostatic hypotension in RA 19% vs 0%, p⬍0.001). No significant differences were found between AN in RA patients and demographic and clinical variables, including disease duration, m-HAQ, joint counts, and Larsen index. Conclusions: The prevalence of AN in RA patients is high. It seems that RA duration does not influence the development of AN. The role of AN in cardiovascular-related mortality in RA patients remains to be elucidated.

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SLEEP DISTURBANCES IN RA: FREQUENCY AND ASSOCIATIONS WITH DISEASE MEASURES. Fathima N Kabir, Beth L Jonas, Jennifer L Dent, Shannon S Currey, Leigh F Callahan Chapel Hill, NC

PATIENTS’ WILLINGNESS TO ACCEPT IMMEDIATE RISK OF DEATH IN THE TREATMENT OF RA WITH HIGH DOSE CHEMOTHERAPY. Robert J Verburg, Saskia D Mahabali, Jacob K Sont, Anne M Stiggelbout, Jacob M van Laar Leiden, The Netherlands

PURPOSE:To assess sleep disturbances in patients with RA using a 4 item sleep scale and to examine associations with clinical variables. METHODS: 100 consecutive patients with RA who came for follow-up were assessed by a physician and completed a self-report questionnaire including a modified health assessment questionnaire (MHAQ), 5 item rheumatology attitudes index (RAI) measuring helplessness, visual analog scales for pain, fatigue and wellbeing and a 4 item sleep scale1. The sleep scale assessed number of times during the last month the subject experienced: (1)Trouble falling asleep (2)Waking up several times per night (3)Trouble staying asleep (including waking far too early)and (4)Waking up after usual amount of sleep feeling tired and worn out. The 4 questions were analyzed individually as well as a computed summary score. The scale showed a high level of reliability (␣⫽0.89). Univariate and multivariate analyses were conducted to examine correlates. RESULTS:The RA patients were 77% female, mean age of 52 years, mean MHAQ of 1.6. 64% were rheumatoid factor(RF)positive, 62% had erosive disease, 42% were currently on low dose (⬍10mg/day) prednisone, and 38% were on sleep meds. 98% of patients reported difficulty with at least one element of the scale and the mean sleep score (0-5) was 2.3(⫹/-1.6). Patients reported most difficulty with frequent nighttime awakenings followed by waking up feeling tired and then difficulty staying asleep. On univariate analysis the mean sleep score correlated with multiple measures of function and disability including the MHAQ, helplessness, pain, fatigue, well-being, prednisone, and sleep medications(p⬍ 0.05 in all). The sleep score was not statistically correlated with erosions. 35% had a mean sleep score ⬎3 indicating sleep disturbances more than 8 days/month. A parsimonious multivariate logistic regression model was used to examine associations of sleep scores ⬎3 with MHAQ, RAI, pain, sleep meds and prednisone as the explanatory variables. In this model, the poorest sleep was associated with helplessness(OR⫽2.1, CI 1.05-4.12), and sleep medications(OR⫽3.8, CI⫽1.30-11.05). There was a trend for patients who used prednisone to report more sleep disturbances (OR⫽ 2.6, CI .93-7.56). When controlling for these other variables, neither pain nor MHAQ were significant. CONCLUSION:Sleep disturbances are frequent in patients with RA and are associated with measures of disease activity, particularly scores for helplessness, even when controlling for sleep med use. 1.Jenkins, CD et al, J Clin Epi, 1988, 41(4), 313-21.

Background: Patients with intractable rheumatoid arthritis (RA) may benefit from treatment with high dose chemotherapy (HDC) followed by rescue with autologous peripheral blood stem cells (SCT). The aim of this study was to establish whether the risks of this approach are acceptable to patients with RA and whether risk taking was associated with disease associated, socio-economic parameters and/or personality traits. Patients and methods: A survey in the outpatient clinic was held. 2 cohorts of 45 (cohort A) and 51 (cohort B) RA patients with active disease enrolled. Patients received information about the potential benefit of HDC (2/3 of patients good clinical response, 1/3 no response), observed toxicities (alopecia, infections and bleedings) and duration of hospitalization. Cure was assumed not te be a realistic perspective. Cohort A was asked to choose between their own disease state for indefinite time or HDC. Nonparametric tests were performed to assess predictive factors for patients to accept TRM: swollen joint count, tender joint count, VAS, ESR, HAQ, socio-economic parameters, RAQoL and the Life Orientation Test. Cohort B was asked to consider a worst case scenario with respect to the disease activity. The minimal duration of benefit was assessed, given a transplant related mortality (TRM) of 0.01% and 2% . Results: In cohort A, VAS disease activity (P⫽0.006), VAS pain (P⫽0.021) and HAQ (P⫽0.05) were signicantly higher in patients willing to accept risk of death. Religiosity (P⫽0.093) and a higher Ritchie articular index (P⫽0.1) showed a trend towards risk taking. In cohort B, 22 of 51 patients (43%) were willing to accept TRM as part of HDC ⫹ SCT. In the 22 patients the mean required duration of benefit given a TRM of 0.01% was 42 months (range 6-180). Given a TRM of 2% the mean duration was 77 months (range 12-180). Conclusions: We evaluated risk taking in patients with RA based on a realistic perpective in which the trade off between short term risks and possible long term gain of HDC was investigated. Based on current efficacy data of HDC ⫹ SCT (2 years improvement in 2/3 patients and a TRM of 2%), a majority of patients in these cohorts did not accept a TRM of 2% . Patients willing to accept TRM had higher VAS disease activity, VAS pain and HAQ. Disclosure:

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HLA-DRB1 ALLELES AND CLINICAL MANIFESTATIONS OF RHEUMATOID ARTHRITIS IN A TRI-ETHNIC SAMPLE. Inmaculada del Rincon, Daniel F Battafarano, Ramon A Arroyo, Frederick T Murphy, Agustin Escalante San Antonio, Texas

DOWNREGULATION OF GLUCOCORTICOID RECEPTORS IN EARLY DIAGNOSED RHEUMATOID ARTHRITIS. A M Huisman, A A van Everdingen, M JG Wenting, D R v Reesema, F PJG Lafeber, J WG Jacobs, J WJ Bijlsma Utrecht, The Netherlands

BACKGROUND: The HLA-DRB1 alleles associated with rheumatoid arthritis (RA) vary between ethnic groups. The degree to which this variation translates into clinical differences is not well known. PURPOSE: To compare HLA-DRB1 alleles and clinical manifestations among people with RA in a sample from three ethnic groups. PATIENTS AND METHODS: We assessed consecutive patients with RA for age at symptom-onset, age at RA-diagnosis, joint tenderness, swelling and deformity, subcutaneous nodules, erythrocyte sedimentation rate (ESR), rheumatoid factor (RF) and treatment with prednisone or methotrexate. We typed HLA-DRB1 alleles by the PCR-sequence specific primer amplification method, and categorized patients according to the number of shared epitope (SE)-containing alleles. We used regression models to adjust ethnic comparisons for age and sex, using White patients as the reference group. To assess the influence of the SE, we fit separate models with and without the SE, as well as models with an ethnic-group X SE interaction term. RESULTS: We studied 777 RA patients, 498 of whom were women (71%), mean age 55.8 years (range 18 to 90). Ethnically, 272 were White, 53 were Black, 432 were Hispanic and 20 were Asians. Black and Hispanic patients were younger than Whites, and were more likely to be men. Age- and sex- adjusted ethnic differences included an older age at diagnosis among Blacks (46 vs. 44, P ⱕ 0.05); higher tender and swollen joint counts among Hispanics (17 vs. 11, P ⱕ 0.001 for tender joints; 8 vs. 7, P ⱕ 0.05 for swollen joints); less frequent subcutaneous nodules among Black patients (18% vs. 28%, P ⱕ 0.05); more frequently positive RF among Hispanics (93% vs. 84%, P ⱕ 0.001); higher ESR among Hispanics (45 vs. 36, P ⱕ 0.05); and more frequent use of methotrexate among Hispanics (62% vs. 56%, P ⱕ 0.05). Hispanic and Black patients were less likely to have two SE-containing alleles (OR 0.59 for Hispanics and OR 0.25 for Blacks, P ⱕ 0.01), and were more likely to be null for the SE (OR 4.59, P ⱕ 0.001 for Blacks; and OR 1.61, P ⱕ 0.05 for Hispanics). Ethnic differences that depended on the SE included the older age at diagnosis and lower frequency of nodules among Blacks. CONCLUSIONS: We observed significant differences between Whites, Blacks and Hispanics in the clinical expression of RA. These differences were accounted for, in part, by differences in the frequency of HLA-DRB1 alleles.

In rheumatoid arthritis (RA) patients with a mean disease duration of 6 years, glucocorticoid receptor (GR) down-regulation has been reported, without change in cortisol levels (Schlaghecke et al., Arthritis and Rheumatism 1992;35:740-44 and 1994;37:1127-31). This phenomenon might play a role in the etiology and/or pathogenesis of RA. In that case, GR down-regulation should also be found in early-diagnosed RA. Therefore, we compared the GR-expression, as well as the cortisol levels of early RA patients with those of sex and age matched healthy controls. In early (disease duration ⬍ 1 yr) RA patients (52F/29M; mean age 63 ⫾ 2 yrs) and in 39 controls (23F/16M; mean age 63 ⫾ 2 yrs) blood samples were taken between 8-10h AM. GR-expression (GR-number; Steiner et al., Clin.Chem. 1984;31:1855-60), cortisol-levels (fluorescence polarization immunoassay, FPIA; Abbott, Illinois, USA), ESR, CRP, painful and swollen joints were determined. RA was diagnosed according to the ACR classification criteria. In early RA a significantly lower GR-expression was found compared to controls (7.7 fmol/million cells ⫾ 0.4 vs. 9.5 ⫾ 0.7, pⱕ 0.02). Interestingly, also cortisol levels were significantly lower (0.24 mg/l ⫾ 0.15 vs. 0.37 ⫾ 0.03, pⱕ0.0001). Both findings were due to the observed differences in the female population (7.0⫾0.5 vs. 9.8⫾0.8, pⱕ0.002 and 0.21⫾0.02 vs. 0.41⫾0.05, pⱕ0.0001). In the male population no differences were found (8.9⫾0.7 vs. 8.9⫾0.4, ns and 0.29⫾0.03 vs. 0.30⫾0.03, ns). No correlations were found with parameters of disease activity nor was there a relation between GR-expression and serum cortisol levels. In early diagnosed female RA patients a decrease in glucocorticoid receptor expression was found as well as a decrease in cortisol levels. Since normal glucocorticoid receptor expression was found in the RA diagnosed early RA male population, it remains questionable whether glucocorticoid receptor expression is causally involved in pathogenesis of RA.

Disclosure: This research was supported in part by NIH grants HD37151, HL04481, AR47530, a Clinical Science Grant from the Arthritis Foundation, and a Beginning Grant-in-Aid from the Texas Affiliate of the American Heart Association.

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HLA-DRB1 ALLELES CONTAINING SHARED EPITOPE ASSOCIATED WITH EARLIER ONSET OF RHEUMATOID ARTHRITIS IN MEN BUT NOT WOMEN. Agustin Escalante, Inmaculada del Rincon San Antonio, Texas

SINGLE VALUE OF SERUM TRANSFERRIN RECEPTOR IS NOT DIAGNOSTIC FOR IRON DEFICIENCY IN ANAEMIC PATIENTS WITH RHEUMATOID ARTHRITIS. Stefan Siebert, Robert Henley, Richard Ellis, Bryan Williams, Ivor Cavill, Mark Worwood Cardiff, United Kingdom BACKGROUND Anaemia is the most common extra-articular manifestation of rheumatoid arthritis (RA). It is often difficult to differentiate between iron-deficiency anaemia (IDA) and anaemia of chronic disorders (ACD) in these patients. It has been suggested by several authors that serum transferrin receptor (sTfR) levels can differentiate between IDA (raised sTfR) and ACD (normal or slightly raised sTfR) in anaemic RA patients. The sTfR level has also been proposed as an indicator of erythropoiesis. We undertook to study the diagnostic value of a single sTfR result in anaemic patients with RA, and the relationship between sTfR and serum erythropoietin (Epo). METHODS Patients with RA attending a monitoring clinic were selected if blood tests at previous visit met WHO criteria for anaemia. Patients were excluded if they had received iron supplementation or blood transfusion within the previous 3 months. Blood samples were taken for CRP, sTfR, Epo, ferritin, transferrin saturation and red cell indices. RESULTS Samples were obtained from 74 patients, of which 44 (65%) were anaemic at time of testing. sTfR concentrations only correlated positively with percentage hypochromic cells (r⫽0.676) but none of the other variables, including Epo. Median sTfR was significantly higher in patients with clear IDA (ferritin ⬍ 50␮g/l and MCH ⬍ 27pg) than in those with ferritin ⬎ 50␮g/l and MCH ⬎ 27pg. Median sTfR did not however differ between patients with adequate iron stores (ferritin ⬎ 100 ␮g/l) and those with indeterminate iron stores (ferritin 50-100 ␮g/l). There was considerable overlap between individual sTfR values of the 3 groups. CONCLUSION While median sTfR concentration was higher in the group with low ferritin and MCH, individual sTfR concentrations did not identify further patients with iron deficiency in the group with indeterminate values of serum ferritin. sTfR concentrations were affected both by iron status and rate of erythropoiesis. This study therefore suggests that single values of sTfR are currently of limited value in determining iron status of individual patients with RA. Determination of percentage hypochromic cells may be a better and simpler indicator of functional iron status in these patients. Disclosure:

Disclosure: Supported in part by NIH grants HD37151, HL04481, AR47530, a Clinical Science Grant from the Arthritis Foundation, and a Beginning Grant-in-Aid from the Texas Affiliate of the American Heart Association

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INTRODUCTION: Both female sex and HLA-DRB1 alleles containing the shared epitope (SE) are associated with increased susceptibility to rheumatoid arthritis (RA). It is not clear how these two factors interact in determining RA susceptibility. OBJECTIVE: To test the hypothesis that women and SE-positive RA patients would have a younger age of disease onset. PATIENTS AND METHODS: A reumatologist or a trained research nurse evaluated consecutive RA patients, inquiring about the age at the time of diagnosis of RA, which we considered as the onset. We typed HLA-DRB1 alleles using PCR sequence specific primer amplification. We considered HLA-DRB1*0101, *0102, *0401, *0404, *0405, *0408, *0410, *1001, *1402 and *1406 as the SE-positive alleles. We categorized patients as null, heterozygous or homozygous for the SE. To assess the effect of sex and the SE, we modeled the age at diagnosis of RA using ordinary least squares regression. The independent variables were sex, the number SE copies, current age, and dummy variables for the recruitment center. We included an interaction term between sex and the SE. RESULTS: We studied 755 RA patients, mean age 59.9, 71% women, 88% rheumatoid factor positive, and 73% SE-positive. The figure plots the adjusted age at diagnosis for men and women, according to the number of copies of the SE. Men were significantly older than women at the time or RA diagnosis only among SE-null patients (49.1 vs. 45.0, P ⫽ 0.02). Among men, having two SE-positive alleles was associated with a reduction in the age at diagnosis of 5.0 years (95% C.I. -9.2, -0.9, P ⫽ 0.01). However, among women, the SE did not significanlty influence age at diagnosis. CONCLUSION: The SE is associated with an earlier onset of RA in men but not women. The interaction between sex and HLA-DRB1 in RA pathogenesis deserves further study.

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IMMUNOHISTOCHEMICAL ANALYSIS OF THE CELLULAR INFILTRATE AND ANGIOGENIC GROWTH FACTORS IN RHEUMATOID ARTHRITIS CORNEAL MELT. Ursula M Fearon, Konstantina Griosios, Alexander Fraser, Pamela F Jones, Martin McKibbin, Douglas J Veale Leeds, United Kingdom

THREE-DIMENSIONAL KINEMATICS AT THE ANKLE JOINT COMPLEX IN RHEUMATOID ARTHRITIS. James Woodburn, Philip S Helliwell Leeds, West Yorkshire, United Kingdom

Rheumatoid arthritis (RA) may be associated with corneal inflammation termed - Corneal Melt (CM), the pathogenesis of which is unclear. We undertook a retrospective pathological study of RA corneas, removed for CM at the time of grafting, compared to control corneal tissues. In particular, we examined for cellular infiltrate and evidence of angiogenesis and neovascularisation. All patients had seropositive RA (n⫽10; 7f:4m), providing corneal tissue from one or more events of CM (n⫽20), the control corneas all had Keratoconus (n⫽5). The RA patients had a mean (range) age of 66.8 (55-73) years, a mean (range) disease duration of 10 (2-45) years. The corneal tissues were stained using routine immunohistochemistry for protein expression of CD3, CD68, CD34, and the vascular growth factors - VEGF and Angiopoietin 1. The cell infiltration and vascularity were assessed semi-quantitatively using a standard 0-4 grading, VEGF and Ang1 expression was initially assessed as present ⫽1 or absent ⫽0. All RA CM showed evidence of increased neovascularisation compared to controls according to CD34 staining. In addition 18 (90%) showed infiltration with CD68 macrophage cells, which was grade 3-4 in more than 50%, while CD3⫹ T cells were observed in only 11 patients. Protein expression of VEGF and Ang1 was observed in 11/14 (78%) of RA CM. There were no active T cells or macrophages and no neovascularisation in the no control corneal tissue. These data suggest that RA CM is predominately a macrophage-driven disease that is associated with profound angiogenesis and neovascularisation stimulated by the growth factors, including VEGF and Ang1 which are overexpressed in the inflammatory corneas.

Objective: Combined ankle/tarsal joint involvement in rheumatoid arthritis (RA) is associated with local joint pain, stiffness and impairment of gait. This study evaluated, at a functional level, the three-dimensional (3D) motion characteristics of the resulting valgus deformity. Methods: Gait analysis was peformed in 50 consecutive RA patients (mean age- 54.0 (SD 11.8) years, median disease duration 3 (IQR 1, 7) years) presenting with painful valgus deformity of the rearfoot. 3D ankle joint complex (tibiotalar and subtalar joint) motion was measured using an electromagnetic tracking system (accuracy- 0.7°) under barefoot and shod walking conditions. Normalised motion curves were derived for dorsi/plantarflexion, inversion/eversion and internal/external rotation. The motion-time integral was derived for use in statistical analyses comparing barefoot and shod conditions from the RA patients with data from 45 age- and sex-matched normal subjects measured using the same gait protocols. Results: Valgus deformity in the RA group was characterised by a 27% reduction in inversion/eversion ROM. The RA group had a large negative motion:time integral (-546deg.s) in comparison with normals (87deg.s), and this was significantly different (P⬍0.0001). From the average motion:time curves the RA patients had a 3.2° everted heel-strike position (3.1° inverted in normals), no period of subtalar joint neutral positioning (2 periods in normals) with all motion occuring about an everted range (inversion and eversion motion in normals). Motion remained significantly different under shod conditions (P⬍0.0001). Motion was coupled hence the RA group showed a large positive motion:time integral for internal/external rotation (695deg.s) in comparison with normals (66deg.s), and this was significantly different (P⬍0.0001). At no period during stance did the mean motion:time curve for the RA AJC show external rotation past the neutral position. The deformity showed no significant deviations in dorsi/plantarflexion motion in either barefoot (P⫽0.17) or shod (P⫽0.24) conditions. Conclusions: Painful valgus deformity of the rearfoot in RA is characterised by abnormal eversion and internal rotation motion about the AJC. Gait analysis may be used to facilitate early detection and to plan and evaluate appropriate mechanical therapy.

Disclosure:

Disclosure: Supported by grants from the Medical Research Council and the Arthritis Research Campaign.

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ASSESSMENT OF PROXIMAL FINGER JOINT INFLAMMATION IN PATIENTS WITH RHEUMATOID ARTHRITIS USING A NOVEL LASER-BASED IMAGING TECHNIQUE. Alexander K Scheel, Andreas Krause, Ingolf Mesecke von Rheinbaben, Georg Metzger, Helmut Rost, Volker Tresp, Peter Mayer, Monika A Reuss-Borst, Gerhard A Mueller Goettingen, Berlin and Munich, Germany

RHEUMATOID ARTHRITIS (RA) AND INTERSTITIAL PULMONARY FIBROSIS (IPF): A FAMILY STUDY. Nuha R Said, Bernard Zimmermann, Edward V Lally Providence, RI

Objective: To evaluate a newly developed laser-based imaging technique for the study of soft tissue changes and acute inflammatory processes of proximal interphalangeal (PIP) joints in patients with rheumatoid arthritis (RA). Methods: A novel imaging device was developed which allows the transillumination of PIP joints using laser light in the near infrared wavelength range. In a first clinical follow-up study a total of 72 PIP joints of 22 patients with RA and 64 PIP joints of 8 healthy controls were examined both clinically and with the new laser device. At baseline and on follow-up after a mean of 6 weeks clinical signs of synovitis, circumference and pain degree were assessed for each PIP joint in order to determine the clinical degree of inflammation. Different features were extracted from the laser images and evaluated by a neural network (NN). Results: At baseline, 72 PIP joints from the RA patients showed clinical signs of inflammation. At follow-up, 45 PIP joints showed clinical improvement whereas 13 showed a steady active inflammation, and 14 a deterioration compared to the first visit. None of the 64 PIP joints from the healthy individuals did show any signs of synovitis. The inflammatory status of 60 of the 72 RA joints examined were classified correctly by laser examination and joint circumference determination, giving a sensitivity of 80%, a specificity of 89% and an accuracy of 83% in detecting inflammatory changes in affected joints. Laser data and joint circumference follow-up determination of healthy joints resulted in an accuracy of 85% in reproducing the image. Conclusion: The new laser-imaging technique allows the transillumination of PIP joints and gives information about the inflammatory status of the joint after processing through a NN. Our data indicate that laser imaging may provide additional information in the early diagnosis of an inflammatory joint process and in particular prove useful in assessing the follow-up of acute joint inflammation.

Interstitial pulmonary fibrosis (IPF) is a significant, yet uncommon, extraarticular manifestation of rheumatoid arthritis (RA). Familial cases of IPF in the context of RA are rare. We describe an extended family with an unusually high prevalence of RA and IPF, which came to our attention when one member presented with an acute respiratory syndrome. The index patient, a 48 year-old male with seropositive RA, had acute dyspnea and hypoxia. An open lung biopsy revealed inflammatory changes diagnostic of bronchiolitis obliterans with organizing pneumonia (BOOP). Therapy with high dose corticosteroids and oral cyclophosphamide resulted in marked improvement in his lung function and HRCT scan. The patient’s family history was remarkable for RA and IPF. The pedigree revealed that his father, paternal aunt and 4 of his 7 siblings had seropositive erosive RA. Interstitial pulmonary fibrosis was the cause of death in the patient’s mother and maternal aunt, neither of whom had RA. One sister had RA and IPF (RA/IPF), which was manifested by rapidly progressive lung disease leading to death at age 50 despite high-dose prednisone therapy. A second sister was recently diagnosed with RA/IPF on the basis of HRCT scan. Review of lung biopsies from 2 other affected relatives showed typical changes of IPF. Idiopathic familial IPF is usually a rapidly progressive disorder, unresponsive to therapy. In the kindred described, a mother with ILD and a father with RA produced 8 offspring of whom five had RA and 4 had RA/IPF. The index patient was diagnosed with BOOP and responded dramatically to immunosuppressive therapy. We believe that this study has important implications for the presence of IPF in the context of RA. Patients with RA and pulmonary symptoms should be fully evaluated since subsets of these patients may be responsive to immunosuppressive therapy. Disclosure:


Disclosure: There has been financial support by SIEMENS Medical AG for developing the prototype.

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DETECTION OF REVERSIBLE MIDDLE EAR INVOLVEMENT IN ACTIVE RHEUMATOID ARTHRITIS BY MULTIPLE FREQUENCY TYMPANOMETRY. Francisco Maceiras, Juan Antonio Garcia-Meijide, Juan Amarelo, Carlos Cea, Antonio Mera, Ruth Lechuga, Carlos Frade, Carlos Martin, Juan Jesus Gomez-Reino Santiago de Compostela, A Coruna, Spain

REFERRALS TO AN EARLY SYNOVITIS CLINIC: ARE THEY APPROPRIATE? Philip Alexander Courtney, Gary David Wright Belfast, Northern Ireland United Kingdom

The incudo-malleolar and incudostapedial joints are true diarthroses. Generally, RA involvement in the ear is atributed to sensorineural damage in the inner ear by vasculitis, neuritis or ototoxic drugs, and less frequently to inflamation of the synovial membrane of the ossicular chain joints which causes stiffnes. Multiple frequency tympanometry (MFT) by assessing the resonance frequency (RFREQ) provides an useful information to detect disorders of middle ear. Objective:To evaluate the middle ear involvement and its response to treatment in active RA by assessing the RFREQ by MFT. Material and Methods: Twenty-eight RA patients aged 20 to 75 years with a mean duration of disease of 6 ⫹/- 2 months were prospectively investigated by assessing RFREQ from 1999 to March 2001 in the Rheumatology Division of our hospital. All patient were with active disease attending clinical (articular index) and laboratory (esr/crp) criteria. No patient had prior otological history or pathological otological examination. The RFREQ values were compared with those obtained in a control group of 135 normal subjects aged from 20 to 80 years. The tympanometric study was made by a Lucas Grason Stadler, GSI-33 model. Statistical analysis was made by SPSS (ANOVA test). Results: The mean value of RFREQ in control group was 1032 Hz ⫹/-302.24. We observed that a total of 14/28 patients and 20/56 ears had abnormal high resonance values (⬎1100 Hz). In active RA, patients mean RFREQ was 1194.5 Hz ⫹/-189.5. In 3 of 4 RA patients, with high RFREQ values at baseline and with clinical RA status improvement after treatment, a 6 month reevaluation showed a decreased of RFREQ to control values. Conclusions: Our study suggests the existence in active early RA of some kind of involvement in the middle ear, causing stiffness of the ossicular chain produced by inflamation of the ear diarthrodial joints. This could be reversible by controlling RA disease activity with treatment.

Background There is mounting evidence that early treatment with disease modifying therapy improves the outcome in patients with rheumatoid arthritis (RA) and that therapy should be commenced before the disease process is established and irreversible damage has occurred. Methods We reviewed all referrals (n⫽156) to our early synovitis clinic at the Royal Victoria Hospital, Belfast, which was established in January 1999, to determine the proportion of appropriate referrals. Referral guidelines to the early synovitis clinic were circulated to all general practioners in the catchment area of the hospital (population 600,000) at three monthly intervals. Prognostic markers were recorded to determine which RA patients should be treated more aggressively with combination disease modifying therapy. Results Fifty-five percent (n⫽84) of the 156 patients were classified as having inflammatory arthritis. Of these patients, 33 were diagnosed as RA and an appropriate disease modifying drug or combination therapy was commenced. Forty-five percent (n⫽72) patients did not have inflammatory arthritis. The median time from symptom onset to referral was 8 weeks and the median time from arrival of the referral letter to attendance at the early arthritis clinic was 4 weeks. Conclusion These results suggest that, although the message regarding early referral appears to have been successful, there were a large number of inappropriate referrals. Factors contributing to inappropriate referrals may include: (a) the low priority of musculoskeletal disorders in undergraduate training, resulting in poor skills at recognising signs and symptoms of inflammatory arthritis. (b) the opportunity for faster access to a specialty with long waiting lists. (c) the broad based referral guidelines which were designed to obtain maximum sensitivity for early RA patients. We are exploring novel methods of triage in primary care groups, by general practioners with a special interest in rheumatology and specialist rheumatology nurses, in an attempt to improve future referral patterns.

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ADENOSINE MEDIATES THE ANTIINFLAMMATORY RESPONSE TO METHOTREXATE (MTX) AND ITS ANALOGUE MX-68; DIFFERENT ADENOSINE RECEPTORS SUPPRESS INFLAMMATION AT DIFFERENT ANATOMIC LOCATIONS. M Carmen Montesinos, Jiang Fan Chen, Avani Desai, Inmaculada Posadas, David L Delano, Marlene Jacobson, Michael A Schwarzschild, J Stephen Fink, Bruce N Cronstein New York, NY; Boston, MA and West Point, PA

TREATMENT OF COLLAGEN INDUCED ARTHRITIS IN DBA/1 MICE WITH L-ASPARAGINASE. Andreas Reiff, Mike Zastrow, Bee-Chun Sun, Syuji Takei, Hiro Misuhada, Bram Bernstein, Donald L Durden

Background: Adenosine, acting at one or more of its receptors (A1, A2A, A2B and A3) mediates the antiinflammatory effects of MTX in both acute and chronic inflammation. To establish which receptor(s) are involved in modulation of acute inflammation by MTX and its non-polyglutamated analogue, MX-68, we studied acute inflammation at two different sites in A2A and A3 receptor knockout (KO) mice. Methods: KO mice and age- and sex-matched littermate wild type (WT) mice received intraperitoneal (ip) injections of saline, MTX (0.75mg/kg/wk X4wks), MX-68 (2mg/kg, 2d and 1h before induction of inflammation) or dexamethasone (DEX, 1.5mg/kg 1h before induction of inflammation). Peritonitis was induced by ip injection of 0.5ml of thioglycollate (10%w/v in PBS). After 4h the animals were sacrificed, their peritoneal cavities lavaged with 3ml of PBS and leukocytes (WBC) counted. Air pouches were induced by injection of 3cc of air (2x/wk for 1wk) subcutaneously on the back. 4h after injection of carrageenan (2mg/ml in 1ml) exudates were harvested and WBC quantitated. Results: Both MTX and MX-68 reduced WBC accumulation in peritoneal exudates of WT mice (21⫾4% and 32⫾5% inhibition, respectively, n⫽18, p⬍0.001) and A3 KO mice (23⫾5% and 28⫾5% inhibition, respectively, n⫽12 p⬍0.001) but not in A2A KO mice (4⫾10% and 2⫾10% inhibition, respectively, n⫽7, p⫽NS). DEX, an agent that suppresses inflammation by a different mechanism, was as effective in A2A and A3 KO as WT mice (38⫾13%, 32⫾4% and 29⫾6% inhibition, n⫽3, 16 and 12, p⬍0.001). In contrast, MTX and MX-68 reduced WBC accumulation in air pouch exudates of WT mice (41⫾4% and 39⫾3% inhibition, n⫽13 and 31, respectively, p⬍0.001) but not A3 KO mice (-5⫾16% and 2⫾7% inhibition, n⫽11 and 20, respectively, p⫽ NS) or A2A KO mice (10⫾9% and 13⫾7% inhibition, n⫽8 and 14, respectively, p⫽NS). As with peritoneal inflammation, DEX was equally anti-inflammatory in WT, A3 KO and A2A KO mice (68⫾6%, 71⫾7% and 59⫾2% inhibition, n⫽8, 4 and 3, respectively, p⬍0.001). Conclusion: Adenosine mediates the antiinflammatory effects of MTX and its analogue MX-68 although the receptor(s) responsible for suppression of inflammation vary with the tissue involved. Disclosure: This work was supported by grants from Chugai Pharmaceuticals and the National Institutes of Health (AR41911, GM56268).

Objective: To evaluate the safety and efficacy of L-asparaginase as an immunosuppressive agent in a mouse model of rheumatoid arthritis. Methods: Male DBA/1 mice with collagen-induced arthritis (CIA) were treated at different intervals with various doses of native and pegolated L-asparaginase from E. coli. The mice were observed for 4 weeks during which time arthritis was scored. Outcome parameters included effect on severity and progression of established arthritis as well as prevention of disease. In addition, X-rays and histology from the affected joints were obtained for comparison. Results: Both native L-asparaginase at a dose of 50 IU/injection intraperitoneally three days a week and pegolated asparaginase (PEG-L-asparaginase) at a dose of 25 IU/injection twice a week, significantly reduced the mean arthritic score (MAS) in mice with established arthritis (p⬍ 0.001 for PEG-L-asparaginase). When native L–asparaginase was administered before the onset of arthritis (days 14-post immunization) the number of mice developing arthritis in the treatment group was significantly reduced (p⬍0.0001). In addition, the number of arthritic paws and the severity of arthritis in the treatment group were significantly decreased (p⬍ 0.005). Treatment with L–asparaginase markedly diminished joint pathology, as determined by the amount of inflammatory cells and the degree of cartilage destruction. Significant differences were found in the X-ray evaluation between treated and control mice. None of the animals died due to drug related events or showed signs of asparaginase induced toxicity. Conclusion: Our data provide the first direct evidence that L-asparaginase is a potent antiarthritic agent and may represent an effective second line agent for future treatment studies in juvenile and adult rheumatoid arthritis. Disclosure:

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THE NOVEL PHOSPHODIESTERASE-4 INHIBITOR ROFLUMILAST SUPPRESSES TNF-␣ PRODUCTION AND EFFICIENTLY PROTECTS MICE AGAINST COLLAGEN-INDUCED ARTHRITIS ALONE AND IN COMBINATION WITH METHOTREXATE. Johannes Barsig, Bernard P Leung, Daniela S Bundschuh, Lutz Wollin, Degenhard Marx, Rolf Beume, Foo Y Liew Konstanz, Germany and Glasgow, United Kingdom

TREATMENT OF ANTIGEN-INDUCED ARTHRITIS WITH LIPOSOMAL SECRETORY LEUKOCYTE PROTEASE INHIBITOR (ANTILEUKOPROTEINASE). Harald Burkhardt, Bettina Sehnert, Christian Schneider, Reinhard Voll, Stefan Soder, Joachim Robert Kalden Erlangen, Germany

Orally available small molecular weight compounds with ability to suppress the proinflammatory mediator TNF-␣ are of high interest for the treatment of rheumatoid arthritis (RA). Roflumilast (3-cyclopropylmethoxy-4-difluoromethoxyN-[3,5-di-chloropyrid-4-yl]-benzamide) is a highly potent and selective inhibitor of phosphodiesterase type 4 (PDE4), that effectively suppresses TNF-␣ production in vitro and in vivo [1,2]. This compound is in late clinical development for asthma and COPD. We investigated the capacity of Roflumilast to reduce the release of TNF-␣ in vivo in mice and in vitro in macrophage cultures. We also examined its antiarthritic potential in murine collagen-induced arthritis (CIA), alone and in combination with methotrexate (MTX). For induction of systemic TNF-␣ release, mice were i.p. injected with lipopolysaccharide (LPS, 5 mg/kg). Mice were orally treated with 1 or 10 mg/kg of Roflumilast 30 min prior to LPS challenge. Plasma was obtained 90 min after challenge for determination of TNF-␣. Macrophage-like RAW 264.7 cells were stimulated with LPS (1 ␮g/ml) shortly after addition of Roflumilast. TNF-␣ in plasma samples and cell culture supernatants was measured by ELISA. CIA was induced in male DBA/1 mice by two intradermal injections of 200 ␮g of type II collagen in Freund’s complete adjuvant on days 0 and 21. Roflumilast (5 mg/kg), MTX (1 mg/kg), or the combination were given once daily p.o. on days 24-35. Arthritic score (0-3) and paw thickness were monitored between days 21-39. Histological analysis was performed on joints taken at day 39 after primary immunisation. Roflumilast dose-dependently suppressed LPS-induced TNF-␣ production in vivo (75% with 10 mg/kg) and in vitro (IC50⫽27 nM). Roflumilast or MTX alone partially protected mice against CIA. However, when animals were treated with the drug combination, protection was nearly complete. The protective effect was also evident histologically by a significant reduction of joint inflammation and bone/cartilage erosion in all treatment groups. These data demonstrate that Roflumilast is a promising candidate for the oral treatment of rheumatoid arthritis. References: 1) Hatzelmann A and Schudt C. J Pharmacol Exp Ther (2001) 297: 267-279. 2) Bundschuh DS, et al. J Pharmacol Exp Ther (2001) 297: 280-290.

Background. Secretory leukocyte protease inhibitor (SLPI), or antileukoproteinase (ALP), is a physiological serine protease inhibitor [1]. Its upregulation in macrophages leads to a reduced LPS response most likely related to an inhibition of NF-kB by interference with the proteolytic degradation of its cytosolic inhibitor IkBbeta [2]. However, the therapeutic use of this principle in vivo is hampered by difficulties to target the molecule into a cytosolic compartment. Objectives. In this preliminary study we applied a liposomal preparation of ALP in the murine antigen-induced arthritis (AIA) model to enhance intracellular accumulation in inflammatory cells to explore whether ALP can reduce inflammation. Methods. Liposomes were prepared by evaporation of a mixture of phosphatidylserine, phosphatidylcholine and cholesterol in chloroform/methanol and addition of ALP solution or PBS. Antigen-induced arthritis was induced in C57BL/6 mice by intraarticular injection of mBSA after prior immunization with mBSA. Liposomal ALP was administered intraperitoneally (5 mg/kg/day) between days 1-4 after disease induction and compared with injections of PBS containing control liposomes or non-liposomal ALP. Arthritis was assessed by measuring knee joint diameter and evaluation of histological alterations in knee joint sections. Results. A marked amelioration of arthritis in mice treated with liposomal ALP was observed, already within 24 hours after the first administration, while non-liposomal ALP or control liposomes did not exhibit any effect. After induction of an arthritis flare on day 28, administration of ALP liposomes following the onset of acute inflammation again led to a marked reduction of joint swelling. Histopathological analysis of knee joints obtained from mice sacrificed at peak flare revealed that signs of acute (cellular infiltrate) and chronic arthritis (synovial proliferation, irregular cartilage surface, cartilage erosions) were exclusively diminished in the group treated with liposomal ALP. Conclusion. Taken together, the data indicate that therapeutic application of liposomal ALP reduces inflammation and consequently cartilage erosion in AIA. Therefore this form of antiproteolytic treatment might be considered as a novel therapeutic option in chronic inflammatory joint disease. References 1. Bohm B et al Biochem J. 1992; 274:269-73. 2. Lentsch AB et al Am J Pathol 1999; 154 (1): 239-47

Disclosure: Drs. Barsig, Bundschuh, Wollin, Marx, and Beume are employees of Byk Gulden Disclosure:

1889

A DEFINITIVE RANK ORDER OF EFFICACY USING DMARD, NSAID AND BIOLOGICAL THERAPIES IN THE MURINE TYPE II COLLAGEN-INDUCED ARTHRITIS (CIA) MODEL. Jacky Buckton, Chris Clarke, Ann Williams, Leo Joosten, Paul Life Stevenage and Ware, United Kingdom and Nijmegen, The Netherlands

AMELIORATION OF ESTABLISHED COLLAGEN-INDUCED ARTHRITIS BY ISATX247, A NOVEL CALCINEURIN INHIBITOR. W P Maksymowych, G Jhangri, L Aspeslet, M Abel, D Trepanier, R Naicker, D Freitag, R Foster, R W Yatscoff Edmonton, Alberta, Canada

It is essential that pre-clinical data generated in animal models of arthritis translate accurately to humans with the ultimate goal of identifying safe and effective therapies for RA. CIA is a T and B cell-dependent model that shares many similarities with RA such as synovial hyperplasia, infiltration of inflammatory cells and erosions of cartilage and bone. In order to evaluate the appropriateness of the CIA model for testing of novel anti-rheumatic compounds we determined the relative efficacy of a broad range of commonly prescribed NSAID, DMARD and biological therapies for anti-inflammatory and disease-modifying activity. The following drugs were investigated: DMARDS: methotrexate, sulfasalazine, dexamethasone, prednisolone; NSAIDS: cyclosporin A, diclofenac, ibuprofen, celecoxib, naproxen, rofecoxib, indomethacin; biologicals: soluble TNFr, anti-murine IL-1 and -CD4 antibodies. Two treatment regimes were used representing early (peri-onset) and established (therapeutic)arthritis. Animals (n⫽8 / group) were monitored for paw swelling for 14 days after which they were sacrificed and hind limbs examined histologically for inflammatory and structural changes. Data were expressed as percentage inhibition relative to vehicle-treated controls. Each drug was allocated to one of 3 efficacy groups: ⬎70%, 50-70% and ⬍ 50%. Using a therapeutic dosing regime, the following efficacies were achieved:- ⬎ 70%: dexamethasone, anti-IL-1, prednisolone, cyclosporin A (sub cutaneous) and indomethacin; 50-70%: cyclosporin A (oral), diclofenac, ibuprofen, celecoxib; ⬍50%: naproxen, rofecoxib, sTNFr, anti-CD4, methotrexate and sulfasalazine. Blinded histological analysis of joint changes agreed closely with the in vivo clinical score. Drug efficacy when dosed peri-onset was as follows: ⬎ 70%: anti-IL-1, prednisolone, cyclosporin A (oral) and methotrexate; 50-70%: sulfasalazine; ⬍50%: sTNFr and anti-CD4. Again, histological data agreed closely with clinical score. In summary, this is the first example where a comprehensive panel of anti-rheumatic drugs have been compared and ranked in the CIA model. We conclude that the CIA model represents a good predictor of activity in human RA for many classes of drug as long as the shortened time-course of CIA is taken into account and appropriate dosing schedules are used.

Objective. To examine the efficacy and toxicity of ISATX247, a novel calcineurin (CN) inhibitor, in comparison to cyclosporine (CsA) and placebo in established collagen-induced arthritis. ISATX247 has up to a 3 fold greater potency than cyclosporine (CsA) in an in vitro CN inhibition assay and in in vivo solid organ and cell transplantation models. Phase I clinical trials show no discernable nephrotoxicity. Methods. Type II collagen-immunized DBA/Lac J mice (8-10 weeks) with established arthritis were treated with ISATX247 (125ug/250ug/500 ug/mouse), CsA (250ug/500 ug/mouse), or drug vehicle by intra-peritoneal injection for 10 days from the onset of clinical arthritis. Results. A significant dose-dependent reduction in clinical severity as well as paw swelling was observed in ISATX247, but not in CsA, treated animals at 10 days. Significant improvement in synovial histology (p⬍0.001) and articular cartilage damage (p⫽0.002) scores was also noted in ISATX247 treated animals, even in the 125 ug dose group (p⬍0.02 for synovial histology; p⬍0.05 for articular cartilage damage). By comparison, neither dose of CsA had any significant impact on articular cartilage damage. ISATX247 (500 ug dose group) was the only agent to significantly decrease the development of proximal interphalangeal joint erosions. A significant reduction in Type II collagen antibody titre was noted in ISATX247 animals in both 250 ug (p⬍0.01) and 500 ug (p⬍0.001) dosage groups but only in the 500ug group for CsA (P⬍0.02). Treatment was well tolerated with no significant toxicity in ISATX247 treated groups. Conclusions. ISATX247 demonstrates efficacy and safety in the treatment of established collagen induced arthritis. Together with its improved potency and nephrotoxicity profile in comparison to CsA, this agent warrants further clinical investigation in autoimmune disease. Phase II studies in RA will commence in 2001.

Disclosure:

Disclosure: The study was supported by Isotechnika Corporation. The first author sits on the medical advisory board of Isotechnika Corporation and holds shares and stock options of the Corporation.

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TACROLIMUS (FK506) BUT NOT METHOTREXATE IS EFFICACIOUS IN RAT ESTABLISHED ADJUVANT-INDUCED ARTHRITIS. Susumu Miyata, Fusako Nishigaki, Katsue Magari, Shozo Sakuma, Toshikazu Ogawa, Yoshitaka Ohkubo, Toshio Goto Osaka, Japan

CHARACTERIZATION OF THE ANTI-INFLAMMATORY EFFECTS OF A SELECTIVE TACE INHIBITOR, DPC 333. Kris G Vaddi, Ron L Magolda, Patrick J Haley, Robert J Collins, Tracy L Taylor, Thomas P Maduskuie, Carl P Decicco, Robert C Newton, Steven M Friedman Wilmington, DE and Ridgefield, CT

Background. Tacrolimus is an immunosuppressive agent that specifically inhibits T cell activation. We have investigated the mechanism of tacrolimus on rheumatoid arthritis (RA) in vitro and found that tacrolimus potently suppresses T cell activation triggered TNF-␣, IL-1␤ and IL-6 production in human peripheral blood mononuclear cells without affecting proliferation and differentiation of normal cells, such as bone marrow cells. The aim of this study is to investigate the antiarthritic properties of tacrolimus compared to methotrexate (MTX), in rat adjuvant-induced arthritis (AIA), an animal model of RA. Methods. AIA was induced in female Lewis rats and the animals were orally administered with tacrolimus (1-5.6 mg/kg) and MTX (0.1-1 mg/kg) from day 15-24. Efficacy was determined on the basis of paw inflammation measured by paw volume and histologic change, hyperalgesia and grip strength. A new technique to evaluate grip strength in arthritic rats was developed. Peripheral white blood cell (WBC) counts and thymus weights were measured mainly as indicators of toxic side effects. TNF␣ in hindpaws was measured by ELISA. Results. Tacrolimus suppressed paw inflammation and hyperalgesia without toxic effects on WBC and thymus in established AIA. Tacrolimus also reduced TNF␣ level in hindpaws. MTX slightly suppressed paw inflammation and hyperalgesia only at the highest dose (1 mg/kg). Toxic effects were observed at lower doses than the effective treatment dose with MTX. Grip strength, an indication of functional integrity of paws in arthritic rats, was found to decrease during development of AIA. Tacrolimus treated rats recovered the grip strength loss in a dose dependent manner. In contrast, MTX treated rats completely failed to recover grip strength loss even at the highest dose. Conclusion. Tacrolimus was more effective and less toxic than MTX in therapeutic effects on advanced stage of AIA. Tacrolimus may be of significant benefit in the treatment of RA.

Tumor Necrosis Factor-␣ (TNF␣) is a pleiotropic cytokine with a well documented role in immune and inflammatory responses. Clinical trials with TNF␣ neutralizing agents such as soluble TNF␣ receptors (etanercept) and anti- TNF␣ antibodies (infliximab) demonstrated significant and sustained disease modifying effect of TNF␣ neutralization in diseases such as rheumatoid arthritis (RA). An alternative approach to modulate TNF␣ production is by inhibiting a metalloproteinase known as TACE (TNF alpha converting enzyme). TACE mediates the cleavage of pro- or membrane TNF␣ (26 kDa) to form a soluble form of TNF␣ (17 kDa), which is released into the extracellular environment. However, inhibition of TACE will not impact the membrane associated form of TNF␣, which is also functionally active. In this report, we describe the in vivo characterization of an orally bioavailable selective TACE inhibitor, DPC 333. This molecule exhibits a significant selectivity for TACE (Ki ⫽ 0.3 nM) over several other matrix metalloproteinases (MMPs) including MMP-1, MMP-3 and MMP-9. When administered orally, DPC 333 inhibits soluble TNF␣ secretion that follows bacterial endotoxin challenge in mice with an ED50 of 6 mg/kg. Treatment of mice with DPC 333 results in a significant suppression of contact hypersensitivity response (CHR) to dinitrofluorobenzene in mice with an ED50 of 10 mg/kg. Anti-arthritic effects of DPC 333 was evaluated in mouse and rat models of inflammatory arthritis. In a passive transfer model of collagen-induced arthritis (CIA) in mice, administration of DPC 333 leads to dose-dependent suppression of clinical and histological signs of disease. When administered in a therapeutic mode in a rat model of established CIA, DPC 333 treatment caused a significant reduction in the clinical signs of arthritis in a manner similar to that achieved by the soluble TNF␣ receptor:Fc , etanercept. In summary, the data presented here suggest that TACE inhibition offers a novel approach to negatively modulate TNF␣ leading to potent anti-inflammatory effects as shown in the preclinical models of inflammation.

Disclosure:

Disclosure:

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INHIBITION OF ESTABLISHED COLLAGEN INDUCED ARTHRITIS (CIA) WITH BIRB 796, A SELECTIVE INHIBITOR OF p38 MAP KINASE. Gerald H Nabozny, Donald Souza, Ernest Raymond, Christopher Pargellis, John Regan Ridgefield, CT

COMPLETE ARREST OF ADJUVANT ARTHRITIS PROGRESS BY A CD64-DIRECTED IMMUNOTOXIN IN HUMAN CD64-TRANSGENIC RATS. Hanneke van Vuuren, Theo Thepen, Vanessa Walraven, Ilonka Stuij, Joel van Roon, Jan van de Winkel Utrecht, Netherlands

It is well known that the pro-inflammatory cytokines TNF-␣ and IL-1␤ play a critical role in the pathogenesis of CIA and human RA. The p38 MAP kinase is central in the regulation of these two cytokines and inhibition of this kinase may prove to be an effective treatment for RA. BIRB 796 is a novel, potent, selective inhibitor of p38 MAP kinase. We evaluated the therapeutic efficacy of this inhibitor in the mouse model of established CIA. Arthritic B10.RIII mice were enrolled into 6 groups (n ⫽ 10/group), treated p.o. 35 days with vehicle, BIRB 796 or ␤-methasone and monitored for CIA progression. Treatment

Dose

CIA Score Day 0 (Mean ⴞSE)

CIA Score Day 35 (MeanⴞSE)

Vehicle ␤-methasone BIRB 796 BIRB 796 BIRB 796 BIRB 796

0.1 ml b.i.d. 2.5 mg/kg b.i.d. 30 mg/kg q.d. 30 mg/kg b.i.d. 10 mg/kg b.i.d. 3 mg/kg b.i.d.

1.1 ⫾ 0.1 1.3 ⫾ 0.2 1.1 ⫾ 0.1 1.0 ⫾ 0.0 1.0 ⫾ 0.0 1.1 ⫾ 0.1

8.6 ⫾ 0.5 1.6 ⫾ 0.5a 3.2 ⫾ 0.7a 3.2 ⫾ 0.7a 6.3 ⫾ 1.1b 7.6 ⫾ 0.8b

ap⬍0.01 vs. vehicle control. bp⬍0.05 vs. vehicle control.

Arthritis progression was significantly inhibited versus vehicle control in mice treated with 30 mg/kg BIRB 796 given q.d. or b.i.d. A slight but significant inhibition of CIA progression was observed in mice treated with 10 or 3 mg/kg b.i.d. BIRB 796. In a separate study, arthritic mice treated with the p38 MAP kinase inhibitor SB 203580 at 50 mg/kg b.i.d. showed no inhibition of CIA development. These results show that BIRB 796 is a potent oral inhibitor of CIA and and is therefore useful in the treatment of human RA and other inflammatory diseases.

Disclosure: CD64-RiA (MDX44) is a research compound provided by Medarex. Supported by Technology Foundation STW, applied science division of NWO and the technology program of Ministry of Economic Affairs

Disclosure:


Activated macrophages are implicated a pernicious role in RA pathogenesis. Specifically inflammatory macrophages show increased expression of Fc␥RI (CD64), the high affinity receptor for IgG, which is highly efficient in endocytosis. The presence of activated CD64 expressing macrophages was observed in biopsies from RA patients both in synovium and lining, while control biopsies from normal individuals showed limited CD64 expression on lining cells only. To deplete inflammatory macrophages we used a CD64-immunotoxin composed of the toxin Ricin A (RiA) linked to a CD64-antibody (H22). In vitro, only activated U937 cells with increased CD64 expression, enhanced by IFN␥, were efficiently killed by CD64-RiA in cytotoxicity assays (80% kill using 10-8 M CD64-RiA). Culturing synovial macrophages of RA patients with CD64-RiA showed complete elimination of CD64 expressing cells, while production of the mediator TNF-␣ strongly decreased. To determine the potential therapeutic effect of CD64-RiA in vivo, the well established adjuvant arthritis model in Lewis rats was used. Arthritis was induced in specifically for this purpose generated CD64 transgenic rats. Animals developed clinical symptoms at day 14 after induction and the degree of inflammation was scored visually for each individual paw. At day 21 animals were intravenously injected with either CD64-RiA or placebo for three consecutive days. In the control group arthritis progressed normally, while complete arrest of disease progress was obtained in the group injected with CD64-RiA. In conclusion, targeting activated macrophages via Fc␥RI may lead to new therapeutic strategies in rheumatoid arthritis.

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TWO IKK2 INHIBITORS ARE ORALLY ACTIVE SMALL MOLECULES DECREASING SEVERITY OF COLLAGEN-INDUCED ARTHRITIS IN DBA/1 MICE. Yves Sagot, Pascale Sattonnet-Roche, Shripad S Bhagwat, Charles E Grimshaw, Michel Dreano, Christine Plater-Zyberk Geneva, Switzerland and San Diego, California

TOTAL ABROGATION OF COLLAGEN II INDUCED ARTHRITIS AND THE B CELL RESPONSE TO TYPE II COLLAGEN USING SUBOPTIMAL DOSES OF A TOPOISOMERASE II ANTAGONIST. Margareta Verdrengh, Ole Zaether, Estelle Bajtner, Rikard Holmdahl, Andrej Tarkowski Goteborg and Lund, Sweden

Introduction: NF-kB activation is the key transcription factor in the synthesis of several pro-inflammatory cytokines (including TNFa and IL-1b) and destructive metalloproteinases (including MMP-1 and 3) by rheumatoid arthritis tissue. In addition, TNFa and IL-1b can directly activate NF-kB pathway thereby establishing a positive loop that can amplify and sustain the chronic inflammation. We have identified, in our proprietary kinase inhibitory library, two potent inhibitors of IKK2 (IC50 ⫽ 60-70 nM in vitro) that we have tested for their therapeutic potentials in a pilot experiment in the collagen-induced (CIA) model of RA. Methods: CIA was induced in DBA/1 mice with native type II collagen. At first signs of disease (3-4 weeks post-immunization), mice received AS 2868 or AS 2910 (15 mg/kg or 30 mg/kg) daily per os. Disease progression was monitored for 8 days by visual clinical scoring and measurement of paw swelling. At termination of the experiment paws were collected for histopathological assessment of disease progression and expression of cytokines at local level. Results: We observed a dose-related reduction of disease severity in the treated animals in comparison to vehicle treated controls. The effect was more pronounced with AS 2920 than with AS 2868. Mice receiving 30 mg/kg of AS 2920 showed a striking reduction in paw edema with a disappearance of the swelling within 5 days of treatment. In addition, an arrest in further spreading of disease was also obtained by the treatment with both molecules at 30 mg/kg. Conclusion: Our results show that a non-peptidique IKK2 inhibitor, orally delivered, can decrease disease severity and block further spreading of arthritis in the murine CIA model. This suggests that IKK2 inhibitors may have potential therapeutic benefits in rheumatoid arthritis. Treatment vehicle AS 2920 AS 2920 AS 2868 AS 2868

(15 (30 (15 (30

Paw edema, (AUC day 1 to 8 mean⫹-sem)

mg/kg) mg/kg) mg/kg) mg/kg)

0.49 ⫾ 0.09 0.29 ⫾ 0.07 - 0.13 ⫾ 0.03 0.33 ⫾ 0.06 0.05 ⫾ 0.02

Mann Whitney test, treated vs vehicle n.s. (P ⫽ 0.06) P ⬍ 0.0001 n.s (P ⫽ 0.09) P ⫽ 0.002

Collagen induced arthritis (CIA) is the most commonly used model of rheumatoid arthritis (RA). In both CIA and RA there is an increase in the cellular content of the synovium being dominated by macrophages. We decided to assess the impact of etoposide, a topoisomerase II antagonist, known to induce monocyte apoptosis, on the development of CIA. Methods: Mice were primed and booster-immunized with collagen II. One group of mice was treated with etoposide two days prior to immunization with collagen and then on four consecutive days weekly until the end of the experiment. The second group of mice was injected with etoposide four days per week starting 40 days after collagen priming. The mice were examined concerning development of arthritis, numbers of circulating leukocytes, serum collagen II antibody and cytokine levels. Results: None of the mice administered etoposide prior to collagen immunization developed arthritis. Serum levels of anti-collagen type II antibodies were undetectable in these mice, while they displayed significantly increased levels of IFN-gamma, IL-6, and MIP-1alpha. In addition, the collagen II specific B cell responses in the draining lymph nodes were highly suppressed. Also mice treated with etoposide at the onset of clinical arthritis showed reduced frequency of their diasease by 50 %. Conclusion: Our study demonstrates a striking disease alleviating impact of topoisomerase II antagonist on the course of collagen II induced arthritis. Disclosure:

Disclosure:

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VALDECOXIB, A NEW COX-2 SPECIFIC INHIBITOR, IS EFFECTIVE IN TREATING THE SIGNS AND SYMPTOMS OF RHEUMATOID ARTHRITIS. William Bensen, Arthur Weaver, Luis Espinoza, William Riley, Beverly Paperiello, David P Recker Hamilton, Ontario, Canada; Lincoln, NE; New Orleans, LA and Skokie, IL

EFFICACY AND SAFETY OF PROSPIDINE VS METHOTREXATE IN ACTIVE RHEUMATOID ARTHRITIS: RESULTS OF A CONTROLLED, RANDOMIZED 12-MONTH TRIAL. Efim Benenson, Boris Nemtzov, Olga Timina, Volker Diehl Germany and Russian Federation

Background: To evaluate the efficacy and tolerability of three single daily doses of valdecoxib, a novel COX-2 specific inhibitor, compared with the conventional NSAID naproxen and placebo. Methods: This 12-week, double-blind, placebo-controlled trial compared the efficacy and tolerability of valdecoxib 10 mg, 20 mg and 40 mg QD with naproxen 500 mg BID or placebo in patients with rheumatoid arthritis (RA). Efficacy was assessed by measuring the responder rate to the American College of Rheumatology-Responding Index (ACR-20). The number of ACR responders were analyzed using the Cochran-Mantel-Haenzel test Stratified by center. Results: The ACR-20- response at weeks 2, 6 and 12 was significantly improved for all doses of valdecoxib compared with placebo (p⬍0.01) and similar to naproxen (Table). All three doses of valdecoxib were well tolerated. Naproxen demonstrated a higher incidence of abdominal pain, dyspepsia and constipation compared to the lower doses of valdecoxib (10 mg and 20 mg QD). Conclusions: This study shows that valdecoxib (10 mg, 20 mg and 40 mg QD) is consistently superior to placebo and similar to naproxen in treating the signs and symptoms of RA over a period of 12 weeks. The lower doses of valdecoxib (10 mg and 20 mg QD) show improved GI tolerability compared with naproxen. Therefore, some extra benefit may be derived from increasing the dose of valdecoxib from 10 mg to 20 mg, but there was no additional benefit in increasing the dose to 40 mg.

Objective: to compare the effects of the new anti-rheumatoid DMARD prospidine (PRO) with methotrexate (MTX) in active rheumatoid arthritis (RA). Methods: 93 patients (mean age 47 years, median disease duration 7 years, RF⫹ 85%) with highly active RA /DAS(28) ⱖ5/ were randomized (ratio 2:1) in two groups: 62 patients were treated with PRO (500 mg intravenously every 5 days, for the first 3-4 weeks and then monthly) and 31 patients with MTX (30 mg/ week for the first 3-4 weeks intravenously, and then 7.5, titrated to 15 mg/week). Results:

mg mg mg mg

Week 2 30 47 ** 46 ** 51 ** 51 **

QD QD QD BID

1

3

6

12

Drugs N ACR 20 ACR 50 ACR 70 TJC SJC

PRO/MTX 62/31 793/48 21/13 -/523/29 613/36

PRO/MTX 60/29 982/72 23/27 153/641/45 812/57

PRO/MTX 59/26 78/71 32/27 13/12 66/53 85/69

PRO/MTX 48/20 73/70 23/15 11/10 63/43 81/63

% response % response % response % decrease % decrease

3⫽p⬍ 0.001, 2⫽p⬍ 0.01, 1⫽p ⬍ 0.05 vs MTX

ACR-20 Responders Index - % Responders (n⫽1089)

Placebo Valdecoxib 10 Valdecoxib 20 Valdecoxib 40 Naproxen 500

Month

Week 6 36 51 ** 49 * 52 ** 50 *

Week 12 32 49 ** 48 ** 46 ** 44 *

Statistically significant * pⱕ0.01 versus placebo, **p⬍0.001 versus placebo Disclosure: Sponsored by Pharmacia Corporation and Pfizer Inc

During the 1st and 3rd months, statistically greater reductions in both ACR 20% and 70% response rates as well as in tender (TJC) and swollen (SJC) joint counts were seen with PRO as compared to MTX. At these treatment stages, CRP and rheumatoid factor showed a significant reduction only in the PRO group. These differences were less pronounced or disapperead after 6 months. Only 4 from 21 PRO-patients with of long standing hormone dependence have discontinued steroids. 76% of PRO patients and 65% of MTX patients completed 12 months of treatment. During the trial we found a drop-out for lack of efficacy of 17.6% (PRO) vs 15.7% (MTX) and for adverse events (AEs), of 6.4% (PRO) vs 19.3% (MTX). The incidence of AEs was for PRO 23 % (transitory cystitis, proteinuria, parasthesia,dizziness, allergy) and for MTX 38% (stomatitis, cytopenia, elevated transaminase level, nausea). Conclusions: PRO compared to MTX seems to be a rapidly acting and well tolerated regime with high antiinflammatory potential. Effective maintaining therapy with PRO has to be improved. Disclosure:

1897

1900

RESULTS OF A PIVOTAL (PHASE III) PLACEBO AND ACTIVE COMPARATOR CONTROLLED EFFICACY TRIAL OF ROFECOXIB 12.5 AND 25 MG IN ADULT PATIENTS WITH RHEUMATOID ARTHRITIS (RA). Kenneth E Truitt, Michael Lee, Lisa M DeTora, Marjorie Anderson, Peng-Liang Zhao Rahway, New Jersey

METHYLPREDNISOLONE PULSE THERAPY VERSUS INFLIXIMAB IN THE TREATMENT OF SEVERE FLARES OF CHRONIC POLYARTHRITIS. Hans C Nossent, Gunstein Bakland, Hanne K Aslaksen, Gunn Olsen, Bjorn Y Nordvag Tromso, Norway

Objective: To determine the efficacy and safety profile of rofecoxib 12.5 mg and 25 mg vs placebo and naproxen in patients with RA. Methods: 909 RA patients were randomized in a double-blind fashion to: placebo (n⫽301), rofecoxib 12.5 mg (n⫽148), 25 mg (n⫽311), or naproxen 1000 mg (500 mg bid; n⫽149) for 12 weeks. Eligible patients were chronic NSAID users and demonstrated a clinical worsening or flare upon withdrawal of prestudy NSAID(s). Efficacy assessments included all components of the ACR core set. Four endpoints were prespecified as primary: tender and swollen joint counts and patient and investigator global assessments of disease activity. The proportion of patients meeting ACR20 criteria, patient assessment of pain, and a modified HAQ were key secondary measurements. Safety was assessed based on investigator-reported clinical and laboratory adverse experiences (AEs). Results: Over the 12-week period, patients receiving rofecoxib 25 mg and naproxen showed similar differences from placebo consistent with improvement: a 3 joint reduction in the number of tender joints; a 1 to 2 joint reduction in the number of swollen joints; a 7 to 10 mm improvement on the patient global assessment (0 -100 mm VAS) and a 0.3 unit reduction on the investigator global assessment (0 to 4 Likert scale); all were statistically significant. In contrast, rofeocixb 12.5 mg was significantly different from placebo only for the patient and investigator global assessments. For key secondary measurements, all active treatments showed a ⬎10% difference (p⬍0.05) versus placebo for the proportion of patients meeting the ACR20 criteria. All active treatments showed a significant difference from placebo, consistent with improvement, for the modified HAQ and the patient assessment of pain. No statistically significant differences were noted between 25 mg rofecoxib and naproxen for any efficacy measurement. All treatments were generally well tolerated, and no unexpected patterns in adverse experiences were observed. Conclusions: Rofecoxib 25 mg once daily and naproxen 500 mg twice daily were similarly effective in the treatment of rheumatoid arthritis, as assessed by primary and key secondary measures. Rofecoxib 12.5 mg once daily showed improvements on two of four primary end points (patient and investigator global assessments). All treatments were well-tolerated.

Background While oral corticosteroid (CS)therapy has a well-established anti-inflammatory effect in patients with chronic polyarthritis, the role of supraphysiological doses (“pulse”) CS is less clear. At our institution intravenous pulse CS has long been used as ’bridging’ therapy for severe polyarhritis. The introduction of the anti-TNFalpha agent Infliximab raised the question if we should continue this practice. We therefore compared the effect of a short course of high dose intravenous (iv) Methylprednisolone (MP) with that of three Infiximab infusions in 19 consecutive patients with severe exacerbations of their polyarthritis . Patients-methods Longitudinal observational cohort-study of 19 consecutive inpatients (67% females) with established polyarthritis (mean disease duration 9,9 years) with severe disease activity. (ⱖ swollen and/or joints, ⬎1 hour morning stiffness,ESR ⬎30 and/or CRP ⬎20) despite previous DMARD therapy. Ten patients received three infusions of MP 1 gram on alternate days at baseline MP-group)and nine patients received three Infliximab infusions(3mg/kg)at baseline and again after two and six weeks (I-group). Disease parameters were recorded at baseline (t⫽0), one (MP-group) to two weeks(I-group)later (t⫽1)and twelve(MP-group)to fourteen weeks later(I-group)(t⫽2). All patients were started on concommitant DMARD medication. Non-parametric tests were used in the statistical analysis. Results Despite the lack of randomisation both groups were comparable in demographic, clinical and laboratory characteristics at baseline. Reductions in disease parameters at both t⫽1 and t⫽2 were similar in both groups. The number of patients reaching ACR 20-50 or 70% response criteria were also similar at both follow-up evaluations. Side effects occurred at similar rates, although none in the MP group resulted in termination of therapy, while 2 patients had to discontinue Infliximab therapy. Conclusion A short course of intravenous MP is as useful as Infliximab therapy in the short-term treatment of severe disease flares in patients with polyarthritis. MP adjuvant therapy may have efficacy up to 3 months, has milder side effects and considerably lower cost than Infliximab therapy. Disclosure:

Disclosure: Research was funded by a grant from Merck & Co., Inc.

1901

EFFICACY AND TOLERANCE A EVERY OTHER WEEK (EOW) INTRAMUSCULAR METHOTREXATE (EOW-IM-MTX) REGIME IN PATIENTS WITH RHEUMATOID ARTHRITIS (FOLLOWED FOR ONE YEAR). JF Moctezuma, C Pacheco-Tena, MA Gonzalez, G Medrano, C Garcia, R Burgos-Vargas Mexico, DF, Mexico

A RANDOMISED PLACEBO CONTROLLED TRIAL OF LOW DOSE ORAL PREDNISOLONE IN THE TREATMENT OF EARLY, ACTIVE RHEUMATOID ARTHRITIS - THE EFFECT ON CLINICAL DISEASE ACTIVITY. David McCarey, Hilary A Capell, Duncan R Porter, E Anne Thomson Glasgow, United Kingdom

Introduction Methotrexate (Mtx), a drug that was seldom given in the early 1980’s , is now the disease-modifying antirheumatic drug most commonly used in RA, intramuscular (IM) administration has been successfully carried out. Objective: To assess the efficacy and the tolerance of IM administration of MTX (IM-MTX) in a every other week regime in patients with RA, after achieving disease control with a IM-MTX weekly regime. Material and methods: We included patients with RA (ARA criteria) treated with 16.6 mg of weekly IM-MTX whom have completed a 4 month long scheme, some patients used aside NSAID’s and corticosteroids (ⱕ10 mg of prednisone). After the completion of the 4 month weekly IM-MTX scheme patients were switched to a (EOW) IM-MTX, each dose remained in 16.6 mg so final dose was reduced to half, the EOW IM-MTX lasted for 12 months. Patients were assessed at baseline, 4 months (weekly IM-MTX completion) and at 12 months (after 8 months of EOW-IM-MTX), in each visit we assessed tender and swollen join counts, pain VAE, AIMS, morning stiffness (in minutes), and general health status (physician and patient). Statistical analysis: Comparisons between the assessments were done with paired t test. Results: 30 patients (28 F/ 2 M), age 38.8⫾9.6 years and duration of the disease of 3.7 ⫾ 1.6 years were included. Relevant aspects of the follow-up are presented on the table, as can be noted the joint counts remained without relevant changes, the comparison of the rest of variables between the 4th and 12th month although statistically significant are clearly clinically irrelevant.

Purpose - Kirwan et al (NEJM 1995 333 p142-146) found that the radiological progression of rheumatoid arthritis is retarded by the use of low dose corticosteroid in early disease. This trial was designed to establish whether these findings could be reproduced within another study population, and to further study the impact of low dose corticosteroid on clinical disease activity. This abstract reports the results of the clinical findings. Methods and patients - 167 patients with rheumatoid arthritis of less than 3 years disease duration, and starting their first disease-modifying anti-rheumatic drug (prior use of hydroxychloroquine was allowed), were randomised to receive prednisolone 7mg daily or placebo, in addition to their other therapy. All patients started sulphasalazine therapy at the same time, but subsequently, disease-modifying therapy could be changed by the patients’ rheumatologists as the clinical need arose. Data was collected on demographics, ESR, C-reactive protein, Ritchie articular index, visual analogue pain score, physician and patient global evaluations of disease activity and Health Assessment Questionnaire, at baseline and annually for 2 years. Analyses used Mann-Whitney, Wicoxon and Chi-squared tests as appropriate. Results - there were no significant differences between the groups at baseline. The group receiving oral prednisolone had a lower ESR at year 1 (borderline significance, P⫽ 0.05),but there were no other significant differences between the groups in any other variable at year 1 or year 2. There was a trend (Chi squared p ⫽ 0.07) for more 20% responders in the prednisolone group at one year, but there was no difference in the 20% response rate at year 2.

Tender joint count Swollen joint count Pain VAS AIMS Morining stiffness Patient global disease activity Physician global disease activity

Baseline

4th month

p (bl -4 mth)

12 month

p (4 -12 mth)

27.0 ⫾ 15.7 14.3 ⫾ 10.5 18.3 ⫾ 8.8 5.6 ⫾ 3.0 108 ⫾ 124 3.2 ⫾ 0.76 3.2 ⫾ 0.93

3.5 ⫾ 4.5 3.2 ⫾ 0.7 7.8 ⫾ 5.2 2.2 ⫾ 1.5 6.2 ⫾ 12.8 2.0 ⫾ 0.6 1.2 ⫾ 0.41

0.001 0.001 0.001 0.001 0.001 0.05 0.001

6.08 ⫾ 8.6 2.1 ⫾ 4.1 8.8 ⫾ 5.7 3.1 ⫾ 1.3 21.7 ⫾ 39.7 2.40 ⫾ 0.57 1.06 ⫾ 0.72

0.108 0.015 0.169 0.005 0.022 0.005 0.003

Differences from baseline to the 4th month were sustained at the end of the EOW-IM-MTX follow-up. Two patients were excluded (1 pregnancy, 1 consent withdrawn), no serious side effects were noted Conclusions: The use of EOW-IM-MTX maintains the improvement achieved by weekly IM-MTX administration, it is well tolerated and an option to ease patients compliance despite repeated puncture when MTX administration is required and oral intake is a non-option.

Prednisolone(n⫽85) Median ESR (mm/hour) CRP (mg/l) Pain score (0-100) Physician global (0-4) Patient global (0-4) Ritchie AI HAQ score (0-3) 20% responders

Year 0 33.5 17.5 54 2 2 13.5 1.75

Year 1 12 10 42 1 1 7 1.375 53%

Placebo(n⫽82) Year 2 14 10 46.5 2 2 8 1.375 41%

Year 0 40.5 25 56 2 2 17 1.625

Year 1 18 10 50 1 2 8 1.5625 39%

Year 2 20 10 46 1 2 7 1.5 43%

Conclusions - low dose oral prednisolone has no impact on the control of clinical disease activity in patients with early rheumatoid arthritis. Disclosure:

Disclosure:

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1898

1902

1905

A PHASE I/II DOUBLE-BLIND, PLACEBO-CONTROLLED TRIAL OF AN EXTRACT OF TRIPTERYGIUM WILFORDII HOOK F SHOWS BENEFIT IN PATIENTS WITH RHEUMATOID ARTHRITIS. Xuelian C Tao, V Pinyopornpanit, Jean Younger, John Jian Cai, Yanping Mao, Li Ma, Sofia Chang, Peter E Lipsky Dallas, TX

B LYMPHOCYTE DEPLETION IN RHEUMATOID ARTHRITIS: EARLY EVIDENCE FOR SAFETY, EFFICACY, AND DOSE RESPONSE. Maria J Leandro, Jonathan CW Edwards, Geraldine Cambridge London, United Kingdom

Extracts of Tripterygium wilfordii Hook F (TWHF)have been reported to be effective in the treatment of autoimmune diseases. To examine the safety and efficacy of TWHF in the treatment of patients with rheumatoid arthritis (RA), an ethanol/ethyl acetate extract was prepared and used in a prospective, double blind, placebo-controlled study in patients with long standing RA who had failed conventional therapy. Thirty-five patients were randomly assigned to receive either placebo or low dose (180 mg per day) or high dose (360 mg per day) of the ethanol/ethyl acetate extract of TWHF for 20 weeks. Patients who completed the full course of the double-blind phase were given an option to continue treatment in an open label extension. Clinical and laboratory variables were examined before and every four weeks after starting the treatment course. Clinical responses were defined as 20% improvement in disease activity according to the ACR criteria. Side effects were actively queried based upon previous reported experience with TWHF treatment in China.Results are shown in the table. Conclusion. Treatment with the ethanol/ethyl acetate extract of TWHF leads to significant therapeutic efficacy. At therapeutic dosages, the ethanol/ethyl acetate extract of TWHF is well tolerated by most RA patients.

Initial Treatment

Placebo (n⫽12)

Low Dose (n⫽12)

High Dose (n⫽11)

4 1 12

2 1 10

0 1 10

0 4 2 0

4 4 4 0

8 7 5 0

Early Withdrawals Disease Worsenning Side Effects Completing more than 4 weeks of Treatment 20% Improved Enrolled into Open-label Extension 20% Improved Side Effects Causing Withdrawals

Purpose: To test the hypothesis that B lymphocytes are essential to the pathogenesis of rheumatoid arthritis (RA), an open study of B lymphocyte depletion in 5 subjects was set up in 1998. Further open treatment has aimed to explore lower doses and safety. Methods: Eighteen further subjects have been treated with more than 6 months (mean 14m) follow up. All had erosive RA, and had failed 2-6 DMARDs (median 4). All but 2 were female. One was seronegative. Median age was 58yrs (33-81) and disease duration 18yrs (5-40). Functional class was III in 11, II in 6 and IV in 1. Extra-articular features were anaemia: 12, nodules: 5, vasculitis: 1, amyloidosis: 1. DMARDs were discontinued at day 0. Subjects were treated in 3 new cohorts (II-IV). Cohort II (n⫽8) rituximab 600-700mg/MSq, cyclophosphamide (CP) 1.5G, oral prednisolone cover (60mg/day over 10 days): Cohort III (n⫽6) rituximab 1200mg/MSq, CP 1.5G: Cohort IV (n⫽4) rituximab 500mg/MSq, CP 1.5G, oral prednisolone cover (60mg/day over 10 days). In all cohorts CP was reduced to 1G for body weight ⬍50Kg. (Cohort I (n⫽5) had received rituximab 1400mg/MSq, CP 1.5G, oral prednisolone cover 60mg/day.) Results: Adverse events attributable to therapy were phlebitis (1) and nausea (7) due to CP and 2 brief respiratory infections. (No rituximab infusion reactions.) ACR grades at 6 months were: Cohort II: 70,70,20,20,70,70,70,50; Cohort III: 70,50,70,0,20,50; Cohort IV: 0,0,0,0. (Cohort I had achieved: 70,70,70,50,50.) Of all 4 cohorts, 6 subjects sustain responses to a single treatment at 10-30 months. All extra-articular features improved. Serum amyloid A normalised in the amyloidosis case. The seronegative case did not improve. Conclusions: B lymphocyte depletion shows promise as a safe, effective therapy for RA. Major improvement occurs in most cases and in some persists well after B lymphocyte repopulation. Most cases are likely to need retreatment with regimens used so far. A major response at 6 months appears to require a minimum of rituximab 600-700mg/M.Sq, with 53% ACR70 and 79% ACR50 in 19 treatments. Four other cases given 300-700mg/MSq rituximab ⫹/- steroid without CP have failed to respond, but major response might occur with higher dose rituximab without CP. Steroids appear to improve outcome but further study of steroid and CP dosage is needed. Failure of cohort IV to respond at 6 months supports evidence that IV CP ⫹/- steroid does not induce sustained benefit. A phase II study is in progress. Further work is needed to establish the mechanism of action of this therapy. Disclosure:


Disclosure:

1903

1906

RECOMBINANT HUMAN SOLUBLE P55-TNF RECEPTOR, ADMINISTERED VIA SINGLE INTRA-ARTICULAR INJECTION, IN RHEUMATOID ARTHRITIS: RESULTS OF A RANDOMISED PILOT STUDY. Rainer Klocke, Deborah Speden, Isabelle Borghini, Alberto Ferreira, Arnaud Ythier, David R Blake Bath, United Kingdom and Geneva, Switzerland

AN AGONISTIC ANTI-HUMAN DR5 ANTIBODY IN TREATMENT OF AUTOIMMUNE DISEASE. Weimin Liu, Limin Zhao, Zheng Wang, Kimihisa Ichikawa, Winn Chatham, Alex Szalai, Robert H Carter, Robert P Kimberly, Tong Zhou Birmingham, AL and Tokyo, Japan

Background: Chimaeric antibodies against tumour necrosis factor-alpha (TNF␣) are beneficial in the treatment of rheumatoid arthritis (RA), but they can be immunogenic. A recombinant form of the natural soluble p55-TNF receptor (TBP-1) should neutralise TNF␣ without inducing immunogenicity. This double-blind study examined the antiinflammatory activity and safety of a single intra-articular (IA) injection of TBP-1 in RA. Method: 23 subjects with RA and active synovitis with effusion of at least one knee joint were randomised to receive one of the following via single IA injection of the inflamed knee: 0.5 mg TBP-1 (n⫽8), 5 mg TBP-1 (n⫽7) or placebo (n⫽8). Recent systemic steroid or regular steroid treatment in excess of 10 mg prednisolone/day was not permitted. Synovial fluid (SF) (from the injected knee) and serum were assessed by ELISA for concentrations of TBP-1, total (i.e. free and bound) TNF␣ and IL-6 at baseline, 4-6, 24 and 144 hours post injection. ACR response criteria were recorded at the same time and 28 days post injection. Adverse events reported here refer to the first 28 days following injection. Results: Baseline characteristics were similar in the 3 groups. The group receiving 5mg TBP-1 had a significant decrease from baseline of median SF IL-6 at 24 hours post injection (-8.8ng/mL vs ⫹2.4ng/mL in placebo; p⬍0.05, non-parametric ANOVA). At the same time point, a significant increase in median SF total TNF␣ was observed (⫹261pg/mL vs ⫹1.2pg/mL in placebo, p⬍0.01). Corresponding trends in the 0.5mg group suggest dose-dependency of these observations. Serum IL-6 and total TNF␣, and ACR response criteria did not change significantly in any group. All adverse events possibly or probably related to the intervention in the active treatment groups were mild or moderate and the frequency was comparable in all groups. Antibodies to TBP-1 were not detected. One subject, who had received 5mg TBP-1, developed increased dsDNA-binding (by ELISA) from borderline values at baseline. However, there were no clinical features to suggest SLE. Conclusion: TBP-1, a novel recombinant soluble p55-TNF receptor, is well tolerated, non-immunogenic and exhibits local anti-inflammatory effects, when given once intra-articularly in RA. Although no significant clinical improvement was observed with this single IA injection, the results warrant further clinical trials in RA, using a systemic route of administration.

Increasing evidence indicates that TRAIL is involved in apoptosis of immune cells. However, the role of DR5 in TRAIL-mediated apoptosis of T and B cells is unknown. Using a novel specific, agonistic anti-human DR5 antibody, TRA-8, expression of DR5 in normal human T cells and B cells was not detectable, and these cells were not susceptible to TRA-8-mediated apoptosis. However, both activated T cells and B cells expressed increasing levels of DR5 and became susceptible to TRA8-mediated apoptosis. Compared to Fas, DR5-mediated apoptosis of T cells appeared to be selective for activated blast cells. Interestingly, the peripheral B cells from approximately 30% SLE patients had increased DR5 expression (n⫽12) and were susceptible to TRA-8-mediated apoptosis. In some RA patients, as much as 50% of the T cells isolated from synovial fluid expressed high levels of DR5 compared to peripheral T cells from the same donors. In vitro treatment of activated T cells with TRA-8 inhibited T cells proliferative response. In vivo treatment of the human PBMC/SCID mice with TRA-8 inhibited activation of human T cells by inducing apoptosis of activated T cells. In murine models of autoimmune disease, inoculation of lpr/lpr mice with adenoviral vector encoding TRAIL (Ad/TRAIL) inhibited the development of lymphadenopathy, and reduced autoantibody production. Treatment of SCID recipient mice with Ad/TRAIL prevented GVHD induced by allogeneic lpr/lpr T cells. Inoculation of NZB/W F1 mice with Ad/TRAIL inhibited anti-dsDNA antibody production. These results indicate that TRAIL modulates autoantibody production and that DR5 is involved in TRAIL-mediated apoptosis of T and B cells. Given the fact that TRA-8 does not induce hepatocyte toxicity, TRA-8 might be a selective immunomodulator for treatment of autoimmune disease.

Disclosure: This work was supported by Serono International SA

Disclosure:

1904

1907

A PHASE I SAFETY STUDY OF COMBINATION TREATMENT WITH PEGYLATED SOLUBLE TUMOR NECROSIS FACTOR RECEPTOR TYPE I (PEG sTNF-RI) AND ANAKINRA (INTERLEUKIN-1 RECEPTOR ANTAGONIST, IL-1RA) IN PATIENTS WITH RHEUMATOID ARTHRITIS. Jacques R Caldwell, Howard Offenberg, Daniel Furst, Nancy P Lam, Gerald Jayne, Tenshang Joh, Mark W Davis, Carl K Edwards III Gainesville and Daytona Beach, FL; Seattle, WA and Thousand Oaks, CA

A PHASE 1, SINGLE DOSE ESCALATION STUDY OF IL-1 TRAP IN PATIENTS WITH RHEUMATOID ARTHRITIS. Hans-Peter Guler, Jacques Caldwell, Thomas Littlejohn III, Harris McIlwain, Howard Offenberg, Neil Stahl Tarrytown, NY; Gainesville, Tampa and Daytona Beach, FL; Winston-Salem, NC

Objectives: The safety of combination treatment with PEG sTNF-RI and anakinra in patients with rheumatoid arthritis (RA) was assessed in a Phase 1, double-blind, placebo-controlled, dose-escalation study at 3 US centers. Methods: Weekly injections of 10, 30, 60 mg, or twice weekly injections of 800 ␮g/kg of PEG sTNF-RI, or placebo, plus daily injections of anakinra (100 mg) were given for 8 weeks following a single-dose treatment phase. Eligible patients had active RA (defined by the ACR criteria) of at least 6 months of duration, ⱖ 6 swollen joints and either ⱖ 9 tender/painful joints, CRP ⱖ 1.5 mg/dL, ESR ⱖ 28 mm/hr, or morning stiffness ⱖ 45 minutes. Patients were required to withdraw from DMARDs for ⱖ 4 weeks prior to entry and throughout the study. Stable doses of corticosteroids and NSAIDs were allowed. Safety was evaluated by summary statistics for adverse events (AEs) and infectious episodes. Results: A total of 16 patients received the combination of PEG sTNF-RI ⫹ anakinra and 4 patients received placebo ⫹ anakinra (control group). Baseline characteristics were similar between the 2 groups. Two patients who received PEG sTNF-RI ⫹ anakinra discontinued early due to joint pain and consent withdrawal. The incidence of AEs observed in the PEG sTNF-RI ⫹ anakinra group and the control group were similar: injection site reactions (62.5 % vs.100 %, respectively), gastrointestinal (GI) complaints (50% vs. 75%, respectively), and respiratory complaints (37.5% vs. 50%, respectively). Four serious AEs were reported among the PEG sTNF-RI ⫹ anakinra treated patients (1 GI hemorrhage, 1 abdominal pain, 1 syncope, 1 fracture due to a fall). None of the events was judged to be related to study drugs or resulted in withdrawal from the study. No serious AEs were reported in the control group. Five (31.3%) patients who received PEG sTNF-RI ⫹ anakinra experienced 6 mild to moderate infectious episodes (1 upper respiratory infection, 1 bronchitis, 1 sinusitis, 2 urinary tract infections, 1 bacterial vaginitis), while one patient in the control group experienced a mild infectious episode (phlebitis). No serious infectious episodes were reported in either of the treatment groups. Conclusions: Mild to moderate infectious episodes occurred in patients receiving the combination of PEG sTNF-RI and anakinra. However, no serious infectious episodes were reported during this 8-week, Phase I study. Larger studies that will further characterize the safety and efficacy of the combination treatment with PEG sTNF-RI and anakinra are ongoing in patients with RA.

Rationale: IL-1 Trap is a fusion protein consisting of IL-1 receptor extra-cellular domains and the Fc portion of human IgG1. IL-1 Trap contains the IL-1 Type 1 receptor and the IL-1 receptor accessory protein. In vitro IL-1 binding studies demonstrate that IL-1 Trap has a very high binding affinity (dissociation constant ⬇1pM) for IL-1␤ and IL-1␣. Methods: This was a placebo controlled, randomized, double blind, single dose escalation study. Twenty patients were enrolled in cohorts (5/group), and received a single sc injection of IL-1 Trap 50, 100, 200, or 400 ␮g/kg, or placebo. Clinical observations and laboratory measurements were obtained. Results:

T max (hrs) C max (ng/mL) Terminal half-life (hrs) Clearance (mL/hr/kg)

50 ␮g/kg

100 ␮g/kg

200 ␮g/kg

400 ␮g/kg

90 218 128 0.93

96 300 162 1.00

96 747 214 0.94

120 1896 182 0.49

The pharmacokinetics of IL-1 Trap are dose dependent. IL-1 Trap was well tolerated. Adverse events were infrequent, did not show a pattern, and tended to be mild to moderate in severity. Conclusions: The IL-1 Trap is a highly specific IL-1 antagonist with an extended serum half-life. Once weekly dosing in RA patients appears adequate. Disclosure: NeilStahl, Hans-Peter Guler

Disclosure: Study was funded by Amgen Inc

S370

1908

1911

IMPROVEMENT IN THREE-DIMENSIONAL JOINT KINEMATICS SUPPORTS THE CONTINUOUS USE OF FOOT ORTHOSES IN THE MANAGEMENT OF PAINFUL REARFOOT DEFORMITY IN RHEUMATOID ARTHRITIS. James Woodburn, Philip S Helliwell Leeds, West Yorkshire, United Kingdom

THERAPEUTIC DELAY AND INDUCTION OF REMISSION IN EARLY RHEUMATOID ARTHRITIS: COMPARISON BETWEEN SINGLE DRUG AND COMBINATION DRUG TREATMENT STRATEGIES WITH DMARDS. Marjatta Leirisalo-Repo, Hannu Kautiainen, Pekka Hannonen, Markku Korpela, Martti Nissila, Jorma Ilonen, Oili Kaipiainen-Seppanen, Per Franzen, Tapani Helve, Juhani Koski, Marianne Gripenberg-Gahmberg, Riitta Myllykangas-Luosujarvi, Timo Mottonen, the FIN-RACo Trial Group Heinola, Jyvaskyla, Tampere, Turku, Kuopio, Tammisaari, Mikkeli and Turku, Finland

Objective: Valgus deformity of the rearfoot in rheumatoid arthritis (RA) is characterised by excessive eversion and internal rotation motion about the ankle joint complex (AJC). The objective was to evaluate three-dimensional (3D) kinematics at the AJC in a prospective clinical trial of custom-designed foot orthoses in RA. Methods: 98 patients with RA and painful correctable valgus deformity of the rearfoot entered a 30 month randomised clinical trial and received either continuous foot orthotic management or no intervention. 3D kinematics at the AJC were measured barefoot, shod and with orthoses using an electromagnetic tracking system at baseline, 3, 6, 12, 18, 24 and 30 months. The AJC (tibiotalar and subtalar joints) were defined using a joint co-ordinate system with sensors placed on the tibia and calcaneus, with in-shoe measurement performed in specially adapted footwear. Movement patterns were compared descriptively and motion:time integrals, by axis of rotation, were derived for statistical analysis using ANOVA techniques. Results: Both RA groups had abnormal 3D kinematics, in comparison with age- and sex-matched normal values. An immediate treatment effect was found at baseline with a significant reduction in frontal plane eversion in the orthotic group (P⬍0.0001). Motion was characterised by restoration of an inverted heel strike position, eversion through midstance, two periods of neutral joint alignment and an inverted toe-off position. There was no coupling effect as internal/external rotation motion was not significantly altered by the orthoses (P⫽0.471). In the ANOVA model footwear provided medial support and partially corrected the deformity in both groups for both frontal plane eversion and transverse plane internal rotation over the 30 months. However, a significantly superior treatment effect was found in the orthotic intervention group in comparison with the control (P⬍0.0001). Partial permanent correction of the deformity under barefoot walking conditions was observed in both groups but the effect significantly greater in the orthotic group (P⬍0.0001). Recoupling of inversion with external leg rotation, affording greater rearfoot stability, was observed in both groups with a significantly greater effect in the orthotic group (P⫽0.007). Conclusions: In a 30 month study of RA, custom designed rigid foot orthoses significantly improved AJC kinematics and this was associated with improvements in clinical symptoms. Disclosure: Supported by grants from the Medical Research Council and the Arthritis Research Campaign

In the FIN-RACo study on patients with early rheumatoid arthritis (RA) (1), combination therapy with sulfasalazine, methotrexate, hydroxychloroquine, and low-dose prednisolone (5-10 mg/day) (COMBI) induced at 2 years more frequently disease remission than did single-drug therapy (SINGLE, initially sulfasalazine with prednisolone if clinically indicated) (37% vs. 18%, p⫽0.011). Purpose To investigate whether a delay from symptoms to the start of therapy with DMARDs would contribute to the frequency of remissions in each treatment arm. Methods The presence of shared epitope allele, sex, age, seropositivity, number of positive ACR 87 classification criteria present at baseline, as well as the duration of symptomatic period were included in the model for logistic regression analysis. Results The duration of symptomatic period was ⬍4 months (short duration) in 33% of COMBI and in 27% of SINGLE group patients. In the COMBI patients, the frequency of patients in remission at the 2-year visit was the same (42%) in those with short or longer duration of symptoms. In SINGLE group, however, the respective figures were 35% and 11% (p⫽0.021). This statistically significant difference did not disappear with adjusting with genetic and baseline variables. In logistic regression analysis, the duration of symptomatic period was the only significant predictor for remission for patients in SINGLE, but not in COMBI arm. Conclusion Diagnostic or therapeutic delay decreases the possibility of induction of remission with traditional SINGLE therapy. Our results encourage to apply DMARD(s) early in the disease and to the use of combination therapy. (1)Mottonen et al: Lancet 353:1568-1573, 1999 Disclosure:

1909

1912

CUSTOM-DESIGNED FOOT ORTHOSES MODULATE PLANTAR PRESSURES IN RHEUMATOID ARTHRITIS. James Woodburn, Philip S Helliwell Leeds, West Yorkshire, United Kingdom

RADIOLOGICAL PROGRESSION RATE AT BASELINE PREDICTS TREATMENT DIFFERENCES: RESULTS FROM THE COBRA-TRIAL. Robert B Landewe, Desiree M van der Heijde, Arco Verhoeven, Annelies Boonen, Maarten Boers, Sjef van der Linden Maastricht, Netherlands

Objective: Plantar pressure/force distribution can be modulated by corrective foot orthoses. This study evaluates the longitudinal pressure modulation effects of custom-designed foot orthoses in rheumatoid arthritis (RA). Methods: 98 RA patients with painful valgus heel deformity were randomised to receive orthotic intervention (Int) (n⫽50) or remain untreated (Con) (n⫽48). Plantar pressure measurement was conducted in-shoe using a flexible capacitance based transducer. Assessments were conducted at baseline, 3, 6, 12, 18, 24 and 30 months. 5 left and right steps were analysed and averaged with peak pressure (kPa), peak force (%BW), contact time (%ROP), contact area (cm2), pressure:time integral (kPas), and force:time integral (Ns) determined for 11 plantar masks incorporating fore-, mid- and rearfoot regions. Individual time:response curves, variable-by-mask (6x11), were constructed for change in score from baseline and summary measures derived (area under the curve), maximum response and time to maximum response, with between group differences determined by Mann-Whitney test, based on the intention-to-treat principle. Results: Over a 30 month period foot orthoses brought about statistically significant reductions in peak pressures at the medial (Int- -844kPa, Con- 2kPa, P⬍0.0001) and lateral (Int- -772kPa, Con- 101kPa, P⬍0.0001) heel sites and significant increases in peak force for the total foot (Int- 191N, Con- 20N, P⫽0.014), medial heel (Int- 116N, Con- 20N, P⫽0.001), and metatarsal segments 3-5 (Int- 51N, Con- 13N, P⫽0.045). Force:time integral was significantly increased for the medial heel mask (Int- 19Ns, Con- -8Ns, P⫽0.036). Contact areas were significantly increased in the orthotic group in comparison with control for total mask (Int- 118cm2, Con- -16cm2, P⫽0.043), midfoot (Int- 91cm2, Con11cm2, P⫽0.002), and the 2nd metatarsal (Int- 23cm2, Con- 2cm2, P⫽0.006) masks. There were no statistically significant group differences for pressure:time integrals or contact times. Peak response and time to peak response mostly occurred within the first 6 months, although individual responses were highly variable. Conclusions: Custom-designed foot orthoses beneficially change foot function in painful valgus heel deformity and thus alter some aspects of foot pressure and force distribution. These findings are associated with changes in gait and improvements in rearfoot pain and related disability.

Background: it is important to predict those patients that will benefit most from particular treatment strategies. Objectives: to determine baseline factors that predict the impact of specific treatment (COBRA) on radiological progression in rheumatoid arthritis (RA). Patients and methods: 5 years follow-up data from the COBRA-trial (a 1 year randomized controlled trial comparing monotherapy sulfasalazine (SSZ) (n⫽74) with combinationtherapy SSZ⫹MTX⫹Pred (prednisolone started with 60 mg/day and rapidly tapered) (n⫽74) were used. X-rays obtained at baseline and after 5 years were scored by two observers (AV & AB) using the van der Heijde-modified Sharp score.Outcome examined was radiological progression rate (progression per year). Radiological progression rate at baseline was estimated by dividing Sharp-score at baseline by disease duration. Results: Only the calculated rate of radiological progression at baseline, divided in tertiles, significantly determined differences in the impact of COBRA on radiological progression rate over 5 years. Well-known baseline predictors of radiological progression itself (rheumatoid factor status, disease activity parameters) did not predict treatment differences. Conclusion: RA-patients can be stratified at baseline by determining the radiological progression rate with respect to the optimal treatment (COBRA or SSZ) they need to limit long-term radiological progression. Importantly, such a stratification with respect to the expected benefit of therapy can limit costs and toxicity.

Disclosure: Supported by grants from the Medical Research Council and the Arthritis Research Campaign

Disclosure:

1910

1913

“STEPPING-UP” FROM METHOTREXATE (MTX): A SYSTEMATIC REVIEW OF PLACEBO-CONTROLLED TRIALS IN PATIENTS WITH RHEUMATOID ARTHRITIS (RA) WITH AN INADEQUATE RESPONSE TO MTX. Marc C Hochberg, J Kathleen Tracy, Raymond H Flores Baltimore, MD

SLOWER RADIOGRAPHIC PROGRESSION IN PATIENTS WITH EARLY RHEUMATOID ARTHRITIS TREATED WITH CYCLOSPORINE AND METHOTREXATE COMBINATION THERAPY. Antonio Marchesoni, Norma Battafarano, Marco Arreghini, Benedetta Panni, Maria Cagnoli, Sergio Tosi Milan, Italy

Etanercept Infliximab Cyclosporine Leflunomide

Placebo

Active Rx

Rate Ratio

Rate Difference

NNT

30 (27%) 88 (20%) 73 (16%) 133 (20%)

59 (71%) 340 (53%) 75 (48%) 130 (46%)

2.7 (1.4,4.9) 2.6 (1.7,4.0) 2.9 (1.7,5.1) 2.4 (1.6,3.5)

45 (25,64) 33 (23,43) 32 (17,46) 27 (16,38)

2 (1,4) 3 (2,4) 3 (2,6) 4 (3,6)

Introduction. Cyclosporine (CsA) and methotrexate (MTX) combination therapy could be proposed as first treatment in early aggressive rheumatoid arthritis (RA). In this randomised controlled study we evaluated the efficacy of the association CsA/MTX to control radiologic damage compared with MTX alone. Patients and Methods. Forty-two patients (39 females and 3 males) (mean age 46.7⫾9.9 yrs) with early RA (mean disease duration 0.85 ⫾0.8 yrs) were randomised to receive either CsA (3 mg/kg/day) and MTX (10-15 mg/week) in combination (20 patients) or MTX alone (22 patients) for 12 months. Hand and foot radiographs were scored blindly using the Sharp/van der Heijde method. Four patients in the CsA/MTX group withdrew because of adverse events but the data analysis was carried out according to the intention-to-treat method. Results. At the end of the study period, in the CsA/MTX group 15 (75%) patients were ACR20 responder, and 11 (45%) both ACR50 and ACR70; in the MTX group 13 (59%) patients were ACR20 responder, 7 (32%) ACR50, and 4 (18%) ACR70. These differences were not statistically significant. Radiologic results were as follows.

CsA/MTX MTX

DS mo 0

DS mo 12

⌬ DS mo 0-12

2.7 ⫾ 1.2 2.2 ⫾ 0.8

3.5 ⫾ 1.5 9.5 ⫾ 3

-0.87 p: ns -7.3 p⬍0.02

EJ mo 0

EJ mo 12

⌬ EJ mo 0-12

0.33 ⫾ 0.1 0.35 ⫾ ⫾0.2

0.53 ⫾ 0.2 1.76 ⫾ 0.6

-0.2 p: ns -1.4 p⬍0.02

DS ⫽ damage score; EJ ⫽ eroded joint count. Adverse events were recorded in 13 (65%) CsA/MTX patients and 11 (50%) MTX patients, but overall the safety and tolerability profile was good in both groups. Conclusions. The results of this study seem to indicate that the association CsA/MTX in early RA is at least as effective as MTX in controlling the arthritic symtpons but could prevent structural joint damage better than MTX alone. Disclosure:

Disclosure: The Division of Rheumatology & Clinical Immunology has received financial support from Aventis Pharmaceuticals Inc., Centocor, Novartis, and Wyeth-Ayerst. Dr. Hochberg is a consultant to Aventis Pharmaceuticals Inc and Wyeth-Ayerst.

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Combination therapy in patients with RA has become increasingly common in recent years. One strategy involves adding agents to patients with an inadequate response to MTX. In a prior systematic review, Verhoeven et al (Brit J Rheumatol 1998;37:612-9) identified 2 placebo-controlled randomized clinical trials (PCRCTs) which used a “step-up” strategy in MTX partial responders; one studied the monoclonal antibody cM-T412 and the other cyclosporine. We updated this sytematic review to test the hypothesis that biologic therapy with anti-TNF agents was more efficacious than either cyclosporine or leflunomide when added to MTX in RA patients with active disease. The MEDLINE database was searched from July 1997 to December 2000, bibliographies of retrieved articles were scanned for additional studies, and abstracts from the 1998-2000 annual ACR meetings were reviewed. Study quality was assessed using the Jadad scale. Relevant data were extracted by two authors. Four PCRCTs of 24-30 week duration that provided strong evidence were included. The 928 RA patients were predominantly white women with a median age and disease duration of ⬇50 and 10 years, respectively. Results for the primary outcome, ACR 20 response at study completion, are shown in the table; data from the 4 different dosage regimens of infliximab were pooled for analysis. Neither the rate ratio nor the rate difference for an ACR 20 response differed significantly between the 4 active treatments. Furthermore, no significant differences were found when results for ACR 50 and ACR 70 responses for etanercept, infliximab and leflunomide were analyzed (data not shown). These data suggest that in RA patients with an inadequate response to MTX, the addition of etanercept or infliximab does not confer a greater ratio of response than the addition of leflunomide or cyclosporine. Decisions of “step-up” combination therapy in MTX partial responders should be based on patient’s and provider’s values regarding route of drug administration, patterns of side effects, costs and insurance coverage.

1914

1917

COMBINATION THERAPY WITH CYCLOSPORINE AND METHOTREXATE IN SEVERE RHEUMATOID ARTHRITIS. A PROSPECTIVE, MULTICENTER, 40-WEEK STUDY. Won Ki Lee, Jisoo Lee, Wan Uk Kim, Chul Soo Cho, Ho Youn Kim, Hong Joon Ahn, Seong Hoon Han, Yoon Woo Lee, Jinseok Kim, Hoon Seok Cha, Eun Mi Koh, Choong Ki Lee, Soo Kon Lee Daejeon, Seoul and Daegu Korea

SUPRATHERAPEUTIC DOSES OF VALDECOXIB HAVE A REDUCED INCIDENCE OF GASTRODUODENAL ULCERS COMPARED WITH CONVENTIONAL THERAPEUTIC DOSES OF NAPROXEN IN OSTEOARTHRITIS AND RHEUMATOID ARTHRITIS PATIENTS. Naurang Agrawal, Beverley Paperiello, William W Zhao, Jeffrey D Kent, David P Recker, Kenneth M Verburg Durham, NC and Skokie, IL

Objectives: To assess the efficacy and safety of cyclosporine A (CsA) and methotrexate (MTX) in patients with severe rheumatoid arthritis (RA) who have only partial responses to MTX. Methods: In a 16-week, multicenter, randomized, double-blind control trial, we compared combination therapy with CsA ⫹ MTX (73 patients) and placebo ⫹ MTX (74 patients) in 147 patients with RA who had active disease despite MTX at maximal tolerable doses. Thereafter in an open-label extension study, patients who had received CsA ⫹ MTX continued this regimen for a further 20 weeks (week 16-40)(group 1), and patients who had recieved placebo ⫹ MTX newly recieved CsA ⫹ MTX for 20 weeks (week 16-40)(group 2). The primary outcome measure was the change in the number of tender joints. Results: In a 16 week study, the CsA group had a significant differences on decrease in tender-joint count (5.7 vs 2.1, p⫽0.003). As well, there were statistically significant difference in the swollen-joint count (4.1 vs 1.6, p⫽0.001), decrease in visual analog pain scale, decrease in modified health assessment questionaire, decrease in patient’s global assessment, decrease in C-reactive protein. Twenty-six patients (35.6%) in the CsA group and 13 patient (17.6%) in the placebo group met the criteria for 20% improvement of ACR (p⬍0.05). Of 147 patients enrolled, 125 (85%) completed the extension study. In patients in group 1 (n⫽62), clinically significant improvement was maintained through week 40 (38.7% at 40 week). In patients in group 2 (n⫽63), the addition of CsA resulted in clinical significant improvement (31.8% at 40 week). A small increase in serum creatinine was observed in CsA treatment. Conclusion: Combination therapy with CsA and MTX in patients with severe RA demonstrated clinically importnat improvement without serious adverse effects. CsA plus MTX seems to be a useful and safe therapy in patients with RA who have only partial responses to MTX.

Background: Conventional NSAIDs are routinely used to treat inflammation and pain associated with rheumatoid arthritis (RA) and osteoarthritis (OA). However, these agents are associated with a high incidence of gastrointestinal (GI) ulcers due to non-selective inhibition of cyclooxygenase (COX). This study compared the incidence of gastroduodenal ulcers in RA and OA patients treated with supratherapeutic doses of the COX-2 specific inhibitor valdecoxib and the conventional NSAID naproxen. Methods: This multi-center, double-blind, parallel group study compared the incidence of gastroduodenal ulcers associated with valdecoxib 20 mg BID and 40 mg BID (2- to 4-fold the optimal arthritis dose) and naproxen 500 mg BID treatment for 14 weeks in 1217 patients with OA and RA. The incidence of gastroduodenal ulcers was determined by pretreatment and Week-14 endoscopies. Patients with less than 10 gastroduodenal erosions and no ulcers at pretreatment endoscopy were included in the study (1064 patients were included in the analysis). Results: The incidence of gastroduodenal ulcers in the valdecoxib groups was significantly lower compared with naproxen in all patients and in the OA and RA patients (table). The incidence of gastroduodenal ulcers was similar for both doses of valdecoxib. There was a statistically higher incidence of ulcers in the valdecoxib 40 mg BID group compared with the 20 mg BID group in the OA subset, although the rate of ulcers in this group were still significantly lower than for naproxen. Conclusion: Long term use of supratherapeutic doses of valdecoxib presents a significantly lower risk of gastroduodenal ulcers in OA and RA patients compared with the conventional NSAID naproxen.

Disclosure: supported by Chong Kun Dang Corporation

Incidence of gastroduodenal ulcers at Week 14 - No. with ulcers/n (%)

All patients OA patients RA patients

Valdecoxib 20 mg BID 15/345 (4%) 6/172 (3%) 9/173 (5%)

Valdecoxib 40 mg BID 27/355 (8%) 16/175 (9%)† 11/180 (6%)

Naproxen 500 mg BID 66/364 (18%)* 36/178 (20%)* 30/186 (16%)*

*pⱕ0.05 versus valdecoxib 20 mg and 40 mg, †pⱕ0.05 versus valdecoxib 20 mg Disclosure: Sponsored by Pharmacia Corporation and Pfizer Inc

1915

1918

FUNCTIONAL STATUS IMPROVEMENT IN SUBJECTS WITH RHEUMATOID ARTHRITIS RECEIVING ANAKINRA THERAPY. George Nuki, Barry Breshnihan, J Michael Woolley Edinburgh, United Kingdom; Dublin, Ireland and Thousand Oaks, CA

REDUCED RISK OF ACUTE THROMBOEMBOLIC EVENTS WITH NAPROXEN IN RHEUMATOID ARTHRITIS PATIENTS IN THE UK GENERAL PRACTICE RESEARCH DATABASE (GPRD). Douglas J Watson, Thomas Rhodes, Richard H Holmes, Harry A Guess West Point, PA

Purpose: To evaluate the effect of anakinra on the functional status of subjects with rheumatoid arthritis (RA). Methods: In a 24-week multicenter trial, 76 subjects who had received placebo for 24 weeks of treatment in a prior study were randomized to receive either 30 mg, 75 mg, or 150 mg of anakinra SC daily. Functional status was assessed at baseline (week 0), after 24 weeks of therapy with placebo (weeks 0 - 24), and after 24 weeks of therapy with anakinra (weeks 24 - 48) using the Health Assessment Questionnaire (HAQ). The HAQ is scored so that lower scores indicate better functional status and negative change scores indicate improvements. Of the 76 subjects, 8 were excluded from the analysis because they did not provide data at week 48. Hypothesis tests were conducted using paired t-tests for the change in HAQ during each time period. Results: As shown in the table, improvements were seen in HAQ scores during the treatment period that exceeded those seen during the placebo period, with two of three doses showing statistically significant and clinically meaningful changes. Analysis of all subjects combined showed that the change in the HAQ during the treatment period (-.25), weeks 24 to 48, was statistically significant, while the change during the placebo period (-.10), weeks 0 to 24, was not.

Purpose: Aspirin irreversibly inhibits cyclooxygenase-1 (COX-1), inhibits platelet aggregation, and reduces thromboembolic risk in patients with acute coronary syndrome, and with prior MI, angina, or stroke. Naproxen, a potent reversible inhibitor of platelet aggregation, could also be antithrombotic. We examined the risk of acute thromboembolic events (MI, stroke, sudden death) with current naproxen vs none in the past year among RA patients in the GPRD. Methods: Patients ⬎⫽40 and ⬍80 years old with RA and treated with NSAID, DMARD, or systemic corticosteroid were studied. Patients with previous thromboembolic event, cancer, vasculitis, coagulopathy, renal disease, liver failure, or alcohol/drug abuse, and those prescribed aspirin/anticoagulants/antiplatelet agents during the year prior to study start were excluded. Up to 4 controls were matched by gender, age, and (when possible) medical practice to cases with first thromboembolic events. The index date was the endpoint date for a given case and the same date for controls matched to that case. Current naproxen use was a prescription for naproxen within 30 days before the index date. Potential confounders evaluated were year of patient start; smoking, DMARD, systemic steroid, estrogen, diabetes; cardiovascular risk, and other medical comorbidity. A matched case-control analysis was conducted using conditional logistic regression. Results: 17,006 patients were eligible for the analysis. Of these 873 had an incident endpoint; 26 with current naproxen, 123 with naproxen ⬎30 days but ⬍365 days prior to the index date, and 694 with no naproxen use for the prior year. Of 16,133 potential controls, 2013 (12.5%) were selected. Cases were more likely to be smokers, have used steroids, have higher CV risk, and have more comorbidity than controls. Current naproxen use was more common in controls (6.5%) than cases (3.6%). The OR and (95% CI) for current naproxen (vs. none in the past year) with matching on age, gender, and practice but without adjustment for other potential confounders was 0.57 (0.36, 0.89). The OR with matching and adjustment (for beginning year of patient start, steroid use, and CV risk score) was 0.58 (0.37, 0.92). Conclusions: These results suggest that RA patients currently treated with naproxen may have a reduced risk of acute major thromboembolic events relative to those with no naproxen in the past year. Sensitivity analyses supported this finding. These results are preliminary; validation of the RA and endpoint diagnoses is ongoing.

Mean Change in HAQ by Dose of Anakinra By Time Period Period

Weeks

Placebo Treatment Total

0-24 24-48 0-48

30 mg (n⫽26) -.03 -.33** -.36**

75 mg (n⫽23) -.12 -.11 -.23

150 mg (n⫽19) -.15 -.35** -.50**

Combined (n⫽68) -.10 -.25** -.35**

* P ⬍ 0.05, ** P ⬍ 0.01 Conclusions: Subjects receiving anakinra experienced improvements in functional status. Observed improvements were of a magnitude considered clinically meaningful. These results are consistent with the hypothesis that anakinra improves functional status of subjects with rheumatoid arthritis.

Disclosure: The authors are employees of Merck & Co., Inc.


Disclosure: Research supported by Amgen, Inc.

1916

1919

CARDIOVASCULAR SAFETY PROFILE OF ROFECOXIB: A META-ANALYSIS. A Reicin, E Barr, D Shapiro Rahway, NJ

TWO CASES OF ETANERCEPT-INDUCED LUPUS-LIKE SYNDROME IN RA PATIENTS. M De Bandt, V Descamps, O Meyer Paris, France

Objectives: To evaluate the cardiovascular (CV) safety profile of rofecoxib, a selective COX-2 inhibitor. Background: Non-selective NSAIDs, selective COX-2 inhibitors, and aspirin each exhibit different patterns of inhibition of platelet COX-1, and chronic use of these agents is likely to be associated with differential risks of platelet-mediated vascular events. Aspirin, which induces irreversible inhibition of platelet COX-1, can be cardioprotective. NSAIDs that mediate near complete inhibition of platelet function throughout their entire dosing cycle may be similar to aspirin and reduce the risk of vascular events; other compounds with less substantial platelet inhibition may have no impact on vascular events. Methods:A meta-analysis of all Phase IIb-V clinical trials was conducted using individual patient data and focusing on the relative risk of CV thrombotic serious adverse experiences in patients taking rofecoxib as compared to placebo, naproxen (an NSAID with near complete inhibition of platelet function throughout its dosing interval), and non-selective NSAIDs that lack potent sustained inhibition of platelet function (diclofenac, ibuprofen, and nabumetone). The primary metric utilized was the Antiplatelet Trialists’ Collaboration (APTC) combined endpoint of cardiovascular or unknown death, stroke, or myocardial infarction. Results: Over 28,000 patients in 19 studies (Osteoarthritis, Rheumatoid Arthritis, Alzheimer’s, and Chronic Low Back Pain trials) representing over 14,000 patient-years at risk were included in the meta-analysis. The relative risk (95% CI) for an APTC combined endpoint was: 0.59 (0.37, 0.94) when comparing naproxen (n⫽7870) vs. rofecoxib (n⫽9083); 1.27 (0.64, 2.50) when comparing non-naproxen NSAIDs (n⫽2755) vs. rofecoxib (n⫽4549); 1.19 (0.73, 1.96) when comparing placebo (n⫽3482) vs. rofecoxib (n⫽6290); and, 1.31 (0.86, 2.01) when comparing non-naproxen NSAIDs or placebo (n⫽6017) vs. rofecoxib (n⫽7675). Conclusions:(1) The risk of sustaining a thrombotic cardiovascular event was similar in patients treated with rofecoxib, placebo, or non-selective NSAIDs without sustained effects on platelet function and, (2) the risk of sustaining a thrombotic cardiovascular event was reduced in patients treated with naproxen compared to rofecoxib. This reduction in events on naproxen is likely due to its ability to maintain near complete inhibition of platelet function throughout its dosing interval. Disclosure: A Reicin, E Barr, and D Shapiro are employees of Merck & Co., Inc.

We report 2 cases of etanercept-induced lupus-like syndrome in RA patients with previous isolated positive ANA. A 38-year-old woman was started on etanercept on February 2000, after a 16-year history of severe seropositive RA unsuccessfully treated with hydroxychloroquine, D-Pen, gold salts, and methotrexate plus prednisone. She had isolated positive ANA. The disease required multiple joint replacement. Under etanercept. RA was remarkably controlled. 3 months later, she presented scattered lesions on the face and the scalp (no sun exposure), fatigue and diffuse pain. Skin analysis demonstrated dermal atrophy with keratinocytes necrosis, perifollicular and perivascular lymphocytic infiltration, with no vasculitis. Biological tests revealed positive ANA, anti-DNA, anticardiolipin antibodies, low C4, negative anti ENA. She had elevated muscle enzymes, normal blood count, liver and renal functions; no proteinuria. Etanercept was stopped. One month later skin lesions had cleared, very active RA was again present. ANA remained positive, anti-DNA decreased as well as muscle enzymes and ACL abs. Biological abnormalities had returned to normal values in September 2000. The patient has not been rechallenged. A 50-year-old woman had active RA despite low dose steroids and sulphasalazine. Methotrexate and ciclosporin were excluded because of chronic alcoholic hepatitis and hypertension. Etanercept was introduced in October 1999. RA was remarkably controlled. She developed 4 months later a diffuse erythematous and purpuric skin eruption with fine scaling (face, hands and fingers) erosions and crusts. Histological analysis demonstrated hyperkeratosis, keratinocyte necrosis and perivascular lymphocytic infiltration without vasculitis. She had lymphopenia, thrombocytopenia, elevated ESR, abnormal liver function test, positive ANA (1/640), negative for anti-DNA and ENA abs. After etanercept stoppage, skin manifestations resolved but synovitis reappeared. The patient has not been rechallenged. Imputability of etanercept is very likely, given the temporal relation between the onset of signs with the initiation of injections, and resolution after withdrawal. Further evaluation is needed to know whether positive ANA or association with other SLE drug inducer are risk factors for these manifestations. Disclosure:

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1920

1923

CHLOROQUINE RELATED CARDIAC TOXICITY: A NOT SO RARE COMPLICATION. Beatriz E Joven, Raquel Almodovar, Rafael Salguero, Alfredo Llovet, Patricia E Carreira Madrid, Spain

DEVELOPMENT OF ACLA (ANTI-CARDIOLIPIN ANTIBODIES) IN PATIENTS TREATED WITH INFLIXIMAB (REMICADE). Thorunn Jonsdottir, Johan Bratt, Lars Klareskog, Ronald van Vollenhoven Stockholm, Sweden

Chloroquine is widely used for rheumatoid arthritis (RA) and lupus patients. Its main toxicity remains retinopathy, but other infrequent complications, myopathy or cardiac dysfunction, have been rarely reported. Cardiac chloroquine toxicity presents as conduction disorders and/or hyperthrophic/restrictive myocardiopathy, both presenting with increased frequency in people over 60 years. We describe 5 RA patients (4F, 1M), who presented cardiac toxicity related to chloroquine. They had a median age of 69 (64-73) years, and had been taking chloroquine for 7 (5-9) years. Drug cumulative doses ranged between 400 and 850 g. All patients had normal EKG before chloroquine treatment, and no risk factors for coronary disease. Clinical presentation was recurrent syncope in all patients, with an EKG showing complete heart block in one case, and right and anterior left blockade in four cases, with atrio-ventricular blockade in two of them. A permanent pacemaker was needed in every case. In three patients chloroquine treatment was continued, as not drug toxicity was considered at that time. Two of these patients developed restrictive cardiac failure after 4 and 6 months, respectively, and endomyocardial biopsy showed in both the characteristic findings of chloroquine toxicity with vacuolar myocardiopathy and curvilinear bodies in electron microscopy. Chloroquine was then discontinued and cardiac failure slowly resolved over several months. In the third patient chloroquine had to be discontinued 2,5 years later because of corneal deposits causing visual alteration. In the other two cases chloroquine was discontinued when the cardiac block was diagnosed, as drug toxicity was suspected. Patients refused endomyocardial biopsies. One of them showed corneal deposits in the ocular exam. Besides the two patients who showed corneal deposits, none showed chloroquine retinopathy or myopathy. Our data suggest that cardiac toxicity may be more frequent than previously considered in long term chloroquine treatment, specially in patients over 65 years. A previous normal EKG does not exclude this possibility. An EKG should be performed before chloroquine treatment is started, and at least once a year afterwards. If cardiac block appears, chloroquine cardiac toxicity should be suspected. Disclosure:

Goal: To investigate the development of ACLA in patients treated with Infliximab. Background. In earlier studies development of ANA has been observed in 5-10% of Infliximab treated patients and anti-DNA in 1-5%. According to one clinical study one out of 20 patients recieving Infliximab developed ACLA. Materials and methods: We studied 82 patients with RA treated with Infliximab. All patients were also treated with MTX 7,5-15mg/week. The dosage of Infliximab was 3 mg/kg given week 0, 2 and 6 and every 8th week thereafter. Analyses for RF, ANA, anti-DNA and ACLA were done at baseline, and after 3, 6 and 12 months. ACLA was done by ELISA. Results: At baseline, 35% of patients had a positive ANA and this percentage was unchanged during treatment. Two patients developed anti-DNA during therapy without SLE-like symptoms. At baseline 10 % of patients had positive ACLA. After 3 months, the frequency of ACLA was 24% (p⬍0,05 compared to baseline) and after 6 and 12 months 22% and 23% respectively. Increases were seen for both IgG and IgM ACLA. No correlation was seen between either ANA or RF and development of ACLA. In this cohort one ACLA positive patient developed lung embolism and one ACLA negative patient developed deep vein thrombosis. Conclusion: We found a statistically significant development of ACLA in patients treated with Infliximab for 3 months or longer. No significant correlation was seen with presence or development of either ANA or RF. The development of ACLA did not appear to increase the risk for thromboembolic events in this cohort. Disclosure: JB, LK, RV speaker bureau various companies

1921

1924

DOES FOLIC ACID (FA) DECREASE THE EFFICACY AS WELL AS THE TOXICITY OF METHOTREXATE (MTX) IN RHEUMATOID ARTHRITIS (RA)? D E Furst, S Cohen, P Emery, K Dorrier, K Simpson Seattle, WA; Dallas, TX; Leeds, UK; Arlington, VA and Bridgewater, NJ

ACCELERATED NODULOSIS AND VASCULITIS FOLLOWING ETANERCEPT THERAPY FOR RHEUMATOID ARTHRITIS. Gaye Cunnane, Martha Warnock, Qaiser Rehman, Ken Fye, David I Daikh San Francisco, CA

Objectives: To examine the effect of oral FA supplementation on the efficacy and toxicity of MTX therapy in RA. Methods: Two completed studies [one conducted in the United States and the other in Europe and South Africa (multinational - MN)] compared leflunomide to MTX (⫾ placebo) in the treatment of RA. The MTX arms of these 12-month, double-blind, randomized trials (US-MTX, n⫽182; and MN-MTX, n⫽498) were examined and compared. In both of these trials, MTX could be increased from 7.5 to 15 mg/wk. Responses and adverse events (AEs) were measured in a standardized manner using the American College of Rheumatology (ACR) response criteria for efficacy and regular laboratory and clinical measures of toxicity. Logistic regression analysis was done to evaluate the effect of FA use, rheumatoid factor (RF) positivity, disease duration ⬍2 years, swollen joint count (SJC), and included interaction terms. Results: Almost all (98%) of the US-MTX group used FA (FA⫹) vs 11% of the MN-MTX group (FA-). The FA⫹ patients had longer disease duration (6.5 yrs vs 3.8 yrs), fewer erosions (66% vs 74%), and a slightly lower percentage of RF positivity (59% vs 76%). Previous DMARD use was similar in the two groups (0.9 vs 1.1 drugs). Mean (median) MTX doses were 11.7 (15.0) mg/wk (FA⫹) vs 11.9 (10.0) mg/wk (FA-). The difference between the FA⫹ and FA- groups in ACR20 response rates was statistically significant, favoring the FA- group [45.6% (FA⫹) vs 65.2% (FA-); P⬍0.001]. Components of the ACR response criteria supported the overall finding: eg, tender joint count, -9.7 (FA-) vs -6.6 (FA⫹); patient global assessment, -3.0 (FA-) vs -1.5 (FA⫹) cm; and ESR, -22.2 (FA-) vs -6.5 (FA⫹) mm/hr. RF positivity, disease duration ⬍2 years, and SJC were not found to be independent factors in ACR20 response. As expected, the use of FA decreased the incidence of ALT elevations ⬎2xULN: 9.3% for FA⫹ vs 31.5% for FA- (P⬍0.001). Conclusions: FA may decrease the response to MTX as well as the incidence of ALT elevations. FA use may be one of the contributing factors for the variability of response to MTX across studies.

Background : Etanercept, a biologic agent which blocks the action of tumor necrosis factor, induces marked clinical benefit and reduced joint damage in patients with rheumatoid arthritis (RA). Injection site reactions are the most common adverse events, but some serious side-effects have been reported with the use of etanercept. Objective : To report the occurrence of accelerated nodulosis and vasculitis in RA patients treated with etanercept and to highlight an adverse event associated with this medication. Results : Three male patients with long-standing refractory sero-positive RA were commenced on etanercept in an attempt to gain better control of their arthritic disease. All 3 patients experienced marked improvement in synovitis, joint tenderness and overall well-being. However, within weeks of commencement of therapy, an increase in nodulosis occurred in all. Patient One noted an increase in nodules on his hands and left elbow. Patient Two developed explosive nodulosis of his fingers in addition to new splinter hemorrhages and evidence of small vessel cutaneous vasculitis. Patient Three who had pre-existing asymptomatic pulmonary nodules developed new cavitating pulmonary lesions associated with necrotizing vasculitis of the adjacent vessels. Continuation of etanercept in patient One did not cause added adverse events. The addition of leflunomide to etanercept in patient Two resulted in slow resolution of the cutaneous vasculitis and a decrease in the size of the nodules while substitution of azathioprine for etanercept in patient Three resulted in complete healing of the pulmonary lesions. Conclusions : This is the first report of accelerated nodulosis associated with vasculitis in RA patients receiving etanercept. There are a number of intriguing mechanisms by which etanercept may induce nodules and vasculitis. Awareness of potentially serious adverse events relating to new biologic agents with immunomodulatory effects should be highlighted.

Disclosure: The research reported in this abstract was supported by Aventis Pharmaceuticals. K. Dorrier is a consultant to, and K. Simpson is an employee of Aventis.

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1925

ANTICARDIOLIPIN ANTIBODIES DEVELOPING DURING INFLIXIMAB THERAPY. Alton J Morris, Christopher R Morris, Clark R Hernandez Kingsport, TN

ANTIBODIES TO INTERFERON-␥(anti-IFN-␥)IN THE THERAPY OF RHEUMATIC DISEASES (RD). Galina V Loukina, Yakov A Sigidin, Boris S Skurkovich, Simon V Skurkovich Moscow, Russia and Rockville, MD

79 patients with inflammatory arthritides, after having failed therapy with methotrexate and other modalities of therapy, were given Infliximab in the usually recommended dose / intervals; 56 had rheumatoid arthritis, 10 had psoriatic arthritis, 10 had other seronegative Spondyloarthropathy; 3 had other collagen vascular disease. One patient, an 85 year old WF with RA, developed a transient ischemic attack 2 weeks after the initial dose of Infliximab; during assessment for an autoimmune cause of her cerebrovascular event presence of positive ANA, as well as Anticardiolipin antibodies (ACL), were demonstrated. Following the identification of ACL antibodies the Infliximab was discontinued. Because of the aforementioned event 45 of the 79 patients were subsequently tested for antinuclear antibodies and anticardiolipin antibodies following administration of the initial series of Infliximab; 24 were found to have developed Antinuclear antibodies. 21/45 have been found to have positive ACL. IgG-ACL was present in 10/45. IgM-ACL was present in 21/45. IgA - ACL was present in 3/45. Apart from the initial patient, other thrombotic episodes have not been observed, nor have other syndromes associated with ACL been seen. Within our clinical practice, 215 other patients who have been checked for ACL because of clinical indication; these antibodies were detected in only 8 patients. Conclusions: A high incidence of ACL has been identified in our our patient population after initiation of therapy with Infliximab. Thus far, their presence has not been associated with clinical manifestations of anti-phospholipid diseases. All patients receiving Infliximab should be monitored for ACL. It may be prudent to start ASA on those patients who become positive.

Purpose. To assess the efficacy and tolerability of anti-IFN-␥ therapy in patients with RD. Methods. Fifty eight patients (pts) with active RD (rheumatoid arthritis /RA/-45, psoriatic arthritis /PA/-5, systemic lupus erythematosus /SLE/-4) received intramuscularly injections of anti-IFN-␥, antibodies to tumor necrosis factor a(anti-TNF) or placebo for 5 consecutive days. The pts were clinically assessed daily for 7 days, then weekly up to the 28-th day. Clinical, laboratory and ultrasound indices were used for the evaluation of the treatment efficacy. Results. Anti-IFN-␥ therapy provided rapid and statistically significant reduction of joint inflammation in RA pts. The results obtained were superior to placebo and comparable to anti-TNF. The thickness of the inflamed synovial membrane assessed by the ultrasound method decreased significantly only on anti-IFN-␥ (both by the 7-th and by the 28-th days). Three remissions were achieved on anti-IFN-␥(4-60 months) and one on anti-TNF (7 months). Articular and skin manifestations improved in all 5 pts with PA, and 2 of them have remissions (30 and 48 months). In 4 pts with SLE anti-cytokine therapy was ineffective, among them 3 pts deteriorated. Tolerability of anti-IFN-␥ was good. The most frequent side effect was mild local itching dermatitis in sites of injections. Conclusion. The results obtained show that IFN-␥ may contribute to the pathogenesis of RA and PA and therefore could represent important therapeutic target. It can be effectively used in treating rheumatoid arthritis (including its treatment-resistant forms) and psoriatic arthritis. Administration of anti-IFN-gamma in SLE is not indicated.

Disclosure: Christopher R. Morris, M.D. has been a part of the Speaker Bureau for Centocor

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1922

1926

1929

REDUCTASE GENE WERE ASSOCIATED WITH BOTH THE EFFICACY AND THE TOXICITY OF METHOTREXATE USED FOR THE TREATMENT OF RHEUMATOID ARTHRITIS, AS EVIDENCED BY SINGLE LOCUS AND HAPLOTYPE ANALYSES. Wako Urano, Atsuo Taniguchi, Hisashi Yamanaka, Eiichi Tanaka, Hiroshi Nakajima, Yuko Matsuda, Hideto Akama, Yutaka Kitamura, Naoyuki Kamatani Tokyo, Japan

FUNCTIONAL AND GENETIC CHARACTERIZATION OF SLE1, A MAJOR SUSCEPTIBILITY LOCUS IN MURINE LUPUS. Laurence Morel, Kim R Blenman, Eric S Sobel, Byron P Croker Gainesville, Fl

Objective: 5,10-Methylenetetrahydrofolate reductase (MTHFR), a key enzyme involved in folate metabolism, has two common polymorphisms that affect enzyme activity. The objective of this study was to examine whether there was a correlation between the genotype or haplotype of the MTHFR gene and the efficacy or toxicity of methotrexate (MTX) in the treatment of RA. Methods: MTX-treated RA patients (n ⫽ 106) were selected from outpatient clinics, and used for a retrospective study to examine the correlation between genotypes or haplotypes concerning polymorphisms of the MTHFR gene, and the efficacy or toxicity of MTX. Estimation of the haplotype frequencies was performed by maximum likelihood estimation based on expectation maximization (EM) algorithm. Results: Single locus analysis examining each locus separately showed that patients with 1298C were receiving significantly lower doses of MTX compared with patients without it (p⬍0.05, RR⫽2.18, 95%CI⫽1.17-4.06), while a higher rate of overall MTX toxicity was observed in patients with 677T than those without it (p⬍0.05, RR⫽1.25, 95%CI⫽1.05-1.49). An estimation of haplotype frequencies showed that there was no 677T-1298C haplotype in the population. Posterior distribution of the diplotype configuration for each individual showed that it was concentrated on a single configuration. Patients with the 677C-1298C haplotype were receiving lower doses of MTX than those without it (p⬍0.05, RR⫽2.14, 95%CI⫽1.13-4.07), while subjects with 677T-1298A had a higher frequency of side effects from MTX (p⬍0.05, RR⫽1.42, 95%CI⫽1.11-1.82). Conclusion: Both single locus and haplotype analyses have shown that polymorphisms within the MTHFR gene are associated with both the efficacy and toxicity of MTX in RA patients.

Sle1 on murine chromosome 1 is a key locus to SLE pathogenesis in the NZM model, and corresponds to loci identified in 2 other murine models, and in multiple linkage analyses of human SLE patients. When isolated on a C57BL/6 background in the B6.Sle1 congenic strain, Sle1 results in the production of high levels of anti-chromatin IgG antibodies specifically directed against H2A/H2B/DNA subnucleosomes, histone-specific T cells, and increased B cell and T cell activation. We have shown that Sle1 is necessary for the full development of SLE in the NZM model by reconstituting the SLE pathology with the combination of the Sle1, Sle2, and Sle3 loci. In addition, we have identified suppressor loci of the Sle1-induced phenotypes. At least one of them, H2-linked Sles1, can abrogate the entire disease process by suppressing Sle1. Taken together, these results indicate that the identification of the genes corresponding to Sle1 and the characterization of their specific contribution to the autoimmune process constitute a major step for our understanding of SLE pathology and the design of new therapeutic interventions. Mixed bone marrow chimeras showed that Sle1 is expressed in both B and T cells. Analysis of B6.Sle1.␮-/- and B6.Sle1.␣␤-/- mice showed that Sle1 independently results in abnormal phenotypes in both B and T cells. Fine-mapping the position of Sle1 revealed that this locus corresponds to a cluster of at least 3 loci, Sle1a, Sle1b, and Sle1c. Each of them independently causes a loss of tolerance to chromatin, indicating that they belong to a same functional pathway. However, each locus displays a distinctive profile of serological and cellular characteristics, with T and B cell functions being more affected by Sle1a and Sle1b, respectively. The distinct functional profile of each of the 3 Sle1 loci is dramatically illustrated in how they interact with 2 known SLE-inducing mutations, Yaa and lpr. The entire Sle1 locus co-expressed with either of these mutations results in a highly penetrant lupus nephritis. Only the Sle1b and Yaa combination results in nephritis, while B6.Sle1a.Yaa and B6.Sle1c.Yaa do not differ from B6.Sle1. On the opposite, both Sle1a and Sle1c interactions with lpr result in lupus nephritis, while B6.Sle1b.lpr is similar to B6.lpr. Finally, Sles1 suppresses the phenotypes from all 3 Sle1 loci. In the process of the molecular identification of the genes corresponding to each of the Sle1 loci, the characterization of their interactions with other SLE loci provides insights into their contribution to SLE pathogenesis. Disclosure:

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1927

1930

MELOXICAM SHOWS NO INCREASED RISK OF CARDIOVASCULAR OR RENAL EVENTS COMPARED TO OTHER NSAIDS: A POOLED ANALYSIS OF 27,039 PATIENTS. Gurkirpal S Singh, Paul Gagnier, David Hall Stanford, CA; Ridgefield, CT

EVIDENCE OF ABNORMAL B CELL SELECTION AND BREAKDOWN OF PERIPHERAL B CELL TOLERANCE IN LUPUS-PRONE NZB AND NZB/W MICE. Valerie Roy, Ralph MacLeod, Joan Wither Toronto, Ontario, Canada

Statement of Purpose: Meloxicam is a novel NSAID used in 100 countries with ⬎40 million prescriptions written over the last 6 years. Clinical trials show meloxicam effectiveness and lower rates of GI adverse events vs other NSAIDs (diclofenac, piroxicam, and naproxen). However, the renal and cardiovascular toxicities of meloxicam were not studied extensively. This is important clinically, because recent studies raised concern about cardiovascular and renal safety profiles of some new COX-2 selective NSAIDs. We wished to document the risk of acute myocardial infarction, congestive heart failure, edema, and hypertension in patients taking meloxicam (7.5-30 mg), and traditional NSAIDs (diclofenac 100-150 mg, naproxen 500-750 mg, piroxicam 20 mg). Methods: We analyzed pooled data from all meloxicam clinical trials lasting ⱖ3 weeks and ⱖ20 patients per arm: 35 trials; 27,039 patients on meloxicam, comparator NSAIDs, or placebo. Nearly 39% (10,680) of patients were ⱖ65. We report event rates as incidence/100 patient-years of exposure because of varying trial duration. Results: Of the 27,039 study patients, 15,071 received meloxicam doses of 7.5-30 mg/d. A total of 11,078 patients took diclofenac, naproxen, or piroxicam; another 736 patients took placebo. Cardiovascular and renal events are summarized below.

WITHDRAWN Cardiovascular and Renal Events: Number* and Incidence Rates**

Patient-Years Events: MI Cardiac failure Peripheral edema Hypertension Hypertension (aggravated) *Intent-to-treat population; **incidence rates/100 patient-years

Meloxicam (n⫽15,071)

NSAIDs (n⫽11,078)

Placebo (n⫽736)

3,129

1,202

113

18 (0.58) 15 (0.48) 98 (3.13) 82 (2.62) 25 (0.8)

8 (0.67) 7 (0.58) 79 (6.57) 32 (2.66) 15 (1.25)

2 (1.8) 0 (0) 1 (0.88) 5 (4.42) 2 (1.77)

Conclusion: Meloxicam in doses of 7.5-30 mg is not associated with increased incidence of cardiovascular or renal toxicity. Disclosure: Consultant: Boehringer-Ingelheim Pharmaceuticals Inc.; Merck; Pharmacia and Upjohn; Pfizer Inc.

We recently characterized a splenic B cell population expressing increased levels of costimulatory molecules in NZB and NZB/W mice. This phenotype was mapped to a NZB chromosomal region associated with development of autoantibodies and nephritis. Here we show that in young NZB and NZB/W mice immature splenic B cells already express high levels of B7.1 (%B220⫹CD21low/-B7.1hi: BALB/c⫽ 6.8⫾1.23, NZB⫽ 20.8⫾3.33 and NZB/W⫽ 17.8⫾5.09). We therefore investigated whether these B7.1hi B cells are generated in the bone marrow. In NZB mice there is an increased proportion of B7.1hi cells in both pre-B and immature B cell bone marrow subsets when compared with BALB/c mice (%B7.1⫹ Pre-B cells: BALB/c⫽ 31.4⫾1.56, NZB⫽ 61.8⫾5.41; %B7.1⫹ immature B cells: BALB/c⫽ 2.1⫾0.81, NZB⫽ 25.2⫾7.29). We reasoned that these B7.1hi B cell populations in NZB mice could be derived from B cell precursors expressing autoreactive immunoglobulin (Ig) receptors and/or having altered immunoglobulin receptor signaling. In order to test these two possibilities, anti-hen egg white lysozyme (HEL) Ig and soluble HEL (sHEL) transgenes (Tg) were crossed onto the NZB background using a speed congenic technique. B7.1 expression was similar on B cell precursors within the bone marrow of anti-HEL Ig (STg) B6 and NZB mice. Surprisingly, we found that levels of B7.1 expression are dramatically increased in anti-HEL Ig/sHEL Tg (DblTg) B6 and NZB mice when compared to their single transgenic counterparts. This suggests that B cell precursors binding self-antigen in the bone marrow upregulate B7.1, and that the increased proportion of these cells in NZB mice reflects altered B cell selection. Although B6 (DblTg) mice have peripheral B cells expressing high levels of B7.1, this is not sufficient to break B cell tolerance. HEL-specific peripheral B cells from DblTg B6 mice are functionally anergic and do not become activated to secrete anti-HEL autoantibodies in vivo. In contrast, while peripheral B cells from NZB DblTg mice appropriately down regulate IgM receptor expression and do not proliferate in response to HEL in vitro, large amounts of anti-HEL autoantibodies are spontaneously secreted in these mice. The results suggest that NZB mice have defects affecting both B cell selection and peripheral tolerance induction.


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1928

1931

CD4⫹ T CELLS FROM LUPUS PRONE MICE ARE HYPER-RESPONSIVE TO TCR STIMULATION WITH LOW-AFFINITY LIGANDS, AN EXPLANATION OF SPONTANEOUS T CELL ACTIVATION IN LUPUS. George Vratsanos, Jin-Young Choi, Joe Craft New Haven, CT

IMPACT OF STAT-4 AND STAT-6 DELETION ON AUTOIMMUNE PHENOTYPE OF LUPUS MICE. Chaim O Jacob, Song Zang, Lily Li, Minoru Satoh, Westley H Reeves Los Angeles, CA and Gaineswille, FL

CD4⫹ T cells are necessary for autoantibody production and disease in SLE in humans and in lupus-prone mice. We have hypothesized that CD4⫹ T cells in lupus are intrinsically hyper-responsive to stimulation with self ligands having a low affinity for binding to the TCR. Indeed, we have found that T cells from Fas-intact lupus-prone MRL mice are hyper-responsive in vitro to stimulation with low affinity peptide ligands, compared to control T cells, as assayed by cell division and IL-2 synthesis (J. Exp. Med. 193:329, 2001). We have now extended these findings in two ways. First, we evaluated antigen-specific signal transduction events in naive MRL T cells after TCR engagement with cognate (high affinity) and low affinity peptide antigens, using a TCR transgenic system. Highly purified naive CD4⫹ MRL T cells, in comparison to control H-2 matched T cells, had similar downstream phosphorylation events after TCR engagement with the cognate (high affinity) peptide ligand. By contrast, naive MRL T cells had hyper-phosphorylation of TCR-associated tyrosine kinases and Erk 1 (extracellular regulated kinase 1) following engagement with low affinity peptide ligands, features absent in non-autoimmune cells. These results suggest a biochemical basis for the enhanced activation of lupus T cells after TCR engagement with low affinity ligands. Such low affinity MHC-peptide-TCR interactions are a pre-requisite for peripheral T cell survival in normal animals. Thus, we next hypothesized that these low affinity interactions, because they are interpreted abnormally by T cells from lupus mice, might promote enhanced survival of the latter compared to control T cells. To address this question, we evaluated survival in vitro of naive T cells from Fas-intact MRL and control mice, using identical antigen presenting cells (APC). MRL T cells had enhanced survival in vitro, compared to control T cells, when allowed to contact MHC-self peptide complexes on APC, conditions that mimic homeostatic survival signals in vivo. In total, these data indicate that CD4⫹ T cells in lupus respond abnormally to engagement by low affinity MHC-self peptide complexes, an engagement that mimics the signals required for T cell homeostasis and survival in vivo in normal animals. Based upon these results, we offer the novel argument that engagement of the TCR with low affinity MHC-self peptide complexes, a survival signal in normal mice, leads to T cell activation in lupus, with resultant polyclonal activation, an early feature of disease in both humans and mice.

Th1/Th2 cytokine balance is believed to be a prominent factor in determining susceptibility, progression and prognosis of autoimmune diseases. To study pathways dependent on Th1 and Th2 in lupus prone mice, we have taken the approach of crossing deletions at Stat-4 and Stat-6 genes onto NZBxNZW derived recombinant inbred NZM mice, using marker-assisted selection strategy to accelerate the development of the desired congenic mice. The resultant mice have been characterized as deficient in the respective Stat gene and in the response to IL-12 (Stat-4 null) or IL-4 (Stat-6 null). Our studies show that the deficiency of Stat-4 and Stat-6 significantly alter the phenotype of the lupus-like disease in NZM 2410 and NZM 2328 congenic mice. Specifically, Stat-4 deficient NZM mice develop accelerated nephritis and mortality in the absence of high levels of anti-ds DNA antibodies. In contrast, Stat-6 deficient NZM mice show significantly reduced kidney disease development with dramatic delay in mortality, despite the presence of high levels of anti-ds DNA antibodies. The lack of correlation between levels of these autoantibodies and kidney disease questions the direct cause-effect relationships between the presence of autoAbs and kidney disease. In addition, we observed a very significant shift in Ig isotype toward IgG1 in the Stat-4 deficient lupus mice, and a major shift towards IgG2a in the Stat-6 deficient mice. This shift was noticed both on total Ig level and also regarding anti-chromatin autoantibodies. Furthermore, the results question the apparent equation of the effect of Stat deficiency with loss of secretion or response to, particular cytokines. These findings may be particularly interesting to human SLE and may reflect different forms of human disease such as glomerulonephritis arising even in the absence of elevated anti-ds-DNA Ab.

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1932

1935

ASSOCIATION AND LINKAGE OF CD72 ON MOUSE CHROMOSOME 4 WITH AN INDUCED MODEL OF SLE. Judith A James, Monica Y Kirby, Timothy F Gross, John B Harley Oklahoma City, OK

HIGH PREVALENCE OF SYMPTOMATIC KNEE OA (SXKOA) IN ELDERLY BLACK WOMEN: ROLE OF OBESITY AND QUADRICEPS WEAKNESS. M Nevitt, B Goodpaster, D Felson, A Newman, K Wildy, F Harris, S Kritchevsky, E Simonsick, T Harris San Francisco; Pittsburgh; Boston; Memphis and Bethesda

Anti-Sm and anti-nRNP autoantibodies are commonly associated with SLE. Previously we have described a peptide-induced model of lupus autoimmunity after immunization with an early target sequence, PPPGMRPP, of SmB/B’. Earlier studies show that only certain strains of inbred mice are susceptible to this method of lupus induction. Experiments testing the AKR/J x C57L/J (AKxL) F1 mice suggest that in this model, one autosomal dominant gene may exist. We have used a recombinant inbred strain of mice to grossly localize a region involved in SLE. Fifteen sets of AKXL recombinant inbred (RI) mice have been immunized with PPPGMRPP constructed on a MAP backbone. In total, nine AKXL RI strains develop evidence of peptide induced lupus autoimmunity (similar to the AKR/J parent strain) and six do not (similar to the C57BL/J parent strain). This strain distribution pattern (SDP) has linkage only with two markers of the 372 markers mapped for this RI set and are adjacent markers on chromosome 4. The closest association with peptide induced lupus autoimmunity has been found with cd72, a B-cell surface marker (odds ratio⫽84, p⬍0.0001). Based upon this preliminary linkage, we then tested association of a specific cd72 allele with the peptide induced lupus phenotype. Significant association was found with the cd72c allele in this model of lupus autoimmunity (p⬍0.01). In addition, a large backcross experiment has confirmed and narrowed this region of interest. AKXLF1 female mice were backcrossed to male L/J to produce 274 female AKxLF2 mice. These experiments confirmed the linkage to mouse chromosome 4 and have narrowed the area to span between 8cM around cd72. Initial experiments with cd72c transgenic animals are substantiating the role of cd72 in autoimmune epitope spreading after immunization with PPPGMRPP. Linkage and association of a region of chromosome 4 containing cd72 with peptide induced lupus autoimmunity has been found. This polymorphic B cell surface marker is involved in B cell-T cell interactions and provides an interesting candidate gene for B cell epitope spreading in SLE.

Race and sex differences in disease prevalence can provide important clues about etiology. We studied 2,732 Black (B) and White (W) men and women (40% B, 49% men) ages 70-79 to examine differences in the prevalence of SXKOA and whether these were related to differences in obesity and quadriceps strength relative to body weight. Subjects were participants in Health ABC, a cohort study of weight-related diseases and disablement in well-functioning elderly recruited from Medicare beneficiary lists in Pittsburgh and Memphis. Knee pain was defined as moderate or worse pain in the past 30 days by WOMAC or ‘pain on most days’ of a month in the past year. Standing PA semiflexed knee x-rays were obtained in subjects with knee pain and a random sample without pain, and scored for K-L grade by a single reader. SXKOA required pain plus x-ray OA (K-L grade ⱖ 2) in the same knee. Quadriceps strength (QUAD) was expressed as peak torque (KinCom isokinetic dynamometer 60°/s) divided by body weight. Leg %fat was measured by DXA (QDR4500A). We assessed the independent effect of race and gender on SXKOA in knee-specific logistic regression analysis (GEE) adjusting for age, covariates and correlation between knees. Results for X-ray OA without symptoms were similar.

SXKOA QUAD (N-M/kg) BMI (kg/^2) %Leg fat

B-F (603)

W-F (788)

B-M (458)

W-M (877)

26.4% 1.13 29.6 43%

13.2%* 1.15 26.0* 44%

13.8%* 1.61* 27.1* 26%*

10.4%* 1.53* 27.1* 26%*

*P⬍.01 vs B-F B-F had a higher prevalence of SXKOA than W-F (OR: 2.1 95%CI: 1.6,2.6), B-M (1.5; 1.1,2.0) and W-M. In each race/sex group, reduced QUAD and higher BMI and leg %fat were associated with increased SXKOA (P⬍.01), as well as with x-ray OA in knees without pain (P⬍.01). In women, the greater risk of SXKOA in B-F was due to a higher BMI (adj OR for B-F: 1.1; 0.8,1.5). In Bs, the greater risk of SXKOA in B-F was largely due to reduced QUAD and higher BMI and leg %fat (adj OR for B-F: 1.2; 0.9,1.7). In conclusion, quad weakness, greater BMI and increased leg fat increase the risk of knee OA in elderly Blacks and Whites, men and women. The high prevalence of SXKOA in B-F may result from a combination of greater body mass, increased leg fat and reduced quadriceps strength relative to weight.

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1933

1936

CD1-REACTIVE T CELLS AND CD1hi B CELLS FROM THE SPLEEN OF NZB/NZW MICE ARE ACTIVATED IN VIVO AND SECRETE CYTOKINES AND AUTOANTIBODIES IN VITRO. Defu Zeng, Andrea Brandolan, James Tung, Yingping Liu, Olga V Naidenko, Mitchell Kronenberg, Samuel Strober Stanford and San Diego, CA

CURRENT HORMONE REPLACEMENT THERARY (HRT) AND RADIOGRAPHIC KNEE AND HIP OSTEOARTHRITIS (OA) IN POST-MENOPAUSAL AFRICAN-AMERICAN AND CAUCASIAN WOMEN. Anca D Dragomir, Joanne M Jordan, Jordan B Renner, Gheorghe Luta, Marc C Hochberg, Charles G Helmick Chapel Hill, NC; Baltimore, MD and Atlanta, GA

We have reported that CD1hi B cells from the non-autoimmune C57BL/6 mouse spleen were almost all marginal zone B cells. The CD1hi B cells from the spleen of autoimmune NZB/NZW mice were the predominant source of IgM anti-dsDNA antibody production by the spleen B cells. The NZB/NZW spleen T cells proliferated vigorously in response to the stimulation by CD1-transfected A20 cells, but not to the non-transfected A20 cells. In vivo anti-CD1 mAb treatment significantly reduced the serum levels of IgG anti-dsDNA antibody, and ameliorated the development of lupus. In the current study, we have found that the percentage and absolute number of CD1hi B cells in the spleen of C57BL/6 and NZB/NZW mice were comparable. However, in NZB/NZW mice, about one half these B cells were CD5⫹ B1 cells and one half were CD21hi marginal zone B cells. While the percentage and absolute number of CD1-reactive T cells from the spleen of NZB/NZW and C57BL/6 mice at 3-month age identified with PE-conjugated CD1-aGalcer tetramer were also similar, the sorted CD1-tetramer⫹CD4⫹ T cells from NZB/NZW mice secreted eight-fold higher levels of IFN-␥ and IL-4 in vitro as compared to those from C57BL/6 mice after 48 hours incubation in tissue culture medium without further stimulation. Sorted tetramer-CD4⫹ T cells secreted no IFN-␥ or IL-4 in the same culture. These results indicate that in vivo activation of CD1hi B cells and CD1-reactive T cells may result in their secretion of autoantibodies and cytokines respectively.

PURPOSE: Current HRT use has been reported to be protective for radiographic knee and hip OA in Caucasian women. Whether these effects are seen in women of other ethnicity has not been studied. We examined the relationship between current HRT use and radiographic knee and hip OA in an ethnically diverse, population-based study. METHODS: All post-menopausal women from the Johnston County Osteoarthritis Project with data on current HRT use and radiographic OA were included. Current HRT use was defined as self-reported post-menopausal use of hormones pills, shots, or implants. Radiographic knee and hip OA was defined by Kellgren-Lawrence (K-L) grade at least 2; moderate/severe OA was defined as (K-L grades 3, 4). Logistic regression models were used 1) to assess associations between current HRT use and knee and hip OA, bilateral OA, and moderate/severe OA, adjusting for ethnicity, age, education (⬍12 vs. 12 or more years), body mass index, previous joint injury, and current smoking; and 2) to assess potential differences in HRT effect between the two ethnic groups. RESULTS: A total of 1,589 post-menopausal women were included. Mean age (SD) was 63 (10) years; 34% were African-American, 23% were current HRT users (12% in African-American women and 29% in Caucasian women). In unadjusted models, HRT use was strongly inversely associated with all knee OA outcomes (p ⬍ 0.0001) and with hip OA and bilateral hip OA (p ⫽ 0.01). After adjustment, the protective effect remained statistically significant for knee OA (aOR⫽0.63, 95%CI: 0.45,0.88) and bilateral knee OA (aOR⫽0.48, 95%CI: 0.29,0.78). In all models there was no evidence of effect modification by ethnicity or any other covariates. CONCLUSIONS: The protective effect of current HRT use upon knee OA and knee OA severity was similar in African-American and Caucasian post-menopausal women. No protective effect was seen for hip OA outcomes in either ethnic group.

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1937

COFFEE, TEA, AND CAFFEINE CONSUMPTION AND RISK OF RHEUMATOID ARTHRITIS: RESULTS FROM THE IOWA WOMEN’S HEALTH STUDY. Ted R Mikuls, James R Cerhan, Linda Merlino, Lindsey A Criswell, Kenneth G Saag Birmingham, AL; Rochester, MN; Iowa City, IA and San Francisco, CA

PREDICTORS FOR THE PREVALENCE AND INCIDENCE OF DEGENERATIVE LUMBAR SPINE DISEASE: THE CHINGFORD STUDY. Geraldine Hassett, Deborah J Hart, David V Doyle, Tim D Spector London, United Kingdom

Purpose: Coffee intake has previously been associated with a positive rheumatoid factor (RF) status and the development of rheumatoid arthritis (RA). We evaluated whether coffee, tea, and caffeine consumption are risk factors for incident rheumatoid arthritis (RA) among older women. Methods: These factors were evaluated in a prospective cohort study that was initiated in 1986, and included 31,336 women aged 55-69 years without a history of RA. Risk factor data were self-reported using a mailed questionnaire. Through 1997, RA cases were identified and validated against medical records. The relative risk (RR) and 95% confidence interval (CI) were used as the measure of association and were calculated using Cox proportional hazards regression. Results: During the 334,463 person-years of follow-up, there were 158 cases of validated incident RA with an average age of onset of 67.8 (⫾ 4.9) years. Compared to those reporting no use, subjects drinking ⱖ 4 cups/day of decaffeinated coffee were at increased risk of RA (RR⫽2.58; 95% CI 1.63-4.06). In contrast, women consuming ⬎ 3 cups of tea daily had a decreased risk of RA (RR⫽0.39; 95% CI 0.16-0.97) compared to women who never drank tea. Caffeinated coffee and daily caffeine intake were not associated with the development of RA. Multivariable adjustments for age, smoking history, age at menopause, marital status, and the use of hormone replacement therapy did not alter these results. The associations of RA onset with the highest categories of decaffeinated coffee (RR⫽3.10; 95% CI 1.75-5.48) and tea (RR⫽0.24; 95% CI 0.06-0.98) consumption were significant in women with seropositive disease. In contrast, women with seronegative disease displayed weaker, non-significant associations with the highest categories of decaffeinated coffee and tea use (RR⫽1.54; 95% CI 0.62-3.84 and RR⫽0.93; 95% CI⫽0.27-3.20, respectively). Conclusions: Decaffeinated coffee intake is independently and positively associated with RA onset while tea consumption shows an inverse association with disease onset. Further investigations of decaffeinated coffee and tea intake as arthritis risk factors are needed to confirm these findings and elucidate their biological basis.

Aims: Little is known about risk factors for lumbar spine degenerative disease in the population. We therefore examined predictors of prevalent and incident degenerative lumbar spine disease over 9 years in a population based cohort of women. Methods: 426 paired lateral lumbar spine x-rays taken at entry to the study and at year 9 were examined. The films were read by one experienced reader for the individual features of anterior osteophytes (AO) and disc space narrowing (DSN) using the Lane grading score and an overall Kellgren and Lawrence grade (KLG) at each lumbar disc space L1-5. Prevalent cases were defined as the presence of KLG 2⫹ or a Lane grade of 2⫹ for either AO or DSN in at least one of the four disc spaces. Incident cases were defined as ⬍ grade 2 at Year 1 and grade 2⫹ at Year 9. Predictors of prevalence and incidence including age, body mass index (BMI), smoking, ERT, physical activity, presence of OA at the hip, knee, and hand were examined using logistic regression. Results: At entry the mean age was 56.4 ⫾ 6 years and BMI 25.7 ⫾ 4.4 kg/m2. There was a significant increase over 9 years in the prevalence of disease in the lumbar spine in both the overall grade and for individual features, ranging from 20-50% (p⬍0.001). Prevalent cases of DSN were predicted by the presence of knee osteoarthritis (p⫽0.002)and by BMI (p⫽0.004). There were 26% of new incident cases defined by KLG, 21% by AO and 15% by DSN giving an average incident rate of 2% per year. Incident cases of AO were associated with baseline hip osteophytes(p⫽0.04) and DSN were associated with baseline BMI (p⫽0.02). Conclusion: This is the first time population based incidence of new degenerative changes of the lumbar spine has been reported. The incidence of degenerative lumbar spine disease is around 2% a year in this population. Factors associated with lumbar spine disease include baseline OA at the hip and knee and BMI, although these factors appear to be site and feature specific.

Disclosure: Supported by a Clinical Science Grant from the Arthritis Foundation and RO1 CA39741 from the National Cancer Institute

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1934

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1941

LIFESTYLE FACTORS ASSOCIATED WITH DEVELOPMENT OF RHEUMATOID ARTHRITIS (RA): RESULTS FROM THE BLACK WOMEN’S HEALTH STUDY (BWHS). Margaret K Formica, Julie R Palmer, Lynn Rosenberg, Timothy E McAlindon Boston, MA

TRANSCRIPTIONAL REGULATION OF COLLAGENASE-3 (MMP-13) BY IL-1 IN CHONDROCYTIC CELLS REQUIRES STABLE CHROMATIN INTEGRATION AND THE TRANSCRIPTION FACTORS RUNX-2 AND AP-1. John A Mengshol, Matthew P Vincenti, Constance E Brinckerhoff Hanover, NH

RA is an important cause of pain and disability in Black women, but most epidemiologic studies of this disorder have used Caucasian samples with prevalent disease. These studies have suggested associations with smoking and with coffee consumption. Therefore, we prospectively investigated the associations of incident RA with lifestyle factors including coffee, tea, alcohol and smoking in the BWHS, a follow-up study of 64,000 black women that began in 1995. The baseline questionnaire collected information on demographic characteristics, reproductive and medical histories, lifestyle factors, and diet (food frequency questionnaire). Cases of RA were women who reported both a physician diagnosis of RA and appropriate DMARD medication use in either the 1997 or 1999 follow-up questionnaire. Controls were frequency matched to cases by 5-year age group with a 10:1 ratio. We classified 71 women (mean age 40.8 ⫾ 10.8 years) as having incident RA. Cases were more likely than controls at baseline to be current smokers (29 vs. 13%, p⫽0.0003), current alcohol drinkers (37 vs. 26%, p⫽0.04) and to drink at least one cup per day of decaffeinated coffee (20 vs. 8%, p⫽0.002). In mutually adjusted analyses, current smoking was significantly associated with development of RA (OR⫽2.6, 95%CI: 1.3, 5.0). The association did not differ according to the amount smoked or the duration and there was no increased risk for those who quit smoking prior to baseline (OR⫽1.3, 95%CI: 0.6, 2.6). There was not a statistically significant increased risk of RA with current (OR⫽1.6, 95%CI:0.8, 2.8) or former (OR⫽1.3, 95%CI: 0.6, 2.8) alcohol consumption. Decaffeinated coffee and tea consumption were strongly associated with RA, while caffeinated coffee was not (see table). Decaffeinated coffee and tea consumption and smoking are modifiable lifestyle factors that may increase the risk of RA among black women. Further study is needed to confirm these findings and to assess whether bias may have contributed to the associations. ORs (95%CIs)

Coffee Decaf Coffee Tea

⬍1 Cup/Week Referent Referent Referent

1-6 Cups/Week 1.0 (0.5, 2.2) 2.4 (1.2, 4.9) 2.2 (1.2, 4.1)

ⱖ1 Cup/Day 1.1 (0.6, 2.1) 3.9 (1.8, 8.3) 2.1 (1.0, 4.2)

P-Value for Trend 0.96 0.0001 0.03

Disclosure:

Collagenase-3 (MMP-13 ) digests type-II collagen in cartilage, and its expression co-localizes with active osteoarthritic lesions. MMP-13 expression is more restricted than the other secreted collagenases (MMP-1, MMP-8), suggesting that tissue specific transcription factors control its expression. Using SW-1353 cells, a human chondrosarcoma cell line, as a gene regulation model for osteoarthritic chondrocytes, we previously determined that IL-1 induced a strong MMP-13 transcriptional response that requires p38, JNK MAPKs and the transcription factor NF-kB . We have now focused on delineating the cis-acting sequences responsible for the regulation of MMP-13 transcription in these cells. First, we studied the tissue specific regulation of MMP-13. Constitutive expression of the transcription factor Runx-2 correlated with the ability of a cultured cell type to express MMP-13; moreover, Runx-2 enhanced IL-1 induction of the MMP-13 gene. Activated MAPK pathways, induced by IL-1 or by constitutively active upstream MAPK activators, synergized with Runx-2 to activate transcription from the MMP-13 promoter. Thus, Runx-2 appears to be an integration point for IL-1 induced MAPK signaling. Transiently transfected MMP-13 promoters with luciferase reporters were not IL-1 inducible. However, -405 base pairs of stably integrated promoter was sufficient for 5-6 fold IL-1 induction of reporter activity, implying that chromatin structure is important for regulation of this gene. The endogenous MMP-13 gene and the integrated reporter gene were inhibited by the same MAPK inhibitors, demonstrating that these MAPK pathways regulate transcription of the reporter via the same regulatory elements as the endogenous gene. Finally, mutation of the proximal Runx binding site and the proximal AP-1 site blunted the transcriptional response to IL-1, further demonstrating the importance of AP-1 and Runx transcription factors in MMP-13 transcription. In summary, our data suggest that IL-1 regulation of MMP-13 requires native chromatin structure, and that the transcriptional MMP-13 response to IL-1 is controlled by MAPK pathways interacting at the MMP-13 promoter through the tissue specific transcription factor Runx-2 and the ubiquitous AP-1 transcription factor. Disclosure:

1939

1942

NUTRITIONAL RISK FACTORS FOR INCIDENT GOUT IN A LARGE, LONG-TERM, PROSPECTIVE COHORT STUDY. Hyon K Choi, Karen Atkinson, Walter Willett, Gary Curhan Boston, MA

MKK4 IS A KEY MAP KINASE KINASE (MAPKK) THAT REGULATES JNK IN RHEUMATOID ARTHRITIS (RA) FIBROBLAST-LIKE SYNOVIOCYTES (FLS). Gary S Firestein, Zuoning Han, Monisha Sundarrajan La Jolla, CA

Background. Various nutrients have been proposed as risk factors for gout but little prospective information is available. While patients with gout are often advised to reduce animal protein and alcohol intakes, the magnitude of their effects on gout are unknown. Methods. We examined the relation between purported nutrient risk factors for gout and incident cases of physician-diagnosed gout in a large prospective cohort over a period of 12 years (the Health Professionals Follow-up Study with 47,909 male participants). There were 1,539 newly diagnosed cases of gout during 454,878 person-year of follow-up. Data were collected by biennial mailed questionnaires and food-frequency (updated every 4 years). Logistic-regression analyses were performed to adjust for known and suspected risk factors for gout. Results. After adjustment for age, the risk of gout was associated with increasing alcohol intake; RR of gout for men in the highest (⬎50g/d) as compared with the lowest group (none) was 2.24 (95% CI, 1.74 to 2.87; P for trend, ⬍0.00001). This increase in risk attenuated but still was significant in multivariate analysis (Table). After adjustment for age, animal protein intake was not associated with increased risk for gout (RR between top to bottom quintile, 1.13; 95% CI, 0.96-1.32). However, in multivariate analysis, only the top quintile of animal protein was significantly associated with increased risk for gout (by 25%) (Table). Interestingly, dietary calcium intake was associated with a decreasing risk of gout (Table).

Introduction. Mitogen activated protein kinases (MAPK) regulate inflammation and joint destruction in RA. Previous studies demonstrated that the MAPK c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) gene expression in RA FLS. JNK phosphorylation is markedly greater in RA FLS compared with osteoarthritis (OA) FLS (J Pharm Exp Ther 291:124, 1999), which contributes to MMP overexpression in RA. At least 2 upstream MAPKK, known as MKK4 and MKK7, are known to activate JNK. However, the primary upstream activator of JNK and its contribution to JNK overactivation in RA has not been determined. Methods. Passage 3-8 FLS from RA synovium were stimulated with 2 ng/ml of IL-1 for 15 min, which is the time point for maximum JNK activation in FLS. JNK, MKK4 and MKK7 expression and phosphorylation in FLS extracts were determined by immunoprecipitation or western blot analysis using anti-JNK or anti-MKK antibodies. Kinase activity was determine in FLS extracts by immunoprecipitating MKK4 or MKK7 and incubating the complex with substrate and g32P-ATP. Results. Western blot analysis demonstrated constitutive expression of JNK, MKK4 and MKK7 in FLS. No difference was observed between OA and RA FLS with regard to basal or IL-1 stimulated levels of these kinases. MKK4 protein levels, however, were greater than MKK7 (p⬍0.001). Immunoprecipitation studies showed that JNK protein co-precipitated with anti-MKK4 but not with anti-MKK7 antibody, indicating that MKK4 and JNK form a signal complex. When FLS were stimulated with IL-1, threonine in the MKK4-JNK complex was rapidly phosphorylated in RA but not OA FLS. However, the MKK7 immunoprecipitate was not phosphorylated. In vitro kinase assays showed that the MKK4-JNK complex phosphorylated recombinant GST-c-Jun, while the MKK7 complex did not. Since MKK4 does not directly phosphorylated c-Jun, cytokine activation of MKK4 in the MKK4-JNK signal complex leads to JNK phosphorylation and subsequent c-Jun phosphorylation. Conclusions. JNK forms a signal complex with MKK4 in FLS, and activation of this complex is greater in RA than OA FLS. Therefore, MKK4 is a key upstream MAPKK in FLS that regulates JNK and MMPs in RA. Overactivation of JNK in RA FLS is mediated by increased phosphorylation of the functional MKK4-JNK complex.

Alcohol intake (g/d) 0 0.1-4.9 5-9.9 10-14.9 15-29.9 30-49.9 ⱖ50

Multivariate RR (95% CI) 1 (referent) 1.08 (0.93 ,1.26) 0.99 (0.82, 1.19) 1.19 (1.00, 1.43) 1.27 (1.07, 1.52) 1.68 (1.39, 2.02) 1.93 (1.48, 2.51)

Animal Protein (g/d) ⱕ 50 51-58 59-66 67-76 ⱖ77

Multivariate RR (95% CI)

Dietary calcium intake (mg/d)

Multivariate RR (95% CI)

1 (referent) 1.05 (0.89, 1.24) 1.03 (0.87, 1.23) 1.12 (0.94, 1.34) 1.25 (1.04, 1.52)

⬍ 605 605-722 723-848 849-1049 ⱖ1050

1 (referent) 0.84 (0.72, 0.98) 0.77 (0.65, 0.91) 0.71 (0.58, 0.85) 0.61 (0.48, 0.77)

Conclusions. High alcohol intake is a significant risk factor for gout. High animal protein consumption may slightly increase the risk for gout. In addition, there may be important protective dietary factors for gout such as dietary calcium intake.

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1940

1943

INHIBITION OF COL2A1 PROMOTER ACTIVITY BY INTERLEUKIN-1 IS MODULATED BY THE EGR-1 TRANSCRIPTION FACTOR. Lujian Tan, Bob K Choy, Makoto Osaki, Mary B Goldring Boston, MA

IMPAIRED ANTI-INFLAMMATORY CYTOKINE SIGNALING IN RHEUMATOID ARTHRITIS SYNOVIUM. Jong Dae Ji, David Deon, Lionel B Ivashkiv New York, NY

IL-1␤ inhibits expression of the type II collagen gene (COL2A1) in chondrocytes following induction and activation of the early growth response (Egr)-1 transcription factor. Transient transfection analysis in chondrocytes using COL2A1-Luc constructs showed that IL-1-induced inhibition was retained with the -131 /⫹127 bp promoter. EMSA analysis of the -120/-107 bp sequence revealed overlapping binding sites for Egr-1 and Sp1. IL-1-induced Egr-1 in nuclear extracts displaced the strong binding of Sp1 or Sp3 that was observed in untreated nuclear extracts. Cotransfection of pCMVEgr1 reduced COL2A1 promoter activity and enhanced the inhibitory effect of IL-1, while dominant-negative Egr-1 mutants reversed the inhibition either wholly or partially. Overexpression of Sp1 increased COL2A1 activity without affecting the IL-1 response, while Sp3 decreased constitutive activity and blocked the inhibition by IL-1. Mutations that blocked binding of both Sp1 and Egr-1 markedly reduced constitutive expression of the -131 bp promoter, whereas mutation of the Egr-1 site only prevented inhibition by IL-1. Substitution of the Egr-1 site with a Gal4 recognition sequence permitted binding of a Gal4/Egr1 fusion protein that, in turn, prevented binding of recombinant Sp1 added at 1:1 concentration. Our results indicate that activation of Egr-1 is necessary for the inhibitory effect of IL-1 on the strong activity of the proximal COL2A1 promoter. Egr-1 appears to reduce constitutive activity by competing with Sp1 for binding and thus permits the direct effects of IL-1-induced transcription factors via sites distal to the proximal promoter. Disclosure:

RA synovial tissue contains high levels of anti-inflammatory cytokines, such as IL-10 and TGF␤. An interesting question is why high levels of anti-inflammatory cytokines that are expressed in RA joints are unable to suppress chronic synovitis in patients. We have tested the hypothesis that the action of IL-10 in RA synovium is suppressed at the level of signal transduction, possibly secondary to antagonism of IL-10 signaling by the inflammatory cytokines IL-1␤ and TNF␣. In addition, we have tested whether the inflammatory synovial environment modulates signaling by the pleiotropic cytokine IL-6. IL-10 signaling results in the activation of transcription factor Stat3 that mediates the antiinflammatory effects of this cytokine. Activation of Stat3 DNA binding by IL-10 in control monocytes and macrophages (fresh blood monocytes, and in vitro primed and activated macrophages) was compared to macrophages derived from RA synovial tissues and fluids. IL-10 activated Stat3 in control cells on all occasions tested (35/35). In contrast, IL-10 activated Stat3 DNA binding activity in RA synovial tissue-derived macrophages in only 1/11 samples (p ⬍ 0.001) and in synovial fluid macrophages in 3/6 samples (p ⬍ 0.01). Initial disease controls suggest that IL-10 signaling is not blocked in Reiters syndrome synovial macrophages. The block in IL-10 signaling in RA macrophages was reversed when cells were cultured in the presence of anti-TNF␣ and anti-IL-1␤ antibodies, suggesting that IL-10 signaling blockade may be induced by these cytokines. Signaling by the pleiotropic cytokine IL-6 was suppressed in freshly isolated RA synovial fibroblasts. This signaling inhibition waned after several passages in culture after removal of IL-1␤and TNF␣-producing macrophages, and could be re-established by adding exogenous IL-1␤ or TNF␣. RA synovial fibroblasts constitutively expressed the inhibitors of Jak-STAT signaling SOCS3 and PIAS3, and inhibition of Jak-STAT signaling correlated with expression of these molecules. Our results demonstrate a novel level of cytokine crosstalk in RA synovium, and suggest that cytokine antagonism at the level of signal transduction contribute to the balance of cytokine activity, and thus pathogenesis. Specifically, suppression of IL-10 activity by IL-1␤ and TNF␣ may contribute to ongoing synovitis. Disclosure:

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1947

STAT4 SERINE PHOSPHORYLATION IS CRITICAL FOR IL-12 INDUCED IFN-␥ PRODUCTION AND TH1 DIFFERENTIATION. Akio Morinobu, Massimo Gadina, Roberta Visconti, Gerald M Feldman, Ryuta Nishikomori, Warren Strober, John J O’Shea Bethesda, MD

GENE EXPRESSION PROFILING OF TIGHT-SKIN MOUSE FIBROBLASTS SUGGESTS DIFFERENTIAL CONTROL OF EARLY AND LATE-STAGE PHENOTYPE. Christopher P Denton, Xu Shi-wen, I Fisher, Lowri A Evans, David J Abraham, Carol M Black London, United Kingdom

IL-12 is a crucial immunoregulatory cytokine that promotes IFN-g production, Th1 differentiation, and cell-mediated immunity. IL-12 activates the transcription factor Stat4, and its essential function is evident in Stat4 deficient mice. Stat4 is phosphorylated on Y693 and S721 upon IL-12 stimulation; however, the biological role of the latter modification is unknown. To investigate the biological importance of Stat4 serine phosphorylation, we reconstituted primary spleen cells from Stat4 deficient mice with wild type and mutated Stat4. We utilized a retroviral gene transduction to express Stat4 and GFP under the control of a bicistronic vector to permit high efficiency of expression in primary lymphocytes and to allow the identification of transduced cells. Mutation of Y693 abolished tyrosine phosphorylation and DNA binding upon IL-12 stimulation. Accordingly, Stat4 Y693F-transduced spleen cells neither produced IFN-␥ nor proliferated in response to IL-12. In contrast, mutation of S721 showed normal tyrosine phosphorylation and DNA binding. However, Stat4 S721A-transduced spleen cells showed markedly impaired IFN-g production upon IL-12 stimulation with decreased mRNA expression. In addition, Stat4 S721A-transduced naive CD4⫹ T cells did not differentiate into Th1 cells while wild type Stat4-transduced naive CD4⫹ T cells did. One potential confounding issue is that IL-12R expression is dependent upon Stat4. To circumvent this problem, we reconstituted cells from Stat4 -/-, IL-12R transgenic mice and obtained the same results. Our results clearly indicate that Stat4 serine phosphorylation is critical for IFN-␥ production in primary T cells. Interestingly, Stat4 S721A-transduced cells showed normal proliferative response to IL-12, illustrating that serine phosphorylation is not required for IL-12-induced proliferation. These results imply the existence of serine phosphorylation dependent-, and independent- gene expression. Thus we conclude that Stat4 S721 phosphorylation plays an essential, but selective role in lymphocyte responses to IL-12. Stat4 S721 is phosphorylated by the MKK6/p38 pathway, and mice with genetic alterations in this pathway have impaired Th1 differentiation. Failure to phosphorylate Stat4 S721 may explain the defective Th1 differentiation seen in these mice. This also has important therapeutic implications in that p38a inhibitors could be used to attenuate IFN-␥ production by interfering with Stat4 S721 phosphorylation.

The tight-skin mouse (Tsk1) is a genetically determined mouse model of fibrosis demonstrating many histological and biochemical similarities to human scleroderma (SSc). The mechanistic link between the underlying partial reduplication of the fibrillin-1 gene in Tsk1 and sustained dermal and visceral fibrosis is unclear, but over-expression of lineage-specific reporter transgenes introduced into Tsk1 mice suggests activation of fibroblast-specific genetic programmes. We have used cDNA microarrays (Clontech Atlas Mouse 1.2) for parallel assessment of gene expression, comparing Tsk1 and non-Tsk1 littermate fibroblast cultures (n⫽6) from skin biopsies at three time points: neonatal (1 week); 6 weeks and 12 weeks old. Neonatal fibroblasts expressed 244 (21%) of the 1176 genes represented on the microarray. At 6 and 12 weeks 147 (13%) and 94 (8%) genes were expressed respectively. Differential expression was defined by 2-fold differences in hybridisation between control or Tsk1 RNA samples. Thus, 51 genes were differentially expressed neonatally but the number of differentially expressed transcripts fell to 25 at 6 weeks and 24 at 12 weeks. Six genes showed sustained overexpression at all time-points (CD44, thrombospondin-1, osteopontin, hypoxia inducible factor-1␣, tissue inhibitor of metalloproteinase-3, and transforming growth factor ␤). Several genes, including biglycan and the b-chemokine MCP-3, were overexpressed neonatally but suppressed in fibroblasts derived from established fibrotic skin. In neonatal cultures MCP-3 was the most highly over-expressed gene, with more than 15-fold greater expression in neonatal Tsk1 compared with non-Tsk1 fibroblasts. This was associated with significant (2.4-fold) overexpression of MCP-3 protein in Tsk1 fibroblast supernatants. In parallel western blot analyses, overexpression (mean⫾SD RLU/ml) of MCP-3 was also observed in fibroblasts cultured from lesional skin of early stage SSc (4.4⫾0.3; n⫽3), compared with matched control skin biopsies (2.1⫾0.6; n⫽3). Together, these data suggest that cDNA microarray analysis of explanted fibroblasts will be valuable in elucidating key factors underlying this mouse phenotype and suggest that genes involved in initiating the tight-skin changes may differ from those which sustain fibroblast abnormalities. We are now examining expression and potential function of the products of other differentially expressed genes.


1945

1948

CD28 TRANSCRIPTIONAL INITIATOR PROTEINS: A NOVEL MODEL OF GENE REGULATION AND ITS IMPLICATIONS ON THE EMERGENCE OF CD4⫹CD28null T CELLS IN RHEUMATOID ARTHRITIS. Abbe N Vallejo, Ewa Bryl, Klaus Klarskov, Stephen Naylor, Cornelia M Weyand, Jorg J Goronzy Rochester, MN

LATENCY ASSOCIATED PEPTIDE PREVENTS TGF-␤1-DRIVEN SKIN FIBROSIS IN SCLERODERMATOUS GRAFT VERSUS HOST DISEASE, A MODEL FOR HUMAN SCLERODERMA. Yan Zhang, Laura L McCormick, Snehal R Desai, Anita C Gilliam Cleveland OH

The T cell repertoire of patients with rheumatoid arthritis includes CD4⫹CD28null T cells, a functionally aberrant, highly oligoclonal lymphocyte subset rarely found in healthy individuals. The loss of CD28 is due to an inoperative transcriptional initiator (INR) element in the gene promoter. The CD28 INR consists of two non-overlapping ␣ and ␤ motifs that have distinct protein-binding profiles but function as a single regulatory element. In CD28null T cells, there is a coordinate loss of ␣-/␤-bound complexes hence the ␣␤-INR is inactive. To identify ␣␤-INR-binding proteins, nuclear extracts from CD28⫹ T cells were subjected to DNA affinity chromatography and mass spectrometry (MS). Tandem MS approaches identified two proteins, namely, nucleolin and hnRNP-D0A as the major components of the site ␣-complex. In DNA-binding assays, specific antibodies confirmed their antigenic presence in a-bound complexes. Moreover, in vitro transcription assays showed that they are both required in the trans-activation of ␣␤-INR-driven DNA templates. Similar MS-based strategies have also led to the identification of a component of the ␤-bound complexes. This ␤-specific protein is potentially a new human DNA-binding protein that has homology to a yet undescribed C. elegans protein. It is proposed that ␣-proteins, specifically nucleolin/hnRNP-D0A complexes, are involved in strand displacement of the CD28 ␣␤-INR allowing for accessibility of the flanking ␤-site for binding of additional motif-specific proteins to initiate transcription. This provides a model for INR-regulated transcription that is independent of the known components of the basal RNA polymerase-transcription complex. Disclosure:

AIM: Murine sclerodermatous graft versus host disease (Scl GVHD) models human scleroderma with respect to skin thickening, lung fibrosis, cutaneous mononuclear cell infiltration and upregulation of cutaneous TGF-␤1 and type I collagen mRNAs, occurring by day 21 post bone marrow transplantation (BMT). Elevated TGF-␤1 appears to be the critical cytokine in Scl GVHD, which can be prevented with antibodies to TGF-␤ administered early in disease. In this study, we tested the efficacy of latency associated peptide (LAP), a naturally occurring antagonist of TGF-␤1, in inhibiting Scl GVHD. METHODS: We produced Scl GVHD by transplanting lethally irradiated BALB/c (H-2d) mice with B10.D2 (H-2d) bone marrow and spleen cells. Syngeneic transplanted BALB/c mice served as controls. Mice were administered 2 doses of 2 ␮g of human recombinant LAP on days 1 and 6 post-BMT. We evaluated skin thickening by image analysis, cutaneous TGF-␤1 and pro(a1) I collagen mRNAs by RT/PCR analysis, and cutaneous infiltrating cell types and their activation status by flow cytometry and immunostaining analysis. RESULTS: LAP effectively prevents Scl GVHD. By day 21 post-BMT, the skin thickness of LAP-treated experimental animals with Scl GVHD is comparable to that of syngeneic BMT controls, while the skin of untreated animals with Scl GVHD exhibits a more than 40% increase in thickness compared to controls. By RT/PCR analysis and immunostaining, LAP treatment also abrogates the upregulation of cutaneous TGF-␤1 mRNA and type I collagen synthesis in Scl GVHD. In contrast to anti-TGF-␤ Ab treatment, which reduces cutaneous immune cell influx and prevents upregulation of activation markers on those cells, LAP at 4 ␮g total/mouse has no significant suppressive effect on immune cell influx or activation. LAP treatment does not prevent influx of T cells and monocytes to skin as demonstrated by FACS and immunostaining analysis. To assay activation, we measured expression of I-Ad, a MHC class II alloantigen that is upregulated during antigen presentation. In Scl GVHD animals by day 21 post-BMT, I-Ad expression on cutaneous infiltrating CD45⫹, CD11b⫹ and CD3⫹ cells is markedly upregulated. I-Ad expression on these cells in Scl GVHD mice receiving LAP treatment remains as high as that in untreated Scl GVHD mice. CONCLUSIONS: LAP may be a new potential therapy for scleroderma and for other TGF-␤-driven fibrosing diseases that targets TGF-␤ more specifically, without affecting other critical roles of TGF-␤ in immune cell function. Disclosure:

1946

1949

CONSTRUCTION OF A BACTERIAL ARTIFICIAL CHROMOSOME CONTIG CONTAINING THE TSK2 GENE. Paul J Christner, Joshua Pelta-Heller, Josephine Peters Philadelphia, PA and Harwell, Oxon, UK

LIGAND-INDEPENDENT ALTERATION OF TGF-␤/SMAD SIGNALING IN SSC FIBROBLASTS. Yasuji Mori, Shinsuke Takagawa, John Varga Chicago, Illinois TGF-␤ plays a central role in the pathogenesis of fibrosis in scleroderma (SSc) by inducing fibroblast activation. Smad3 mediates stimulation of collagen synthesis induced by TGF-␤, whereas Smad7 suppresses this response. Here we examined the expression and activity of Smads signaling in SSc fibroblasts. The results indicate that the levels of Smad3 mRNA and protein are selectively elevated in SSc fibroblasts. In the murine model of skin fibrosis induced by bleomycin, elevation of Smad3 expression in vivo was demonstrated in the lesional dermis. Treatment with TGF-␤ results in delayed suppression of Smad3, and rapid induction of Smad7 expression in both control and SSc fibroblast. Whereas in control fibroblasts Smad3 and Smad4 are predominantly cytoplasmic in distribution, in ⬎40% of SSc fibroblasts these Smads are located within the nucleus even in the absence of TGF-␤. Blockade of TGF-␤ signaling failed to down-regulate nuclear accumulation of Smad3 and Smad4, indicating that nuclear import of cellular Smads in SSc fibroblast was not caused by autocrine TGF-␤ stimulation of these cells. Transcriptional activity of a Smad-regulated minimal promoter was enhanced in transiently transfected lesional SSc fibroblast. These results indicate signal-independent activation of the TGF-␤-Smad axis in SSc fibroblasts. Alterations of the Smad pathway may contribute to fibroblast activation in the pathogenesis of fibrosis. Disclosure:

Disclosure:

S377


Systemic Sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by the deposition of excessive amounts of collagen and other connective tissue macromolecules in the skin and various internal organs. The Tsk2 mouse has been shown to display cutaneous fibrosis similar to that seen in SSc and other fibrotic skin diseases. The position of the Tsk2 mutation with respect to visible mutations and molecular markers has been established in the proximal region of mouse chromosome 1. We performed an intersubspecific back cross with C57BI/6J mice carrying the Tsk2 mutation bred to Mus Castaueus. Over 300 tight N2 animals were produced. Four possible candidate genes encoding extracellular matrix proteins, Fn1, Des, Inha and Col3a1 were mapped with respect to Tsk2. The results showed that three of the genes, Fn1, Des or Inha, could be excluded as candidates for Tsk2 because they each showed multiple recombinations with Tsk2. However, the Tsk2 mutation cosegregates with Col3a1 as well as with D1Mit174, D1Mit175, D1Mit236 and D1Mit248. The Tsk2 mutation has been mapped to a 1.6 cM region, flanked on the proximal side by D1Mit233 and on the distal side by D1Mit213. We report here the construction of the physical map which spans the interval from D1Mit233 to D1Mit 213. The map was constructed from bacterial artificial chromosomes (BAC’s). The BAC-contig consists of 18 individual BAC clones from the RPCI-23 library and has a total size (without subtracting overlap regions) in excess of 5 million base pairs. Over 90% of the base sequences are published for this contig; however, the sequences exist as 12 to 15 unordered pieces of DNA of approximately 25,000 base pairs for each piece. We, as well as others, are in the process of ordering the pieces of the BAC’s in the contig containing Tsk2. From this physical map we have identified several possible candidate genes for Tsk2. These genes include but are not limited to Col3a1, Pou3f3, Stat1, Stat4, Aox1 and Zap70.

1950

1953

INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS CONTRIBUTE TO THE INCREASED EXTRACELLULAR MATRIX PRODUCTION BY FIBROBLASTS FROM PATIENTS WITH SYSTEMIC SCLEROSIS (SSc). Alycia V Knauer, Thomas A Medsger, Jr, Timothy M Wright, Carol A Feghali Pittsburgh, PA

TIME TRENDS IN CUMULATIVE DISABILITY IN RHEUMATOID ARTHRITIS 1960-1998. Eswar Krishnan, James F Fries Palo Alto, CA

Insulin-like growth factor binding proteins (IGFBPs) are secreted proteins which bind IGF-I in the extracellular milieu and modulate its action on cells. IGFBP-3, -4, and -5 are produced by fibroblasts, bind IGF-I and enhance its actions in a cell-type specific manner. IGF-I’s biological actions on dermal fibroblasts which are potentiated by IGFBP include stimulation of collagen production, suppression of collagenase production, increase of TGF-␤ mRNA levels, and stimulation of cellular proliferation. We have recently detected elevated mRNA levels for IGFBP-5 in fibroblasts from clinically involved skin of patients with SSc. IGFBP-5 mRNA levels were up to 20-fold greater in involved compared to uninvolved skin fibroblasts and healthy donor fibroblasts. We are currently examining the role that such elevated expression may play in the fibrosis seen in SSc in a unique sample of dermal fibroblasts obtained from identical and fraternal twins discordant for SSc. IGFBP protein levels were examined by Western Blot (WB) analysis and immunohistochemistry, and IGFBP functional levels by Western Ligand Blot (WLB). The ability of IGFBP to bind extracellular matrix (ECM) components was examined by immunoprecipitation and WB. Our results indicate that, compared to healthy twin dermal fibroblasts, SSc fibroblasts from involved and uninvolved skin secrete into their culture medium increased amounts of IGFBP. Much greater amounts of IGFBP are found in the ECM of the SSc fibroblasts where they are bound to collagen type I and fibronectin. This increased IGFBP expression is detected in early passage (P3) fibroblasts and is maintained over 20 passages in vitro. In summary, our results demonstrate increased IGFBP production and binding to ECM components by SSc fibroblasts. IGFBPs may contribute to the development and maintenance of fibrosis by binding collagen and fibronectin in the ECM and protecting them from degradation and by potentiating the actions of IGF-I on fibroblasts. Finally, our results also suggest that fibroblasts from uninvolved SSc skin are not an appropriate control since these cells also had increased IGFBP levels in their ECM. Rather, dermal fibroblasts from a healthy twin of a SSc patient provide an ideal control.

Year-onset

Number

Median ADD(IQR)

1960-1964 1965-1969 1970-1974 1975-1979 ADD: Annual Disability Density

201 302 404 417

1.6 (1.0-2.1) 1.6 (1.0-2.1) 1.3 (0.7-1.9) 1.2 (0.7-1.8) IQR: Interquartile Range

Disclosure:

Disclosure:

1951

1954

NOVEL INTERACTION BETWEEN IL-1␣ AND THE NUCLEAR PROTEIN NECDIN IN SYSTEMIC SCLEROSIS FIBROBLASTS. Bo Hu, Grace Wang, Yingze Zhang, Carol Feghali, Jeffrey Dingman, Timothy M Wright Pittsburgh, PA

JOB LOSS INCREASES IN EARLY RA DESPITE CONTROL OF DISEASE ACTIVITY: RESULTS FROM A LARGE SECONDARY CARE MULTI-CENTRE STUDY USING STEP-UP COMBINATION THERAPY. Mark A Quinn, Philip G Conaghan, Paul Astin, Michael J Green, Bridget Giffiths, Zunaid Karim, Heather Lancaster, Paul Emery

Year-onset Number 1980-1984 1985-1989 1990-1994 1995-1998

314 310 234 102

Median ADD(IQR) 1.2 (0.5-1.7) 1.0 (0.4-1.5) 0.8 (0.4-1.4) 0.6 (0.2-1.1) p value for trend⬍0.001

Systemic sclerosis (SSc) is characterized by excessive matrix production and proliferation of fibroblasts in the skin and internal organs. We previously found that the 31-kDa IL-1␣ precursor protein (preIL-1␣) was highly expressed in fibroblasts from lesional skin of SSc patients and was localized primarily in the nucleus. We also showed that stable overexpression of preIL-1␣ in normal fibroblasts induced IL-6, PDGF, and collagen production, and proliferation. To identify the protein(s) that associate with preIL-1␣ in the nucleus and potentially mediate(s) its effects in SSc fibroblasts, yeast two-hybrid screening was performed. cDNA libraries were constructed using RNA from fibroblasts from lesional and uninvolved skin of a SSc patient. These libraries were screened using the two-hybrid method with preIL-1␣ as the “bait”. Twenty-three cDNA clones encoding potential preIL-1␣ interacting proteins were identified from screening 2x106 independent colonies. Eight of these clones encoded full-length necdin protein. Necdin was previously identified as a cell growth suppressor in neural and fibroblast cell lines. It is believed to function through interaction with cell cycle regulatory proteins such as SV40 T antigen, E1A, E2F, and p53. We found that necdin was constitutively expressed in both lesional and uninvolved skin fibroblasts from SSc patients and in healthy control fibroblasts, as determined by Northern hybridization. Deletion mutant analysis of preIL-1␣ in the yeast two-hybrid system revealed that the amino-terminal region of preIL-1␣ interacted with necdin. Interestingly, this region is not present in the secreted mature 17-kDa IL-1␣ cytokine. Using immunofluorescence staining, we found that necdin and preIL-1␣ co-localized in the nuclei of fibroblastic cell lines (Cos-7, NIH-3T3, and Saos-2) that were transiently transfected with expression constructs encoding human preIL-1␣ and human necdin. Association of preIL-1␣ and necdin in Cos-7 cells was confirmed by co-immunoprecipitation of these proteins from nuclear extracts of transfected cells. Stable expression of preIL-1␣ reversed the growth inhibitory effect of necdin in Saos-2 cells tested by colony formation assay. Our results suggest that the constitutive upregulated expression of preIL-1␣ in SSc fibroblasts may stimulate proliferation and matrix production through binding necdin and counteracting its growth suppressor activity.

Background: Work impairment and job loss is widely reported in RA. Important factors identified in determining employment status in RA are age, social class, type of work and functional score (HAQ). YEAR is a region wide register of new RA patients in secondary care. Patients follow a standardised step-up treatment protocol reflecting modern use of combination therapy. Methods: Patients with a rheumatologist diagnosis of RA, ⬍12mo symptoms and DMARD naive receive standard assessment and follow up. All patients commence sulphasalazine mono-therapy with addition of methotrexate to non-responders. IA and IM corticosteroid are used as indicated but not within 1mo of assessment and NSAID as per routine practice. Employment status was recorded and compared to conventional outcome measures. Results: 310 patients have been recruited, mean age 57yrs, 70% RF⫹, 18% erosive, F:M ratio 1.8:1, and median symptom duration 6mo. 42% of patients were in employment and 54% of these were impaired in their work, which improved to 47%, 32% and 31% at 3, 6 and 12mo respectively (p⬍0.05). 6% had lost their jobs at baseline, increasing to 10%, 16% and 19% over 12mo with no patients returning to full time employment. At 6mo and 12mo respectively 51% and 61% of patients achieved ACR 20, 30% and 44% ACR50 and 16% and 36% ACR remission. There was a reduction in mean Disease Activity Score (DAS28), from 6.0 (severe) at baseline to 4.0 and 3.4 (moderate) at 6 and 12 months (p⬍0.05). There was no significant difference in ACR responder status or DAS in the employed vs job loss group at any time point. Job loss was significantly associated with RAQoL score (OR⫽1.08 95%CI 1.00-1.16) and documented work impairment (OR 5.89 95%CI 1.78-19.57). Conclusions: Despite overwhelming data, from ACR improvement and DAS, to support therapeutic success, job loss continues to progress throughout the first 12months of therapy. The majority of jobs are lost in the first 6 months in patients with poor quality of life scores and work impairment. The data suggests conventional therapies are ineffective in maintaining employment in early RA.

Disclosure:

Disclosure: We acknowledge the Arthritis Research Campaign for funding this project

1952

1955

PREDICTORS OF IMPAIRMENT, DISABILITY AND HANDICAP IN EARLY RA. Angela Zink, Rolf Rau, Rieke Alten, Erika Gromnica-Ihle, Joachim Sieper, Joachim Listing Berlin, Ratingen and Berlin-Buch Germany

RHEUMATOID ARTHRITIS IS AN INDEPENDENT RISK FACTOR FOR ACCELERATED CORONARY ARTERY DISEASE. Kenneth J Warrington, Peter D Kent, Takako Nakajima, Stephen L Kopecky, Robert L Frye, James F Lymp, Jorg J Goronzy, Cornelia M Weyand Rochester, Minnesota

Objective: To compare candidate risk factors for impairment, disability and handicap in recent onset rheumatoid arthritis (RA). Methods: Baseline data of an inception cohort of 139 patients with early RA (⬍ 2 years) and initial prescription of a DMARD were used to assess the risk for erosive RA, functional disability and work disability after 3 years of follow up. Candidate risk factors included age, sex, years of education, HLA-DRB1 *04,*01, rheumatoid factor, radiographic score, tender and swollen joint counts, DAS28, ESR, CRP, physician’s global assessment of activity, patient’s global assessment of health status, disability, pain, psychological well being, helplessness. “Impairment” according to the WHO classification was measured by the Ratingen radiographic score (RS⬎5), “disability” by the FFbH, a HAQ-like instrument (⬍60% of full function corresponding to ⬎1.5 HAQ), and “handicap” by permanent work disability in n⫽68 pts. employed at baseline. Univariate and multivariate logistic regression were applied to assess the prognostic value of the candidate risk factors. Results: The table gives the results of the univariate logistic regression. The figures are results of the likelihood ratio test (LRT). Higher numbers indicate stronger predictive value (*** p⬍0.001; ** p⬍0.01; * p⬍0.05)


Since 1980’s disease modifying drugs have been used earlier and more consistently in patients with Rheumatoid Arthritis (RA). While theoretically disability should therefore have decreased over time this has not been documented. We examined the cumulative disability of adults with RA with symptom onset between 1960 and 1997, followed at eight data bank centers. Annual disability density (ADD) was calculated from the Health Assessment Questionnaire disability index (HAQ) by time-integrating the area under the disability curve for each patient and dividing by number of years of observation. Numbers therefore may be considered roughly as on the HAQ 0-3 scale. Median ADD was plotted across the calendar years for patients in different time of onset groups. An extended Wilcoxon rank-sum test was used to test for trend. The effect of calendar year was examined in general linear and logistic models after adjusting for potential confounders. 2284 RA patients with at least 3 HAQ disability measurements were included in this study (Male⫽ 23 %. non-white race⫽ 12%) Median age at onset was 58 years, Median duration of disease was 12 years (IQR 6-20). The median ADD showed almost a 3-fold drop from 1960 to 1998 (Table). ADD fell in both men and women, whites and non-whites and across all levels of education. In both linear and logistic multivariate models, the downward trend persisted after adjusting for age, sex, center, education and HAQ score at entry into the study, duration of RA at baseline and length of follow-up(p⬍0.01). In this established multi-center cohort, cumulative disability in RA has fallen very substantially, particularly from 1978 to 1997. The reduction persists after adjusting for socio-demographic variables and duration of the disease. Since similar initial HAQ values were seen over time, disease severity or patient mix does not appear to provide an explanation. Changes in treatment and in treatment strategies are the likely cause of improved disability outcomes in RA.

Impairment Risk factor at t0 Erosive at t0 HLA- DRB1*01,*04 ESR RF

Disability LRT 36.1*** 14.4*** 13.5*** 10.7**

Risk factor at t0 FFbH patient global tender joints yrs. of education

Handicap LRT 25.5*** 10.7** 10.6** 10.1**

Risk factor at t0 FFbH age helplessness tender joints

LRT 8.4** 6.2* 5.6* 4.8*

Impairment was not significantly influenced by any patient assessment. Functional disability and work disability were not significantly influenced by ESR, CRP, or RF. In addition, no association between work disability and HLA or erosion status at baseline was found. Conclusion: The data show that the three dimensions of the consequences of a chronic disease have very different predictors. Disability and handicap were strongly influenced by patient behavior and education whereas erosion was associated with biological processes only. Disclosure: The study was funded by the German Federal Ministry for Research (Grant No. 01EF9403/6)

Patients with rheumatoid arthritis (RA) have a reduced life expectancy, predominantly due to an excess of deaths from cardiovascular disease. It has been proposed that inflammatory mechanisms are involved in atherosclerotic disease, in particular, acute coronary syndromes share common pathogenetic pathways with RA. Evidence has been presented that CD4⫹CD28null T cells have a direct role in both disease processes. We hypothesized that the immune dysregulation in RA contributes to an increased risk of accelerated atherosclerosis in this population. We conducted a retrospective case-control study of Olmsted County residents carrying a diagnosis of RA and new-onset coronary artery disease (CAD) between 1985 and December 1999. Patient records were reviewed to confirm the diagnosis of RA according to ACR criteria. Angiographic scores of the first coronary angiogram for coronary vessel involvement were obtained from the Mayo Clinic Coronary Care Unit database. Data on cardiovascular risk factors including smoking, hypertension, hyperlipidemia, and diabetes mellitus was obtained by chart abstraction. Controls were age and sex-matched individuals from Olmsted County with newly diagnosed CAD. Coronary angiograms from 75 cases (RA with CAD) and 130 controls (CAD) were compared for number of diseased vessels. Patients with RA were more likely to have multi-vessel coronary involvement at first coronary angiogram compared with controls (P ⫽ 0.02). Risk factors for CAD including age, male sex, diabetes, hypertension, hyperlipidemia, and smoking history were not significantly different in the two cohorts. After adjustment for age and hyperlipidemia, the difference in angiographic scores between the two groups remained statistically significant (P ⫽ 0.006). In a nested study, cases (RA with CAD) (n ⫽ 27), stable angina controls (n ⫽ 24) and unstable angina patients (n ⫽ 22) were compared for the frequencies of CD4⫹CD28null T cells in the blood. The population of CD4⫹CD28null T cells was significantly expanded in the RA with CAD cohort compared with the stable angina cohort (P ⫽ 0.001), and was similar to a subset of CAD patients who presented with acute coronary syndromes (P ⫽ 0.9). In conclusion, patients with RA are at increased risk of multi-vessel coronary artery disease. In addition, they exhibit the immunological phenotype of individuals with acute coronary syndromes, and this may explain, at least in part, the increased cardiovascular mortality in RA. Disclosure:

S378

1956

1959

DISEASE SEVERITY, TREATMENT, DEMOGRAPHICS AND THE RISK OF HOSPITALIZATION IN RA. Frederick Wolfe, Jodi Messer Wichita, KS

DOES HUMAN PARATHYROID HORMONE (hPTH 1-34) TREATMENT INCREASE VERTEBRAL SIZE IN POSTMENOPAUSAL WOMEN WITH GLUCOCORTICOID-INDUCED OSTEOPOROSIS (GIOP). Q Rehman, T Lang, G W Modin, H K Genant, N E Lane SF, CA

Background: Hospitalization is the most expensive direct cost of RA, and represents failure to control illness and/or the result(s) of treatment toxicity. But precise understanding of factors associated with hospitalization remains uncertain because of interactions with demographic and severity factors, and the requirement for large sample sizes to detect drug effect. Objectives: To determine the risk factors for hospitalization using a comprehensive set of demographic, severity, and contemporary treatment variables. Methods: 10,832 RA patients were surveyed during 4 consecutive 6 months periods over 14,474 patient years (PY) of observation. The incident rate ratio (IRR) or the increased risk of hospitalization was determined using multivariate Poisson-based generalized estimating equations. Results: The percent of observations associated with specific treatments was MTX 50.7%, prednisone (PRED) 42.7%, hydroxychloroquine (HCQ) 25.0%, leflunomide (LEF) 27.3 %, etanercept (ETAN) 12.2%, sulfasalazine (SSZ) 7.4%, and infliximab (INFL) 2.4%. The median age and disease duration was 60.5 and 13.6 years, respectively. The strongest correlates of hospitalization were HAQ disability (IRR 1.50 (95% CI 1.39, 1.61), and the number of comorbid and limiting comorbid conditions 1.33 (1.26, 1.39). Other demographic factors that contributed significantly to the risk of hospitalization included age, sex, disease duration, and reduced income. Not significant in these analyses were education, marital status, and ethnic origin. A wide series of clinical variables contributed to hospitalization, including HAQ, pain, global severity, and joint count. Strong surrogates for HAQ were SF-36 and WOMAC variables, though HAQ performed best. The number of lifetime DMARDs predicted hospitalization 1.03, (1.001, 1.05). Among treatment variables, the IRR for the risk of hospitalization was PRED 1.39 (1.29, 1.50), ETAN 1.10 (.98, 1.23), LEF 1.02 (.94, 1.11), HCQ .93 (.85, 1.02), INFL .88 (.71, 1.09), MTX .86 (.80, .93), and SSZ .84 (.73, .98). Conclusions: The factors that emerge from these analyses are the importance of comorbidity, functional status, disease activity, and treatment. Even after controlling for demographic and severity factors, prednisone emerged as a strong factor relating to hospitalization while MTX, HCQ, and SSZ relate to reduced risk. There is a strong suggestion of a risk associated with etanercept, but additional users will be required to measure that effect with more confidence.

hPTH (1-34) is an anabolic agent that stimulates bone formation and increases trabecular bone mass but has less of an effect on cortical bone mass. PTH has been found to reduce vertebral fracture risk (Lindsay R et al. Lancet 1997, Neer RM et al. N Engl J Med 2001). Although hyperparathyroidism is associated with cortical bone loss through cortical tunneling, an increase in the vertebral size was observed in patients concurrently on an anti-resorptive agent and daily injections of PTH (1-34). Increased periosteal bone apposition may partly explain this increase in cortical thickness and bone strength. The purpose of this study was to determine if hPTH (1-34) therapy is associated with an increase in vertebral size using DXA and QCT scan in postmenopausal women on HRT with GIOP. Method: 51 post menopausal osteoporotic (T-score of hip or spine ⱕ-2.5) women, mean age 62⫾10 years were treated concurrently with prednisone (mean 8⫾4 mg/d) and estrogen (duration 14.⫾10yrs) were recruited. 28 patients were randomized to receive hPTH (1-34) injection therapy 40 mg/d for 1 year with another year of follow-up and 23 control patients were continued on hormone replacement therapy (HRT). Baseline DXA (Hologic QDR 1000) and QCT (GE 9800) scan of the spine and DXA hip were obtained annually. All patients were followed for two years. Analysis: Lumbar spine BMC and area from the DXA scan and vertebral cross sectional area (VCSA) of L1 and L2 from the QCT were compared between PTH ⫹ HRT and HRT only groups using repeated measures ANOVA and post-hoc tests. A linear regression analysis was performed to determine independent predictors of change in vertebral size. VARIABLE LS AREA (cm2) LS BMC (gm) QCTLSVCSA(cm2)

Baseline 1 Year Baseline 1 Year Baseline 1 Year

PTHⴙHRT (MeanⴞSD)

HRT ONLY (MeanⴞSD)

55.56 (ⴞ5.7) 53.06 (ⴞ5.1) 47.62 (ⴞ9.7) 47.88 (ⴞ9.8) 18.94 (ⴞ2.8) 19 (ⴞ2.7)

52.67 (ⴞ5) 53.06 (ⴞ5.1) 47.62 (ⴞ9.7) 47.88 (ⴞ9.8) 18.94 (ⴞ2.8) 19 (ⴞ2.7)

P