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POSTER DISCUSSION SESSIONS Poster Discussion Session 1 Progress in Hymenoptera Venom Allergy

170 Sublingual immunotherapy with honeybee venom reduces large local reactions: a randomised, double blind controlled study Severino, M1; Cortellini, G2; Bonadonna, P3; Francescato, E4; Panzini, I5; Macchia, D1; Campi, P1; Spadolini, I6; Passalacqua, G7; Canonica, G7 1 Allergy Clinic, Nuovo Ospedale San Giovanni di Dio, Florence, Italy, 2Internal Medicne and Rheumatology, Rimini Hospital, Rimini, Italy, 3Allergy Unit, Verona General Hospital, Verona, Italy, 4Entomon sas, Florence, Italy, 5Research and Innovation Unit, Rimini Hospital, Rimini, Italy, 6Anallergo, Florence, Italy, 7Genoa University, Allergy and Respiratory Diseases, Department of Internal Medicine, Genoa, Italy

Introduction: Hymenoptera venom immunotherapy (VIT) is highly effective, since it confers a clinical protection against hymenoptera sting, including honeybee (HB) in about 90% individuals (1). The effectiveness of VIT can be easily demonstrated by the reduction of the intensity and size of large local reactions (LLR)(3, 4). VIT is currently given only by subcutaneous injections. Sublingual immunotherapy SLIT proved effective and safe in respiratory allergy, therefore its use in hymenoptera allergy can be hypothesized. We aimed at evaluating the clinical efficacy of SLIT with HB venom in beekeepers or their family members, by assessing the effect on LLR to HB stings. LLR was chosen as evaluation parameter for safety reasons, as this is a pilot study. Methods: This was a randomised, double blind, placebo controlled study. Patients with LLR due to HB sting and skin tests/ CAP-RAST assay solely positive to HB were enrolled. They were randomised to receive either HB SLIT (15 patients) or undistinguishable placebo (15 patients) for 6 months. The HB SLIT (Anallergo, Florence, Italy) involved a 6-week build-up, followed by a maintenance phase where 400 mcg were given monthly. LLR on HB sting were measured before SLIT and after no less than 3 months of maintenance. Treated patients and placebo were compared by Pearson test. P values less than .05 were considered statistically significant. Results: Thirty adult patients were enrolled and 26 completed the study, with 1 dropout in the active and 3 dropouts in the placebo 78

group. In SLIT patients the peak diameter of LLR was reduced 450% in 8/14 (57%). No change in LLR was seen in 11 of 12 placebo patients, and a systemic reaction (generalised urticaria) occurred in a patient on re-sting. The difference was significant (P 5 0.004) between the 2 groups. No adverse event was reported and no discontinuation of SLIT was required. Conclusion: HB-SLIT significantly reduced the extent of LLR in HB allergic subjects, and its safety profile was satisfactory. This pilot study envisages the possible use of SLIT also in hymenoptera allergy, and trials in patients with systemic reactions should be the next step to confirm our observations.

171 Usefulness of the basophil tests in monitoring the immune response to bee venom immunotherapy controlled by sting challenge 1

2

2

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Hausmann, O ; Schneider, M ; Weber, J ; Helbling, A ; Mu¨ller, U4; Pichler, W3 Inselspital, University of Bern, Department of Rheumatology, Division Allergology, Bern, Switzerland, 2 Buehlmann Laboratories AG, Allschwil, Switzerland, 3 Inselspital, University of Bern, Department of Rheumatology, Division of Allergology, Bern, Switzerland, 4Department of Internal Medicine, Division of Allergology, Spital Netz Bern Ziegler, Bern, Switzerland

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Background: For life-threatening hymenoptera venom allergy a 5 year-long specific immunotherapy with insect venom (VIT) is the treatment of choice. However, in 5 to 15% of VIT-treated patients no full immunologic protection can be achieved. As no reliable in-vitro marker for the evaluation of a successful VIT exists, a sting challenge with the relevant living insect is still recommended before VIT is stopped. In this study we investigated whether a change in basophil activation and degranulation assays helps in identifying bee venom tolerant patients. Methods: Thirty bee venom allergic subjects were examined. Twenty subjects after VIT (just before sting challenge, group 1) and 10 subjects before starting VIT (group 2). Basophil tests were performed with commercially available kits (CAST 5 sulfidoleukotriene release, Flow CAST 5 anti-IgE/CD63 Journal Compilation

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and Flow2 CAST 5 anti-CCR3/CD63, Buehlmann Laboratories) at varying in vitro conditions ( 7 IL-3, whole blood vs. washed leukocytes). Basophils were incubated with various bee venom concentrations (2– 10.000 ng/mL) to investigate whether the basophils of patients before or after VIT show a different dose response to bee venom. Results: Nineteen of 20 VIT-treated patients tolerated the bee sting provocation. The bee venom concentrations ranging from 2 to 250 ng/mL were found to differentiate between subjects before and after VIT, while higher concentrations consistently activated the basophils of the bee venom allergic patients. The VIT-treated patients showed a significantly lower basophil activation and degranulation at a given concentration compared to patients before VIT (s. Table). Bee

Anti-IgE/ Anti-IgE/ Anti-

Anti-

CAST 1

CAST

venom CD63 1

CD63

CCR3/

CCR3/

IL-3

IL-3

conc.

IL-3

CD63 1

CD63

IL-3

IL-3 NS

NS

250

IL-3

P50.0031 NS

P50.0061 NS

ng/mL 50

P50.0009 P50.0056 P50.0003 P50.0121 NS

P50.0335

ng/mL 10

P50.0003 P50.0017 P50.0018 P50.0121 P50.0252 P50.0168

ng/mL 2

ND

ND

P50.0455 P50.0061 P50.0098 P50.0098

ng/mL

P values (Mann-Whitney-U-Test) ns 5 not significant nd 5 not done Conclusion: A decreased basophil sensitivity could be demonstrated in bee venom allergic patients after VIT compared to patients before VIT. This finding correlates to the unproblematic sting challenge in 19/20 VITtreated patients, suggesting that the higher concentration of bee venom to elicit a basophil activation or degranulation may serve as an in vitro surrogate marker for bee venom tolerance. The use of basophil activation tests with different bee venom concentrations is potentially able to identify those bee venom allergic subjects who need higher VIT doses and/or a longer duration of VIT for full protection.

r 2008 The Authors 2008 Blackwell Munksgaard Allergy 63 (Suppl. 88): 78–157

Poster Discussion Session 1 – Progress in Hymenoptera Venom Allergy

172 Cross reactivity between European hornet and yellow jacket venoms

are primarily sensitised to yellow-jacket, and the cross-reactivity is responsible for the severe reactions to hornet.

Severino, M1; Caruso, B2; Bonadonna, P3; Macchia, D1; Campi, P1; Dama, A3; Schiappoli, M3; Senna, G3; Passalacqua, G4 1 Azienda Sanitaria di Firenze, Allergy and Clinical Immunology Unit, Florence, Italy, 2Verona General Hospital, Laboratory of Clinical Chemistry and Haematology, Verona, Italy, 3Verona General Hospital, Allergy Unit, Verona, Italy, 4University of Genoa, Allergy and Respiratory Diseases DIMI, Genoa, Italy

173 Screening for carbohydrate determinants as a cause for cross-reactivity in double sensitive patients

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Ermen, R1; Korogec, P2; Sˇilar, M2; Mugie`, E1; Kognik, M1 Hospital Golnik, Department for pulmonary diseases and allergy, Golnik, Slovenia, 2Hospital Golnik, Immunology Laboratory, Golnik, Slovenia

Background: Allergen specific immunotherapy is the treatment that lead to the longterm tolerance to allergen, previously causing severe allergy symptoms mediated by IgE. T-regulatory cells are known to be involved in the induction of peripheral tolerance – they influence either other T cell subsets either effectors cells of hypersensitivity reaction. Most Tregs are defined based on expression of CD4, CD25 and FoxP3. Recent reports showed, that a combination of CD4, CD25 and CD127 can result in significant identification of cells with regulatory and suppressive effect. We focused on the presence of Treg in patients during the course of allergen immunotherapy due to Hymenoptera venom (HV) allergy. Methods: Thirty-one patients with a history of insect venom allergy (systemic reactions – anaphylaxis) were included in the study (11 men, age range 24–72, mean age 52). An allergy history was taken, skin prick tests and determination of specific IgE (Phadia ImmunoCAP FEIA) were performed. Patients underwent treatment by standardized aluminum based allergen extract. Five healthy subjects with negative history of insect venom allergy were enrolled as negative controls. Leukocytes from fresh blood were incubated with human monoclonal antibodies (PE conjugated antiCD127, PC5 conjugated anti-CD25 and FITC conjugated anti-CD4), and measured by flow cytometry. Linear regression and t-test were used for statistical evaluation. Results: The range of duration of venom immunotherapy (VIT, maintenance dose) was 1–60 months (mean 23.55, SD 18.01). The mean proportion of CD4 1 CD25 1 and CD127 negative cells was higher in treated patients (1.897 of all lymphocytes, SD 0.519), than mean proportion of Treg in healthy controls (1.02, SD 0.43, P 5 0.0011). We did not find significant correlation between the duration of VIT and the proportion of Treg (r 5 0.162, 95% CI 5 0.488 to 0.204, P 5 0.3841). Also the correlation between the level of sensitisation of patients (specific IgE to relevant allergen) with Treg population was not significant (r 5 0.158, 95% CI 5 0.485 to 0.208). Conclusion: In our group of patients on VIT, the mean of T regulatory population was higher than in nonallergic controls, with no correlation of treatment length and sensitisation. It seems that the major influ-

Background: The choice of the vaccine for immunotherapy (IT) is crucial in hymenoptera venom allergy (HVA). European Hornet (Vespa crabro) is largely present in many European countries and is now recognized as an important cause of severe reactions in patients with HVA. We evaluated the presence and extent of cross-reactivity between the venoms of Vespa crabro and Vespula germanica (Yellow Jacket) in patients with severe reactions to Vespa crabro stings. The cross reactivity was evaluated with CAP-inhibition techniques. Methods: Sera from patients with severe reactions (grade III and IV), and who unequivocally recognized Vespa crabro as the stinging insects were collected for the CAP-inhibition experiments. All patients underwent the standard diagnostic workup, including clinical history, skin prick test, intradermal tests and specific IgE measurement. The CAP inhibition test was carried out with a specific program in UniCap100 (Phadia, Uppsala, Sweden). The extent of homologous (blockage of venom-specific IgE by the same venom) and heterologous (blockage of the venom-specific IgE by the other venom) inhibition was computed with the following formula: %inhibition 5 100[IgE inhibited sample (kU/L) 100/IgE antivenom (kU/L) at zero concentration]. An inhibition higher than 75% was considered indicative of cross-reactivity. Results: Seventeen patients (12 male, mean age 45.3 years) had a severe reaction (10 grade IV and 7 grade III) and unequivocally identified the responsible insect as Vespa crabro. All had skin and CAP positivity to both Vespa crabro and Vespula germanica, with specific IgE of 3.98 7 2.55 and 10.2 7 19.6, respectively. In 11/17 patients, vespula venom efficiently bound the Vespa Crabro specific IgE, whereas the opposite did not occur. In 6 subjects the CAP-inhibition test provided inconclusive results, therefore hornet venom was used for vaccination, based on the identification of the stinging insect. Conclusion: In our experience, the positivity to both yellow jacket and European hornet, often makes difficult the choice of the vaccine. The CAP-inhibition assay, indeed evidenced that the two venoms extensively cross-react in 67% of patients. It can be hypothesized that the majority of patients r 2008 The Authors Journal Compilation

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Background: Double sensitivity (presence of specific IgE to honey bee and wasp venom) is frequently encountered diagnostic and therapeutic problem in patients with stinging insect allergy. True double sensitisation or cross-reactivity must be considered and diagnosed in this group of patients. Crossreactivity through venom hyaluronidases or carbohydrates epitopes is possible. In our study role and frequency of carbohydrates epitopes was evaluated. Material and methods: A group of double sensitive patients admitted to our hospital in period 2003–2007 was analysed. Presence of sIgE to honey bee and wasp venom was confirmed with FEIA test. FEIA inhibition test was performed for diagnosing true double sensitivity or cross-reactivity. All patients were investigated for the presence of specific IgE to carbohydrates epitopes containing allergen of oilseed rape (OSR). Results: Sixty-six double sensitive patients were included, 41 men and 25 women, aged 16–66 years. Inhibition tests revealed true double sensitisation in 37 patients (56.1%) and cross-reactivity in 29 patients (43.9%). Five of cross-reactive patients were sensitised to honey bee and 24 to wasp venom. Median value of OSR sIgE in patients sensitised to honey bee was 4.350 IU/mL, in patients sensitised to wasp 0.605 IU/mL and in group of double sensitised patients 0.25 IU/mL. The difference between groups was statistically significant (Po0.02, KruskalWallis test). Conclusion: The values of sIgE to OSR in a group of double sensitised patients were significantly lower than in a cross-reactive group with either honey bee or wasp venom primary sensitisation. This results confirm data that carbohydrates epitopes are frequent cause for double sensitivity.

2008 Blackwell Munksgaard Allergy 63 (Suppl. 88): 78–157

174 T regulatory cells and immunotherapy in patients with Hymenoptera venom allergy Kucera, P1; Cvackova, M1; Heroldova, M1; Vavrova, H2; Ort, J2 3rd Medical Faculty, Charles University, Department of Immunology, Prague, Czech Republic, 2University Hospital KV, Department of Allergy Immunology, Prague, Czech Republic

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ence of venom-specific immunotherapy happen during the early treatment phase and the tolerance is maintained thereafter.

175 Effect of complement on basophil histamine release (HR) in patients with mastocytosis: evidence for an altered signal transmission Rue¨ff, F; Mehrle, P; Przybilla, B Germany

Background: Complement is a non-IgE-dependent inducer of HR in basophils. Complement receptor CD88 was found to be overexpressed on the surface of mast cells in patients with systemic mastocytosis. The reason for this change is unclear. The present study wanted to examine the effect of complement on HR in mastocytosis patients. Methods: Basophils were isolated from blood of three groups of subjects: mastocytosis patients with Hymenoptera venom allergy (HVA) who were simultaneously on venom immunotherapy, group M-HVA-pos. (n 5 9); mastocytosis patients without a history of Hymenoptera sting anaphylaxis, group MHVA-neg. (n 5 10); healthy controls, normal serum tryptase o11.4 mg/L (n 5 10). All mastocytosis patients showed an elevated baseline serum tryptase (19.6–226 mg/L, median 58.2 mg/L). Basophil histamine content (HC) was measured spectrofluorometrically after cell fragmentation, and absolute HR into the supernatant was determined with (HR/C5 1 ) and without C5a stimulation (HR/C5 ). % HR was quantified by the formula [(HR/C5 1 ) (HR/C5 )]/HC. Results: Controls Total HC (ng/mL), mean (confidence interval, CI) % HR upon C5a stimulation, mean (CI)

M-HVA-neg. M-HVA-pos.

47.7 51.2 (30.7–64.8) (43.9–58.6)

29.9 (16.2–43.5)

11.7 2.0$ (2.8–20.7) (1.1–2.9)

3.0y (0.0–6.2)

() Po0.001 vs. M-HVA-neg., P 5 0.11 vs. controls, $) Po0.001 vs. C HC was only lower in M-HVA neg. patients, whereas C5a effects were reduced in all mastocytosis patients. Conclusion: The exclusive decrease of basophil HC in M-HVA-pos. patients can be most likely attributed to the ongoing venom immunotherapy in these patients. In contrast, the reduced effect of C5a on HR was seen in all mastocytosis patients and did not depend on the individual histamine content. We speculate that this finding is due to an altered CD88 signalling in basophils from patients with mastocytosis.

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176 Hymenoptera venom allergy: identification of a new venom allergen responsible of Xylocopa violacea anaphylaxis Pravettoni, V1; Mauro, M2; Bertolotti, F1; Primavesi, L1; Piantanida, M1; Labardi, D3; Spadolini, I3 1 Foundation Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, IRCCS, Unit of Clinical Allergy and Immunology, Milan, Italy, 2S. Anna Hospital, Allergy Unit, Como, Italy, 3Anallergo Laboratories, Florence, Italy

Background: Carpenter Bee (Xylocopa violacea) is a solitary, fast flight, ubiquitous bee usually non aggressive and rarely stinging. Up to now, in medical literature Carpenter bee anaphilaxis has never been reported. In this study we described two cases of Xylocopa violacea venom anaphylaxis and we determined the relevant allergens of Xylocopa violacea venom. Methods: We report the case history of two patients (A, 26 y.o and B, 39 y.o.) with anaphylactic systemic reaction after Xylocopa violacea sting. All the two patients were also stung by yellow jacket before and after anaphylaxis, well tolerating the stings. At the clinical visit we performed serum venom specific IgE antibodies and venom skin prick test and intradermal tests with yellow jacket YJ, paper wasp W, European hornet EH, honeybee HB and Polistes dominulus venoms. Five patients with negative allergenic history were used as negative controls. Xylocopa violacea extract was supplied by Anallergo (Florence, Italy). SDS-PAGE and Immunoblotting: SDSPAGE was performed with Xylocopa violacea and all the above mentioned venoms. The gel was blotted on a nitrocellulose membrane, cutted into strips and matched with the sera of the two patients. After incubation with anti-IgE antibodies, the allergenic bands were detected by a chemiluminescence revealing system. Results: In the two allergic patients skin tests and serum IgE detection were positive only for yellow jacket extract. In both the patients in vitro analysis did not show any IgE binding in the honeybee immunoblotting, while IgE reactive bands at 25.2 kDa, 19.1 kDa and 6.5 kDa were found in the Xylocopa violacea extract. The 25.2 kDa allergen was also recognised in EH and YJ venoms, representing a possible cross reactive allergen between Xylocopa and Vespid venoms, surely not responsible of anaphylaxis, as the two patients were stung by yellow jacket without experience of systemic reactions. No response was found in control sera. The band at 19.1 kDa seemed to be specific of Xylocopa violacea venom. The band at 6.5 kDa, corresponding to mellitin, surprisingly presented an IgE reactivity only in Xylocopa violacea extract, suggesting a difference in HB and Xy mellitin, despite the

Journal Compilation

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belonging of these two Hymenoptera to the Apidae family. Conclusion: In these two cases of Xylocopa violacea venom anaphylaxis the responsible allergen seems to be the protein at 19.1 kDa, only cross inhibition study could confirm this finding

177 Rush hymenoptera venom immunotherapy: a means for reduce sICAM-1 levels and allergic inflammation in hymenoptera allergic patients? Ricciardi, L1; Patella, V2; Saitta, S1; Marotta, G1; Saija, A3; Gangemi, S1 1 Allergy and Clinical Immunology Division University of Messina, Human Pathology, Messina, Italy, 2U.O of Allergy and Clinical Immunology, Department of Medicine ASL-SA3, Salerno, Italy, 3Universita` di Messina, Dipartimento Farmacobiologico, Messina, Italy

Specific venom immunotherapy is the only causal treatment of hymenoptera venom allergy. Rush induction venom immunotherapy protocols have proven to be safe and are considered of great importance in the treatment of hymenoptera allergic patients with a positive hystory of severe systemic reactions, as they allow to rapidly reach the maintenance dose.The aim of our study was to evaluate serum ICAM-1 levels in wasp venom allergic patients treated with a rush induction protocol.ICAM-1, or intercellular adhesion molecule 1, is a membrane glycoprotein member of the immunoglobulin superfamily. Adhesive interactions between endothelium and circulating cells are crucial for the development of inflammatory reactions; furthermore, adhesion molecules have shown to play a crucial role in recruiting and activating inflammatory cells during allergic reactions and high serum levels of ICAM-1 are present in allergic rhinitis and asthma correlating with the severity of the disease and can be considered a useful marker of the presence of allergic inflammation.We report our experience on 13 patients with a positive history of systemic field sting reactions of Mueller grade III-IV, a positive skin reactivity and proven serum specific IgE antibodies against wasp venom.In these patients sICAM-1 levels before immunotherapy treatment were higher than in control subjects (316.47 7 78.27 vs. 230.89 7 43.92 ng/mL) even if the last reaction to an hymenoptera sting had occurred at least 3 months before and only 2 of the 13 patients suffered from other atopic diseases.The rush wasp venom immunotherapy protocol was well tolerated in all patients and allowed to reach the maintenace dose of 100 mcg.Serum ICAM-1 levels, evaluated one our after the rush protocol was finished, showed a decrease in 12 of the 13 patients (295.70 7 77.94 ng/ mL).The results of this study in our opinion highlight that, as patients with hymenoptera



venom allergy had sICAM-1 levels higher than non allergic healthy donors, hymenoptera venom allergy causes a condition of allergic inflammation even in periods without any hymenoptera stings just as it occurs in patients with allergic rhinitis and asthma, where a minimal persistent inflammation is present even when symptoms are absent. Furthermore, the rush venom immunotherapy protocol managed to reduce the sICAM levels in all patients but one, probably reducing the levels of allergic inflammation which may cause an hyperreactivity condition in patients.

178 Hymenoptera venom immunotherapy and autoimmune diseases Koschel, D; Hoeffken, G Fachkrankenhaus Coswig, Zentrum fu¨r Pneumologie, Beatmungsmedizin, Thorax- und Gefa¨Xchirurgie, Pneumologie, Coswig, Germany

Background: Severe autoimmune diseases are still considered as a contraindication for specific immunotherapy. However in hymenoptera venom allergy the advantage of the immunotherapy may be considered to be superior to the potential negative effects. Method: Four patients with different autoimmune diseases and who were treated by specific immunotherapy using an ultra-rush protocol were analysed with respect to side effects of specific immunotherapy and changes in their autimmune diseases. Results: Patient No. 1 had rheumatoid arthritis without medication because of stable disease and a systemic reaction of grade II (Ring & Messmer) after a wasp sting. Pat. No. 2 had an overlap syndrome of systemic lupus erythematodes and rheumatoid arthritis treated by methylprednisolon and methotrexate and a systemic reaction of grade III after a wasp sting. Pat. No. 3 had a psoriatic arthritis treated by methotrexate and reacted with a systemic reaction of grade II after a honeybee sting. His father was a beekeeper. Pat. No. 4 had multiple sclerosis treated by beta-interferon and a history of at least four severe systemic reactions of grade III after wasp stings and a mastocytosis was detected. In all patients specific immunotherapy was initiated with an ultra-rush protocol without any acute systemic side effects. The duration of immunotherapy is actually between 3 and 8 years. In patients No. 2, 3 and 4 no systemic reaction was observed in a sting challenge test. All autoimmune diseases remained stable without any unusual progression. Conclusion: In 4 patients with autoimmune diseases and immunosuppressive therapy and with hymenoptera venom allergy specific immunotherapy to wasp or honeybee venom was safe. Treatment was effective r 2008 The Authors Journal Compilation

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and did not induce any progression of the underlying diseases. Specific immunotherapy to insect venoms may be applicable also in patients with autoimmune diseases.

179 Anxiety and depressive disorders in Hymenoptera venom allergic patients compared with controls Gawlik, R; Badura-Brzoza, K; Kasperska-Zaj 7 c, A; Brzoza, Z; Matysiewicz, J; Hesse, R; Rogala, B Poland

Background: The serious, occasionally even fatal systemic allergic reaction following insect sting are relatively rare but has high impact on patient condition. Clinical manifestation of systemic allergic reaction to insect stings could affect the patients psychological status. They are afraid of a risk at re-exposure. The study’s objective was to examine depression and anxiety according to severity of anaphylactic reaction caused by Hymenoptera insect sting. Methods: We included in the study 81 patients, 45 insect venom allergic patients and 36 controls. Mean age of Hymenoptera allergic patients was 39.79 7 13.65 (mean 7 SD) years, controls 36.33 7 6.20 years. All of allergic patients has severe systemic reaction after wasp (n 5 31) or honey bee (n 5 14) sting with respiratory and cardiovascular symptoms. All investigated patients completed the Beck Depression Inventory (BDI) and State Trait Anxiety Inventory (STAI) questionnaire. Results: Patients presented systemic allergic reaction classified to grade 3 and 4 according to H.L.Mueller, respectively n 5 24 and n 5 21. Hymenoptera venom allergic patients scored 5.53 7 6.28 points in the Beck Depression Inventory questionnaire. This result was higher than in control group (3.86 7 4.90) but without statistical significance (P 5 0.086). We also didn’t observe significant difference between groups in trait anxiety questionnaire (A- 39.89 7 8.42; C- 37.41 7 10.55; P40.05). The only reported statistical significant feature of Hymenoptera venom allergic patients was increased state anxiety: A- 40.08 7 9.96 vs. C- 34.86 7 10.55. Conclusion: The psychological status of Hymenoptera venom allergic patients after systemic reaction is impaired by means of increased anxiety perception.


180 Basal serum tryptase and age as crucial risk factors for severe Hymenoptera sting reactions Guenova, E1; Herberts, T2; Hoetzenecker, W3; Genova, S4; Burow, G5; Mitev, V6; Biedermann, T3 1 Eberhard Karls University, Department of Dermatology, Tu¨binen, Germany, 2Department of Medical Biometry, Eberhard Karls University, Tu¨bingen, Germany, 3 Eberhard Karls University, Department of Dermatology, Tu¨bingen, Germany, 4Medical University, Clinical Center of Allergology, Sofia, Bulgaria, 5Sweden Diagnostics, Medical Research, Freiburg, Germany, 6Medical University, Department of Biochemistry, Sofia, Bulgaria

Immediate type allergic reactions to hymenoptera venom are common and reactions may vary ranging from urticaria to life threatening anaphylactic shock. The causes for these different reaction patterns are not completely understood. Recently, several reports have shown that basal serum tryptase levels correlate significantly with sting reaction severity. Furthermore, it seems that severe anaphylactic reactions to Hymenoptera stings are often associated with mastocytosis for which elevated tryptase levels are one important diagnostic marker. As serum tryptase seems to be a useful parameter for individual risk evaluation, we measured tryptase concentrations in venom allergic patients and linked these findings to severity of sting reaction (grading according to Mu¨ller), the patients’ age and sex. Two hundred and seventy-four consecutive patients visiting the Department of Dermatology, Tu¨bingen, Germany, who were diagnosed with honeybee or wasp venom allergy were included in the study. Sting reaction severity increased significantly with increased age and tryptase levels (univariate logistic regression with P 5 0.001 and P 5 0.0003 for Odds Ratio (OR) level 1 versus level 4, for age and tryptase, respectively) showing that elevated tryptase concentrations and old age are individual risk factors. Interestingly, we observed a general increment in tryptase level in elderly people (P 5 0.0001). Thereby, a continuous increase of tryptase concentrations even below the cut-off (11.4 mg/L) with increasing age (P 5 0.0026) was detectable. Gender did not seem to influence the tryptase level (twosample t-test; P 5 0.8).As anaphylactic sting reaction to honeybee or wasp venom can be a life threatening condition, it is of great clinical importance to identify those patients at risk for developing severe reactions. In our study basal serum tryptase was elevated in 10.9% of venom allergic patients. Thus, it seems that elevated serum tryptase levels even in the absence of mastocytosis are a useful index to assess the individual risk for severe sting reactions. As those patients are at risk for severe sting reactions we propose increased venom doses during venom im-

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munotherapy and a life-long continuation as recommended for mastocytosis patients.

181 Basophil activation test is more sensitive than intradermal testing in patients with convincing histories of systemic reactions to a Hymenoptera sting, but negative other diagnostic tests Korogec, P; Sˇilar, M; Ermen, R; Bajroviæ, N; Kognik, M University Clinic of Respiratory and Allergic Diseases, University Clinic of Respiratory and Allergic Dise, Golnik, Slovenia

Background: Current diagnostic guidelines do not adequately address the question how best to manage the patients with convincing history of insect allergy, but negative venom specific IgE and skin test results. Methods: Twenty-nine patients were recruited from the University Clinic of Respiratory and Allergic Diseases over a 3-year period. All patients had a convincing history of a severe or life-threatening anaphylactic reaction of Mueller grades II to IV (median grade IV) after a Hymenoptera sting, but negative venom-specific IgE and skin prick test results. Diagnostic work-up was prospectively followed up by intradermal skin testing and CD63 basophile activation test. We also included a control group of 25 subjects without a history of systemic reaction to Hymenoptera sting. Results: Nineteen of 29 (69%) patients had positive CD63 response after stimulation with bee and/or wasp venom. All these patients were positive with the cut-off of 15% of CD63 positive basophils. Thirteen of them had positive response to one venom (5 bee and 5 wasp venom) and 7 to both venoms. Only 8 of 20 patients (40%) had a positive intradermal skin test results. In control group only 2 of 25 (4%) subjects had positive basophile response and/or intradermal testing. Conclusion: We showed that in the complex cases with inconclusive diagnostic results, CD63 activation test could be a particularly useful and more sensitive then intradermal skin testing. Both test showed comparable high specificity.

182 Basophil reactivity (CD63 expression) in patients with Hymenoptera venom allergy

tion on peripheral blood basophils, has been proposed for in vitro diagnosis of IgEmediated reaction. The usefulness of BAT in prognosis of clinical symptom severity and monitoring of allergen immunotherapy efficacy is not completely elucidated. The purpose of this study was to investigate the relationship between basophil activation in patients with Hymenoptera allergy and reaction severity and duration of venom immunotherapy (VIT). Methods: Thirty patients (age range 24–72 years, mean age 52) with insect venom anaphylaxis were selected for the study on the basis of clinical history, skin prick tests and serum level of specific IgE. Patients underwent treatment with standardized aluminium based allergen extract. Five healthy subjects without clinical reactions were enrolled as negative controls. BAT using CD63 expression on CD203c-identified basophils was performed after stimulation of blood samples with different concentrations of bee and wasp venom (0.01–1 mg/mL). The data were expressed as the percentage of CD63 1 cells. Linear regression and t-test were used for statistical evaluation. Results: In the group of patients with the most severe reaction (grade IV) the mean proportion of CD63 1 cells at the highest allergen concentration was 30%, SD 20.95, compared to 31.67%, SD 21.34 (P 5 0.8331) in the group of patients with less severe reaction (grade I to III). At intermediate allergen concentration, the mean percentage of CD63 1 cells was 17.3, SD 20.32 and 21.34, SD 17 (P 5 0.5634). Basophil activation at the lowest venom concentration also did not significantly differ in both groups. The range of VIT duration (maintenance dose) was 1–60 months (mean 23.55, SD 18.01). We did not find any significant correlation between VIT duration and basophil activation (r 5 –0.0363, 95% CI 5 0.391 to 0.328, P 5 0.849). Conclusion: In our group of patients, basophil activation correlated neither with reaction severity nor VIT duration. This lack of relationship can be possibly caused by basophil response heterogeneity. The question if the induction phase of VIT is more responsible for basophil tolerance induction than maintenance phase remains unresolved.

183 Correlation between basophil specific sensitivity and efficiacy of venom immunotherapy Peternelj, A1; Silar, M1; Erzen, R2; Kosnik, M2; Korosec, P1 University Clinic of Respiratory and Allergic Diseases, Golnik, Laboratory for Clinical Immunology and Molecular Genetic, Golnik, Slovenia, 2University Clinic of Respiratory and Allergic Diseases, Golnik, University Clinic of Respiratory and Allergic Dise, Golnik, Slovenia

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Background: Treatment failure of venom immunotherapy (VIT) is not rare and the risk and pathogenic factors for those failures are so far poorly understood. For that reason we evaluated allergen-specific basophil sensitivity in patients, who did not tolerate field re-stings after completed VIT treatment. Methods: Basophil responsiveness was evaluated by flow cytometry analyses of basophil CD63 surface expression induced by different concentrations of bee or wasp venom (1, 0.1 and 0.01 mg/mL) in 14 treated patients who had experienced systemic allergic reactions (Muller grades II-III) and 17 treated patients who had no reactions after the field re-stings. We also included a group of 28 Hymenoptera venom allergic patients who have not received VIT. Results: In 14 patients who still reacted to bee or wasp sting, basophil response at a venom concentration of 0.1 mg/mL was significantly higher then in patients who tolerated field re-stings (P 5 0.03; t-test). Basophil response was also slightly higher at a concentration of 1 mg/mL, but not to statistical significance (P 5 0.12; t-test). There was no difference in the response to direct cross-linking of the IgE and in venom specific IgE and IgG4 serum concentrations between those two groups (P40.8; Fischer exact test, t-test). Patients who tolerated field re-stings have also significantly lower basophil response in comparison to patients who have not received VIT, both at 0.1 and 1 mg/mL of venom concentrations (Po 0.001; t-test). Conclusions: The results suggest that basophil venom specific sensitivity is associated with the efficiency of venom immunotherapy.

184 Treatment of mosquito-bite allergy with rupatadine

1

Karppinen, A1; Brummer-Korvenkontio, H2; Reunala, T3 Tampere University Hospital, Department of Dermatology, Tampere, Finland, 2National Public Health Institute, Helsinki, Finland, 3Tampere University and University Hospital, Department of Dermatology, Tampere, Finland

Background: Basophil activation test (BAT), which is based on flow cytometric quantification of allergen-induced CD63 upregula-

Background: People frequently experience whealing and delayed papules from mosquito bites. Whealing is mediated by antisaliva IgE antibodies and histamine. Rupatadine is

1 1

2

2

1

Hulikova, K ; Kucera, P ; Cvackova, M ; Wolfova, E Third Faculty of Medicine, Charles University, Immunology Department, Prague, Czech Republic, 2 University Hospital KV, Department of Allergy and Clinical Immunology, Prague, Czech Republic

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a new antihistamine and PAF antagonist effective in allergic rhinitis and urticaria but the effect on mosquito-bite allergy is not known. Methods: A double-blind, placebo-controlled, cross-over study was performed with rupatadine 10 mg and matched placebo in 30 mosquito-bite-sensitive adults. The mean age was 37 years and the subjects had suffered from harmful mosquito bite reactions for a mean of 15 years. Either rupatadine or placebo was taken at 8 am for 4 days, followed by a 5 day wash out period and then alternative treatment was given for 4 days. On day 3, in the both drug periods the subjects received two Aedes aegypti mosquito-bites on the forearm. The size of two 15 min bite lesions and intensity of accompanying pruritus (VAS) were measured. Results: Twenty-six subjects could be analysed for the efficacy. The size of the 15 min bite reaction was under placebo 106 mm2 and rupatadine 55 mm2. This decrease (48%) was significant (P 5 0.0003). The accompanying pruritus decreased from 60 (VAS; median) under placebo to 47.5 under rupatadine, which also is a significant

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(P 5 0.019) difference. There was no significant (P 5 0.263) difference in the adverse events under rupatadine and placebo. Conclusion: The present double-blind, placebo-controlled study in mosquito-bite-sensitive adults shows that prophylactically given rupatadine 10 mg is an effective treatment for the mosquito-bite whealing and accompanying pruritus.

185 Allergy to bedbug bites Reunala, T1; Ma¨kinen-Kiljunen, S2; Laine, M1; Hulden, L3 Tampere University and University Hospital, Department of Dermatology, Tampere, Finland, 2Hospital for Skin and Allergic Diseases, Tampere, Finland, 3 University of Helsinki, The Finnish Museum of Natural History, Finland

1

Background: Common bedbug (Cimex lectularius) declined in Europe after World War II but is again invading homes and hotels in the UK, Germany, Italy and elsewhere. This nocturnal insect feeds blood from humans and the bites can cause wheals, bullous reactions and long-lasting bite papules. Reactivity to bedbug bites


seems to differ between people but whether an IgE-mediated hypersensitivity plays a role is not known. Methods: We studied 3 adults who had been exposed to bedbug bites in their homes and had suffered 1–3 months from intense local swellings and papules on various parts of the body. A non-reactive husband and student fellow served as controls. Antigen was prepared from the head and body of the bedbugs and IgE immunospot was performed with the sera of the patients and controls. Results: Two bedbug-reactive subjects showed intense and one moderate positive reaction in the IgE immunospot. The exposed nonreactive subjects and a control sensitised to house-dust mite remained negative in the IgE immunospot. Conclusion: The present study showed that IgE-antibodies can be found in bedbug-bite reactive subjects. This suggests that bedbug bites sensitize people similarly to flea and mosquito bites. Whether the IgE antibodies are directed against the bedbug saliva proteins seems probable but needs to be confirmed.

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Poster Discussion Session 2 – Treating Allergic Rhinitis

Poster Discussion Session 2 Treating Allergic Rhinitis 186 Changes in the allergic rhinitis severity according to the treatment management: a cohort of a Spanish study Bartra, J1; de Castellar, R2; del Cuvillo, A3; Cola´s, C4; Ante´para, I5; Herdman, M6; Ferrer, M6; Mullol, J7; Torrens, A8; Mola`, O8; Izquierdo, I8; Valero, A1 1 Hospital Clıńic, Allergy Unit. Pneumology Department, Barcelona, Spain, 23D-Health Research, Innovation and Development, Barcelona, Spain, 3Clıńica Dr Lobatoń, Respiratory Service, Cadiz, Spain, 4Hospital Clıńico ‘‘Lozano Blesa’’, Servicio de Alergia, Zaragoza, Spain, 5 Hospital de Basurto, Servicio de Alergia, Bilbao, Spain, 6 Institut Municipal d’Investigacio´ Me`dica, Health Services Research Unit, Barcelona, Spain, 7Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Unitat de Rinologia i Clinica de l’Olfacte, Barcelona, Spain, 8J Uriach y Compan˜ıá S.A., Clinical Development and Medical Advice, Palau solita i Plegamans, Spain

Background: A stepwise therapeutic approach has been proposed by Allergic Rhinitis And Its Impact on Asthma (ARIA) Workshop group depending on the severity of allergic rhinitis (AR). Valero et al. modified the ARIA classification including a new criterion to discriminate moderate and severe from moderate/severe RA. Our aim was to observe the AR severity changes and the therapeutic management according to AR severity classification: mild, moderate and severe. Methods: Using data from Esprint-V study (longitudinal observational multicenter Spanish study with 4 weeks 7 1 week of follow-up, conducted by allergologists) we analyzed a cohort of patients aged 18 years or over with AR clinically diagnosed 2 years before, and with a total nasal symptom score (TNSS)5 (0 5 no symptoms, 12 5 maximum symptom intensity). Exclusion criteria were having received antihistaminic drugs (Ah) within the previous week or corticosteroid (Cs) therapy the 2 previous weeks. The modified ARIA classification was based on the number of affected healthrelated quality of life items: mild (not affected items), moderate (1–3 affected items), and severe (4 affected items). Results: 707 AR patients were recruited. 58% were women, TNSS mean was 8.2 (standard deviation, SD:1.7), a 40.2% of patients had asthma and a 61.5% had conjunctivitis. The AR was persistent in 60.8% and intermittent 39.2%. At baseline, the AR severity classification showed: 3.1% of patients with mild, 55.4% with moderate, and 41.5% with severe AR. A 62.0% of patients were treated with second generation Ah, 30.9% with Ah 1 Cs, 5.3% with Cs

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only, and 1.7% with Ah 1 others (all of them, with or without immunotherapy). Statistically significant improvements from baseline to 4 weeks were found in TNSS (3.5, SD : 2.3), and in AR severity (mild:55.6%, moderate:39.7%, and severe:4.7%). After 4 weeks of treatment, patients with Ah showed a lower AR severity (mild:62.4%, moderate:34.5%, and severe:3.0%), and also patients with Ah 1 Cs (mild:45.6%, moderate:47.2%, and severe:7.1%). Conclusions: Although ARIA propose intranasal corticosteroids as the first-line therapy in patients with moderate to severe disease AR, second generation antihistamines (added to corticoid therapy or alone) were considered by the Spanish allergologist as a useful agent to reduce AR severity.

187 A good clinical outcome following therapy with a polyvalent mechanical bacterial lysate (PMBL) correlates with the capacity of inducing a specific locoregional immunoresponse in patients with recurrent upper respiratory tract infections Braido, F1; Villa, E1; Schenone, G2; Canonica, G1; Melioli, G3 University of Genoa, Department of Internal Medicine, Genova, Italy, 2Azienda Ospedaliera Universitaria San Martino, ENT Department, Genova, Italy, 3Gaslini Pediatric Institute, Gaslini Pediatric Institute, Genova, Italy

1

Background: Despite the large possibilities offered by modern medicine, several treatments for recurrent upper respiratory tract infections (RURTI) are often unsuccessful. Polyvalent Mechanical Bacterial Lysate (PMBL) has been shown to have the capacity of inducing the maturation of dendritic cells and the secretion of proinflammatory cytokines and to directly activate B cells, having significant effects on the evolution of respiratory tract infections. The aim of this open study was to evaluate whether therapy with PMBL could be associated to the enhancement of the locoregional immunoresponse in patients with recurrent upper respiratory tract infections. Methods: Forty patients were enrolled, of which 33 were followed-up. The duration of the study was 6 months, and every patient underwent five visits. Results: Twenty-three patients were females and 17 males. Twenty-six patients had an objective improvement in clinical and medJournal Compilation

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ical locoregional conditions, while in 7 patients the treatment did not result in an objective amelioration. Twenty-five out of 27 patients with clinical response were characterized by an increase in antibodies concentration against bacterial antigens. Only 2 patients, with a non significant clinical result, had a weak increase in the concentration of locoregional specific IgG. The associations between PMBL-specific immunoglobulins titers and clinical results were significant for IgG and IgA, but not significant for IgM. Th1 switch was detected only in patients with clinical amelioration, while the Th0 phenotype was observed in 3 ‘‘responder’’ and 4 ‘‘non responder’’ patients. Weak Th2 polarization was observed also in 1 clinical responsive patient. The capacity of effectively opsonizing living bacteria was detected in samples derived from ‘‘responder patients’’. Conclusion: The administration of PMBL in patients with RURTI is effective in controlling the disease, provided that a significant IgA immune-response is elicited at mucosal level, since secreted IgA have the capacity of favoring the killing living bacteria. Nevertheless, this specific immune response is achieved by strict collaboration between innate immunity (represented by dendritic cells and granulocytes) and acquired immunity, represented by specific B and T cells. On these bases, it can be hypothesized that PMBL is able to trigger an efficient and well targeted immune-response resulting in positive clinical outcome of treated patients.

188 Can oral lactobacillus paracasei improve grass pollen allergic rhinitis? A randomised, double-blind, placebocontrolled cross-over study Wassenberg, J1; Nutten, S2; Audran, R1; Barbier, N1; Chenevard, J1; Aubert, V1; Moulin, J2; Corthe´sy-Theulaz, I2; Mercenier, A2; Spertini, F1 1 CHUV, Immunology and Allergy Department, Lausanne, Switzerland, 2Nestle´ Research Center, Lausanne, Switzerland

Background: Lactobacillus paracasei strain NCC2461 (ST11) has been found to exert anti-allergic effects in animal models. Objective: To investigate the effect of a shortterm oral administration of ST11 prior to a nasal provocation test (NPT) with grass pollen on manifestations of allergic rhinitis. Methods: 31 subjects (18–35 years-old) were enrolled in a randomised, double-blind,



placebo-controlled, cross-over study. The study consisted of a first 4 weeks period in which subjects consumed either ST11 fermented milk (ST11 group) or placebo (acidified milk) (placebo group), a washout period of six to eight weeks and a final 4 weeks period cross-over of the first treatment phase. The entire study was performed out of the pollen season. Clinical symptoms were analysed after a NPT using grass pollen allergens, and immunological parameters (specific immunoglobulins in serum, cytokines and differential cell count in nasal brushes and cytokines secreted by allergen stimulated peripheral blood mononuclear cells (PBMC)) were compared between the two treatment periods. Results: No significant change of the nasal reaction threshold was observed between the end of the first and second treatment periods, whatever the sequence (active product before or after placebo) of product consumption. However, ST11 group declared having globally less congestion than placebo group (visual analogical scale; Po 0.05). Nasal pruritus followed the same trend. This result was associated with a lower secretion of IL-5 by PBMC stimulated with grass pollen in ST11 group as compared to placebo group (Po0.03). IL-8 and IL-10 secretion by allergen stimulated PBMC followed the same trend. Finally, allergen specific IgG4 were significantly reduced in plasma, favouring probiotic consumption. No effect on mucosal samples was observed. Conclusion: Despite a short term treatment prior to NPT, the effect of the fermented milk containing the L. paracasei NCC2461 (ST11) reached significance for a clinical marker (subjective nasal congestion) and for systemic immune markers (IL-5 and IgG4). This was supported by the non significant trend for a decrease of other markers such as IL-10 and IL-8 secreted by allergen stimulated PBMC and nasal pruritus.

189 Lack of significant effect of bilastine on ventricular repolarization. A thorough QT/ QTc study Sologuren, A1; Valiente, R1; Allison, M2; Tyl, B3 FAES FARMA, S.A., Clinical Research Department, Bilbao, Spain, 2MDS Pharma Services, Clinical Research Center, Tempe, AZ, United States, 3MDS Pharma Services, Telemedicine, Centralized Cardiac Services, Baillet-en-France, France

1

Background: Safety of a new antihistamine drug includes rigorous characterisation of its effects on the QT/QTc interval (ICH E14 guideline). The objective of our study was to evaluate the effect of bilastine 20 and 100 mg once daily and the effect of co-administration of 400 mg ketoconazole on ventricular r 2008 The Authors Journal Compilation

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repolarization administered for 4 consecutive days in healthy subjects. Methods: A multiple-dose, randomised, triple-dummy, double-blind, 5-way crossover design with placebo and active control (400 mg moxifloxacin) with at least 7-day washout between periods. At day 1(baseline) and day 4 of each treatment period, three 10 sec, 12-lead ECG were recorded at 0 (predose), 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, 72 h postdose. All triplicate ECG recorded at baseline were used to calculate the individual correction factors that were applied to all ECG. All tracings were digitally transmitted to the central ECG lab who provided, for each time point, the average of heart rate, RR, PR, QRS, and QT, as well as the RR corrected QT intervals: individual QT correction (QTcNi 5 aQT [1-RR]), non-linear correction (QTcNnl 5 QT/[RR]a), Bazett’s correction (QTcB), and Fridericia’s correction (QTcF). For each subject, time-matched baseline-adjusted QT/QTc intervals on Day 4 were calculated as the difference between the values on Day 4 and the corresponding on Day–1. The time-matched baseline-adjusted QT/QTc intervals were subjected to ANCOVA for repeated measures. QTcNi was the primary variable. Estimates of differences (Diff) in least-squares means between each placebo and active treatment were provided with the 95% upper one-sided confidence limit (UCL). Results: Thirty healthy subjects (14 female, 16 male) 20 to 44 years old were enrolled.Table: Largest Placebo-Corrected Change From Time-Matched Baseline (msec) Parameter

Hour 20 mg Bilastine

100 mg Bilastine

20 mg Bilastine 1keto

Diff UCL Hour Diff UCL Hour Diff UCL QT QTcNi QTcB QTcF QTcNnl

3.0 1.0 1.0 1.0 1.0

4.09 3.47 4.07 3.31 3.54

8.95 6.30 7.58 6.20 6.42

3.0 1.0 1.0 1.0 1.0

6.98 5.02 6.53 4.88 4.97

11.89 7.87 10.07 7.79 7.87

3.0 3.0 1.0 3.0 3.0

12.32 9.31 9.45 9.17 9.61

17.22 12.16 12.09 12.08 12.52

No significant increases in LS means for change in QTcNi from time-matched baseline for bilastine 20 and 100 mg at any time postdose, no placebo-corrected change from time-matched baseline QTcNi prolongations postdose with 95% upper CI bound above 10 msec were found. Co-administration of 400 mg ketoconazole there was a prolongation in QTcNi ranging from 4.0 to 9.3 msec (UCL 12.16 msec) likely due to ketoconazole. Conclusion: The results indicate lack of significant effect of bilastine 20 and 100 mg on ventricular repolarization.


190 A combination of desloratadine 2.5 mg and pseudoephedrine 120 mg BD is more effective than the individual components for the treatment of allergic rhinitis: evidence from randomised, double-blind clinical trials Grubbe, R Allergy and Asthma Center, Oxford, Alabama, United States

Background: Nasal congestion is rated by patients as the most troublesome symptom of allergic rhinitis (AR). Desloratadine (DL), a potent, non-sedating, selective H1receptor antagonist reduced nasal congestion in clinical trials; but in patients with nasal congestion that is not adequately controlled by DL therapy alone, the combination with pseudoephedrine (PSE) can improve symptomatic control. Such combination can provide an additive, greater therapeutic response than either component alone. Methods: Two multicenter, double-blind, randomised, parallel-group studies compared the efficacy and safety of a combined DL 2.5 mg/PSE 120 mg tablet (DL-D12) BID for 15 days versus the individual components alone in the treatment of subjects with symptomatic seasonal AR. Subjects aged 12 years of either sex or any race, with a history of seasonal AR for ¡Y´2 years confirmed by a positive skin-prick test. Symptom scores were recorded each AM and PM in patient diaries. The primary endpoint for the antihistamine component (DL-D12 vs. PSE) was the mean change from baseline in the average AM/PM total symptom score (TSS), excluding nasal congestion, over the 2 weeks of the study. The primary endpoint for the decongestant component (DL-D12 vs. DL) was the mean change from baseline in the average nasal congestion score over the 2 weeks of the study. Adverse event (AE) reports and changes in vital signs and ECGs were noted. Results: The studies included 1248 subjects (64.3% female); the treatment groups were similar at baseline. In both studies, the antihistaminic effect (TSS excluding nasal congestion) of DL-D12 was significantly greater than that of PSE alone (Po0.001), and the decongestant effect of DL-D12 was significantly greater than that of DL alone (Po0.005). From Day 1 onwards the antihistaminic and the decongestant effects of DL-D12 at the end of the 24-h dosing interval were significantly greater than those for PSE and DL, respectively (Pr0.019). DL-D12 was well tolerated. Overall AEs rates were similar in the three study groups in both studies; AEs with DL-D12 were similar to those with PSE (headache, insomnia). Minor increases in mean heart rate

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occurred in the DL-D12 and PSE groups. No clinically relevant changes in any ECG parameters were noted. Conclusion: DL-D12 was well tolerated and was significantly more effective than either component alone in the treatment of all AR symptoms. For patients with nasal congestion, DL-D12 represents a useful therapeutic option.

191 Efficacy and safety of levocetirizine in the treatment of intermittent allergic rhinitis in children Zielnik-Jurkiewicz, B1; Jurkiewicz, D2 Children’s Hospital, Otoalryngology Department, Warsaw, Poland, 2Military Institute of the health Services, Otolaryngology Department, Warsaw, Poland

1

Background: Levocetirizine is an antihistaminie drug used in the treatment of allergic rhinitis, also in children. The aim of the study was to evaluate nasal symptoms, nasal airflow and nasal cytology in children with intermittent allergic rhinitis (IAR) before and after treatment with levocetirizine. Methods: Two center study consisted of 32 children (14 girls and 18 boys), mean age 9.2 7 2.7 years with IAR to grass and/or weed. Diagnosis of IAR based on ARIA recommendations. All children received levocetirizine 5 mg/daily for 4 weeks. Before the start in the study all children did not use any medication. Total nasal symptom score (including rhinorhea, nasal ichting, sneezing and nasal obstruction), nasal airflow and nasal cytology were assessed before and after treatment. Nasal airflow was measured by active anterior rhinomanometry, using Rhinotest 1000. Nasal cytologic specimens were obtained by scraping the inferior turbinate with oese. After preparation smears were analysed by optic microscope. Results: Levocetirizine treatment was significant in reducing symptom severity of IAR. Statistically significant decrease (Po 0.01) compared to baseline were observed for following individual symptom: rhinorhea, nasal itching, sneezing, as well as for the global TNSS. Levocetirizine treatment statistically significant (Po0.05 increased value (1 17%) of nasal airflow. Compared to baseline levocetirizine treatment statistically significant reduced amount of eosinophils in nasal smears. In 2 children adverse events as somnolence and dry mouth were observed. No child discontinued treatment because of adverse events. Conclusion: Levocetirizine is a useful drug in the symptom control for IAR and demonstrates effectiveness in improving nasal airflow and reducing eosinophil infiltration. Levocetirizine is well tolerated.

192 Influence of drug convenience on the satisfaction with ebastine FDT in allergic rhinitis patients Roger, A1; Fortea, J2; Plazas, M3; Mora, S2; Ibarz, R4 Hospital Germans Trias i Pujol, Badalona, Spain, 2 Almirall, S.A., Global Medical Affairs, Barcelona, Spain, 3 Almirall, S.A., Medical Department, Barcelona, Spain, 4 Adelphi Targis, Barcelona, Spain

1

Background: The objective of the present study is to evaluate the influence of the convenience of the fast-dissolving oral formulation (FDT) of ebastine in the global satisfaction with treatment. In previous studies, patients, physicians and pharmacists considered convenience as one of the key attributes for preference of ebastine FDT in comparison to current formulations. Methods: An international, multicenter, observational, retrospective study was conducted in Belgium, Mexico and Spain. Patients with Intermittent and Persistent Allergic Rhinitis (IAR and PAR) were included. Patient’s satisfaction with ebastine FDT was collected using the Treatment Satisfaction Questionnaire for Medication (TSQM), 14 items covering: effectiveness (EF), side effects (SE), convenience (C), global satisfaction (GS). Results: Four hundred and sixty-one adult patients were included (251 IAR; 210 PAR). 62.3% females and 37.7% males, being the age average 35.5 7 11.8 years. The TSQM scale (range 0–100: being 100 the best) showed [mean (95%CI)] a high satisfaction with ebastine in all its dimensions: E [74.2 (72.4–75.9)], SE [95.3 (93.9–96.6)], C [87.9 (86.7–89.1)] and GS [78.6 (76.7–80.4)] without statistically significant differences between IAR and PAR. When comparing the C with E and GS, significant and positive correlation was showed in all countries (Table 1). Conversely, the SE dimension was not correlated with C in any country. When comparing diagnostic groups, the correlations between C and GS was bigger in PAR than in IAR group in Belgium and Spain (RPAR 5 0.47, P 5 0.051, vs. RIAR 5 0.28, P 5 0.29; and RPAR 5 0.51, Po0.0001 vs. RIAR 5 0.47, Po0.0001, respectively). In Mexico, as well as in the whole sample, the trend was the opposite (RIAR 5 0.66, Po0.0001 vs. RPAR 5 0.55, Po0.0001), with no significant differences between groups (P 5 0.69).

Conclusion: Convenience of ebastine FDT was one of the best ranked dimensions of the TSQM, and was well correlated with global satisfaction with treatment. This tendency was shown in all countries. The other TSQM dimensions demostrated a high satisfaction with ebastine FDT, consistent with previous studies with the ebastine FDT formulation.

193 Glucocorticoid treatment reduces secreted mucin expression, goblet cell number, and rhinorrhea in nasal polyp patients Martinez-Anton, A1; de Bolo´s, C2; Garrido, M2; Alobid, I3; Benı´tez, P3; Roca-Ferrer, J4; Picado, C5; Mullol, J3 1 CIBERES. IDIBAPS. Hospital Clinic, Immunoal le`rgia Respirato`ria Clıńica i Experimenta, Barcelona, Spain, 2 Institut Municipal d’Investigacions Me`diques (IMIM), Hospital del Mar., Biologia Cel lular i Molecular, Barcelona, Spain, 3Hospital Clıńic, Servei d’Otorinolaringologia, Barcelona, Spain, 4IDIBAPS. Hospital Clıńic, Immunoal le`rgia Respirato`ria Clıńica i Experimenta, Barcelona, Spain, 5Universitat de Barcelona. Hospital Clıńic, Departament de Medicina. Servei de Pneumologia, Barcelona, Spain

Background: Mucus hypersecretion is a hallmark of nasal polyposis (NP). Corticosteroids constitute the first-line treatment for NP, decreasing their size and inflammatory component. However, their effect on mucin production is not well understood. The aim of this study was to investigate the effect of corticosteroids on mucin expression and their correlation with mucin cell origin and nasal symptoms in nasal polyposis. Methods: Patients were randomised in control (N 5 9) and treatment (oral prednisone for 2 weeks and intranasal budesonide for 12 weeks; N 5 23) groups. NP from non-asthmatic (NP-NA; N 5 13), aspirin-tolerant (NP-ATA; N 5 11) and aspirin-intolerant (NP-AIA; N 5 8) asthmatics were studied. NP biopsies were obtained before (w0) and after 2 (w2) and 12 (w12) weeks of corticosteroid treatment. Immunohistochemistry for MUC5AC and MUC5B mucins, and alcian blue-PAS staining for the detection of goblet cells were performed on paraffin-embedded tissue sections. Mucins and goblet cell percentages were quantified in both epithelium and glands. Rhinorrhea and nasal obstruction were also assessed. Data are reported as median of positive cells among total cells. Statistical significance was set at Po0.05.

Table 1. Spearman Correlations between convenience and the other TSQM satisfaction dimensions in all countries in the whole sample (for abstract 192).

Effectiveness Side effects Global satisfaction

Belgium

Mexico

Spain

Global

R 5 0.35 R 5 0.12 (P 5 0.49) R 5 0.45

R 5 0.52 R 5 0.11 (P 5 0.06) R 5 0.62

R 5 0.43 R 5 0.08 (P 5 0.26) R 5 0.50

R 5 0.48 R 5 0.10 R 5 0.57

Po0.05;Po0.0001

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Results: At w2, MUC5AC and MUC5B mucins showed no significant variations compared to baseline. After 12 weeks of corticosteroid treatment there was a decrease in epithelial MUC5AC (from 40 to 5, Po0.05) and gland MUC5B (from 45 to 2.5, P 5 0.06) expression in NP-ATA patients compared to baseline. Glucocorticoids decreased goblet (from 20 to 10) and mucous cell (from 40 to 10) numbers, correlating with MUC5AC (R 5 0.725; Po0.01) and MUC5B (R 5 0.782, Po0.01) reduction. Glucocorticoids also decreased nasal obstruction (from 3 to 0) and rhinorrhea (from 3 to 1) scores, the latter correlating with MUC5AC decrease (R 5 0.403, Po0.05). Conclusions: These results suggest: 1st) corticosteroids down-regulate secreted mucins MUC5AC and MUC5B; 2nd) there exists a link between secreted mucin expression and goblet cell hyperplasia; and 3rd) MUC5AC decrease may be translated into rhinorrhea reduction.

194 Long-term ocular safety of mometasone furoate nasal spray during treatment of perennial allergic rhinitis Bernstein, D University of Cincinnati, Department of Internal Medicine, Cincinnati, United States

Background: Mometasone furoate nasal spray (MFNS) has a demonstrated clinical efficacy/safety profile in the reduction of perennial allergic rhinitis (PAR) symptoms (congestion, rhinorrhea, sneezing, itching). Few studies have evaluated its effects on ocular safety parameters. Two studies assessed long-term ocular safety of MFNS in adult subjects with PAR. Methods: Two multicenter studies examined MFNS ¡Y´200 mcg QD in 783 symptomatic subjects ¡Y´12 years old with ¡Y´2-year history of PAR. Double-blind, parallelgroup study randomised 490 subjects to 12 weeks of MFNS 200 mcg QD, active comparator beclomethasone dipropionate (BDP) 168 mcg BID, or placebo. Another 52-week, open-label study randomised 293 subjects to MFNS (200 mcg QD or variable dose; 200 mcg QD titrated to 100–400 mcg QD based on therapeutic response) or active comparator BDP 168 mcg BID. Ocular safety in both studies was assessed through ophthalmic evaluations (tonometry/slit lamp procedure) at baseline/study end. Results: Baseline characteristics were similar for all groups. No clinically significant changes in ophthalmic examinations from baseline to study end were observed in either study. In the 12-week study, maximum change from baseline in intraocular pressure (IOP) was in placebo group ( 1 0.41 mm Hg) (Table). Trace posterior subcapsular catarr 2008 The Authors Journal Compilation

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acts were detected in 1 placebo subject at study end but not at screening and in 1 subject in BDP group at screening and study end. No posterior subcapsular cataracts/ increased IOP were observed in MFNS group. These results are supported by data from a 1-year open-label study involving 293 subjects (see Table). Changes in IOP from baseline to study end were 0.7 mm Hg (MFNS 200 mcg QD), 1 0.7 mm Hg (MFNS 100–400 mcg QD; 10.5%, 60.0%, and 29.5% of subjects received 100, 200, and 400 mcg QD, respectively, as last dose), and 0.2 mm Hg (BDP 168 mcg BID). One subject in MFNS 100–400 mcg QD group had IOP of 21/22 mm Hg (left/right eye) at baseline and 26/28 mm Hg at study end. One subject in MFNS 200 mcg QD group had 24/26 mm Hg at baseline which decreased to 18/18 mm Hg at study end. No posterior subcapsular cataracts were observed in any group. Conclusions: MFNS 200 mcg QD and variable (100–400 mcg QD) dosage had a positive ocular safety profile with no evidence of clinically significant increased IOP or increased risk of cataracts in a large population of adult subjects with PAR. Patients with glaucoma should be monitored for changes in IOP. MFNS 200 mcg QD

Visit

n

Left

Right

Double-Blind Study

Screening Week 12 Baseline Week 52

164 141 100 79

15.2561 15.1986 15.4100 14.8354

15.1829 15.1135 15.5100 14.7975

n

Left

Open-Label Study

MFNS 100–400 mcg QD Visit Double-Blind Study

Right

Screening N/A N/A

N/A

N/A N/A

N/A

Week 12 Open-Label Study

Baseline 95

15.0737 14.9684

72

15.4722 15.6806

Week 52

BDP 168 mcg BID

Visit

n

Left

Right

Double-Blind Study

Screening Week 12 Baseline Week 52

163 145 98 81

15.2883 15.1172 15.4694 15.3333

15.2761 14.2483 15.4694 15.5185

Placebo

Visit

n

Left

Right

Double-Blind Study

Screening 163

15.1166

15.1227

141

15.5248

15.4681

N/A

N/A

N/A

N/A

N/A

N/A

Open-Label Study

Week 12 Open-Label Study

Baseline Week 52


195 Mometasone furoate nasal spray improves congestion in subjects with seasonal allergic rhinitis as measured objectively with acoustic rhinometry Salapatek, A1; Patel, P1; Gopalan, G2; Varghese, S2 Allied Research International – Cetero Research, Mississauga, Canada, 2Schering-Plough Corp., Kenilworth, United States

1

Background: Patients report that nasal congestion is the most bothersome symptom of seasonal allergic rhinitis (SAR). This study of mometasone furoate nasal spray (MFNS) in subjects with symptoms of SAR induced in an environmental exposure chamber (EEC) evaluated improvement in nasal congestion by measuring nasal patency objectively with acoustic rhinometry (AcR). Methods: In a double-blind, placebo-controlled study, 310 ragweed-sensitive subjects were exposed to a controlled level of ragweed pollen 500 particles/m3) in an EEC for 2 h pre-dose, given MFNS 200 mcg or 7 (3500 placebo, and studied for 6 h post-dose (Day 1). A subset of 155 subjects received MFNS or placebo for 7 additional days, and returned to the EEC on Day 8 for 4 h of pollen exposure. AcR measurement of nasal congestion was performed before subjects entered the EEC; at 4 time points in the EEC during Day 1: 0, 2, 4, and 6 h; and 3 time points during Day 8: 24, 26, and 28 h post-dose when MFNS treatment levels were at trough value. Results: The minimal cross-sectional areas (MCA) measured with AcR for either nostril before EEC entry were not different in either treatment group. On Day 1 after 2 h of EEC pollen exposure pre-dose, MCA decreased significantly for all subjects. At 6 h, MFNS significantly reduced congestion (increased nasal patency) from baseline after 2 h of pollen exposure, pre-dose (Pr0.00001) and compared with placebo (P 5 0.005). On Day 8, after 7 days of treatment, congestion with MFNS was significantly lower than with placebo pre-EEC pollen exposure (P 5 0.04) and at 2 (P 5 0.01) and 4 h (P 5 0.0008) post-exposure. MFNS-treated subjects remained protected against pollen-induced nasal congestion throughout EEC exposure on Day 8, while congestion continued to increase in subjects receiving placebo. Conclusion: AcR is a sensitive, objective measure of nasal patency and nasal congestion. AcR demonstrated that MFNS significantly reduced nasal congestion after a single treatment. Further, MFNS provided a therapeutic effect even at trough levels, protecting subjects against allergen-induced nasal congestion.

87


196 Fluticasone furoate nasal spray has a favourable safety and tolerability profile across three large clinical trials in children aged 6–11 years Meltzer, E1; Tripathy, I2; Ma´spero, J3; Wu, W4; Philpot, E5 Allergy and Asthma Medical Group and Research Center, Consultant, San Diego, United States, 2Phelps County Regional Medical Center, Allergy and Immunology, Rolla, United States, 3Fundacion CIDEA, Allergy and Respiratory Research Unit, Buenos Aires, Argentina, 4GlaxoSmithKline, Statistics and Programming, Research Triangle Park, United States, 5 GlaxoSmithKline, Clinical Development, Research Triangle Park, United States

Conclusion: FFNS has a favourable safety and tolerability profile across the three analysed studies in children aged 6–11 years.

197

1

Background: Fluticasone furoate nasal spray (FFNS) has recently been approved in Europe for use in children aged 6–11 years with allergic rhinitis (AR). In the US, FFNS has been approved for use in children as young as 2 years. Data from three Phase III trials have been combined in an integrated sub-analysis designed to evaluate the safety of FFNS in the 6–11 year-old subgroup of patients with seasonal (SAR) and perennial AR (PAR). Methods: All three trials included in the integrated analysis were randomised, double-blind, placebo-controlled, parallel-group studies: a 2-week study in US patients with SAR, a global 12-week study in patients with PAR, and a 6-week safety/HPA axis study in patients with PAR. Randomised patients (n 5 948) received once-daily treatment with either FFNS 55 mg (n 5 297; excluding the 6-week study), FFNS 110 mg (n 5 321) or vehicle placebo nasal spray (n 5 330). Safety assessments included the frequency and type of clinical adverse events (AEs), clinical laboratory tests, detailed nasal examinations (turbinates, mucosa, septum, nasal secretions), vital signs, and 12-lead ECGs. Urinary cortisol (6-week and 12-week study) were measured over a 24-h period at randomisation (baseline) and final treatment. In the 12-week study, specialist ophthalmic examinations were conducted by an ophthalmologist or optometrist before the randomisation and final treatment visit. Results: FFNS had a favourable safety and tolerability profile in patients aged 6–11 years. Drug-related AEs were observed in 10%, 7% and 8% of the FFNS 55 mg, FFNS 110 mg and placebo group, respectively. The most common AEs were epistaxis, pyrexia and sinusitis which, with the exception of pyrexia, were similar across all treatment groups. Urinary cortisol excretion over 24 h at baseline and end of treatment was similar between treatment groups; no patients had 24-h excretion levels below normal range at treatment end. Findings from nasal examinations were similar across the treatment groups. No differences between the groups were noted for mean change from baseline in intraocular pressure in each eye.

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Abstract withdrawn.

198 A pilot environmental exposure chamber (EEC) study indicates rapid and prolonged relief of nasal symptoms after a single dose (110 mcg) of fluticasone furoate nasal spray (FFNS) in patients with seasonal allergic rhinitis (SAR)

0.5, 0.75, 1.0 and 1.5 h) for FFNS were 1.50, 1.61, 2.36, 2.32, and 2.46, compared with placebo scores of 0.43, 0.76, 1.17, 1.54, and 1.80. This trend in efficacy continued with the 24 h and 25 h MCFB TNSS for FFNS being 1.70 and 1.37, respectively, compared with placebo scores of 1.13 and 0.50, respectively. Conclusions: This pilot EEC study illustrates trends in efficacy up to 24 h after a single 110 mcg dose of FFNS, confirming results from field trials. Importantly, the EEC model indicates a faster onset of action than previously reported, with reductions in TNSS occurring 15 min post-dose, the earliest timepoint tested. These data indicate that FFNS can provide rapid and prolonged relief of the nasal symptoms of SAR.

Salapatek, A; Bates, M; Patel, P Cetero Research, Research and Development, Mississauga, Canada

Background: Allergic rhinitis (AR) is a serious disorder increasing in prevalence in North America and associated with considerable cost and co-morbidity. FFNS is a novel corticosteroid administered in a unique side-actuated device and recently approved in the US for treatment of AR based on efficacy data from multiple field studies. The objective of this study was to examine the efficacy, onset and duration of action of a single dose of FFNS using the EEC model. Methods: This was a randomised, doubleblind, placebo-controlled, parallel study of 55 patients, aged 18 to 65, with a clinical history of SAR due to ragweed. The study consisted of 2 to 4 priming visits followed by a treatment visit [110 mcg total dose (4 27.5 mcg/spray)] in an EEC where patients were exposed to ragweed pollen at an average concentration of 3500 particles/ m3. Patients remained in the EEC for 12 h post-dose and recorded nasal symptom scores on diary cards at 15, 30, 45, 60, 90 and 120 min post-dose; then every hour up to 12 h. They returned to the EEC the next day for a further 12 h of symptom scoring. The primary endpoints were onset of action and efficacy based on mean change from baseline (MCFB) in instantaneous total nasal symptom scores (TNSS). Changes from baseline were compared between treatment groups at each timepoint using ANCOVA with baseline TNSS as a covariate. Results: Mean baseline TNSS scores for each group were similar [FFNS 5 9.28 (n 5 28); Placebo 5 10.20 (n 5 27)]. Considerably larger declines in TNSS MCFB were observed for FFNS over placebo beginning with the first timepoint after dosing (0.25 h) and were sustained, suggesting an onset of action as early as 15 min.The MCFB TNSS for the first five post-dose timepoints (0.25, Journal Compilation

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199 Effects of proinflammatory stimuli on glucocorticoid receptor translocation in airway fibroblasts Fernandez-Bertolin, L1; Pujols, L1; Fuentes, M1; Isam, A2; Embid, C3; Roca-Ferrer, J1; Mullol, J1; Picado, C3 1 IDIBAPS, Hospital Clinic, IRCE, Barcelona, Spain, 2 Hospital Clinic, Rhinology unit, ENT Department, Barcelona, Spain, 3Hospital Clinic, Pneumology and Allergy Departments, Barcelona, Spain

Background: Poor response of nasal polyps to glucocorticoids (GC) may be due to an abnormal glucocorticoid receptor (GR) translocation to the nucleus. The aim of this study was to evaluate the effect of dexamethasone (Dex) on the GR translocation kinetics and its possible modulation by proinflammatory stimuli. Methods: Primary lines of fibroblasts were obtained from nasal polyp (NP, N 5 11) tissues from asthmatic patients. Fibroblasts from healthy nasal mucosa (NM, N 5 9), obtained from patients undergoing nasal corrective surgery, were used as controls. Fibroblasts were cultured in DMEM 10% charcoal-treated FBS for 2 days in 8-well chamber slides. LPS (10 mg/mL) or cytokine mix (IL1b, TNF-a and IFN-g; 10 ng/mL each) was added for 24 h previous to Dex (100 nM) stimulation for 1, 2 or 3 h. GR translocation to the nucleus was analysed by fluorescent immunocytochemistry using a GR-specific antibody. Results are expressed as mean nuclear fluorescent up-fold compared to non-stimulated cells. Data is shown as median and 25th–75th percentile. Results: At 1 h, Dex induced maximal GR translocation in both NM (1.81; 1.52–2.27 Po0.01) and NP fibroblasts (2.02; 1.48–2.60 Po0.01). At 3 h, translocation was still significantly higher compared to nonstimulated cells in both cell types (Po 0.01). In NP but not in NM fibroblasts,



LPS increased GR translocation at 1 h (2.8; 1.4–3.67 Po0.05), 2 h (2.74; 1.36–3 Po 0.01), and 3 h (1.97; 1.42–2.28 Po0.05) compared to cells treated with Dex alone. Incubation with cytokine mix did not alter Dex-induced GR translocation. Conclusion: The finding of a greater GR translocation after LPS stimulation in fibroblasts from NP, suggests that the underlying inflammation present in NP may render these fibroblasts more susceptible to Dexinduced GR translocation when submitted to an additional inflammatory insult. The mechanisms involved in the priming effect of NP inflammation remain to be clarified.

200 Evaluation of second generation antihistamines following a 2 or 4-weeks treatment by means of a pooled responder analysis in patients suffering from seasonal allergic rhinitis Mullol, J1; Bousquet, J2; Bachert, C3; Canonica, W4; Kowalski, M5; Marti-Guadanõ, E6; Picado, C7; Scadding, G8; Van Cauwenberge, P3; Izquierdo, I9 1 Hosp Clinic, Unitat de Rinologia Servei ORL, Barcelona, Spain, 2Hoˆpital Arnaud de Villeneuve INSERM, Service des Maladies Respiratoires, Montpellier, France, 3Ghent University Hospital, Department Oto-Rhino-Laryngology, Ghent, Belgium, 4Genoa University, Department of Internal Medicine, Genoa, Italy, 5Lodz Medical University,


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Immunology, Rheumatology and Allergy, Lodz, Poland, Fundacio´ Sant Pere Claver, Allergy Service, Barcelona, Spain, 7Hosp Clinic, Pneumology and Respiratory Allergy, Barcelona, Swaziland, 8Royal National Throat, Nose and Ear, Rhinology, London, United Kingdom, 9J Uriach y Compan˜ia, S.A., Clinical Development and Medical Advice, Palau-Solita´ i Plegamans, Spain 6

Background: Second generation antihistamines are the first choice treatment option for allergic rhinitis, as long as they fulfill the ARIA/EAACI requeriments for modern anti-allergic drugs. In controlled clinical trials the efficacy of treatment is evaluated by means of symptoms reduction. However, a responder analysis to identify clinically meaningful differences in response in patients with Seasonal Allergic Rhinits (SAR) has never been reported previously. Aim: To quantify the improvement of patients suffering from SAR treated with different second generation antihistamines using a responder analysis. Methods: The pooled data from seven randomised, double-blind, placebo-controlled, 2 or 4-week multicentre studies were used for this analysis. In all trials, total symptom scores (6TSSs) were calculated by adding the 6 individual symptom scores (rhinorrea, nasal itching, nasal obstruction, sneezing, ocular itching, and watery eyes). Symptom scores were graded according to a conventional 4-point severity scale (0 to 3). The evaluated antihistamines were: Rupatadine 10 mg (RUP 10) and 20 mg (RUP 20),


Cetirizine 10 mg (CTZ 10), Ebastine 10 mg (EBA 10), Loratadine 10 mg (LOR 10), and Desloratadine 5 mg (DL 5). Response rates (50% and 75% improvement) were calculated based on patients’ TSS levels after treatment as compared to baseline symptoms. The percentage of patients over the Lower 95% Confidence Interval of mean change with Placebo (LCI Pl) was also calculated. Results: The pooled analysis is given below: 6TSS

Placebo

RUP 10 RUP 20 CTZ 10

EBAS 10 LOR 10 DL 5

(n 5 334) (n 5 665) (n 5 479) (n 5 115) (n 5 82)

450%

(n 5 227) (n 5 117)

36.5

54.0

63.1

51.3

63.4

61.2

41.0

9.3

22.1

28.9

13.9

29.3

26.0

12.8

47.3

61.1

66.4

46.9

65.8

63.0

53.9

Reduction from baseline 475% Reduction from baseline LCL Pl criteria

Po0.05 vs. placebo

Conclusion: Based on the responder analysis, we conclude that both doses of RUP, EBA and LOR once daily provide a significant and relevant benefit in SAR patients for the responder criteria assessed. Responder analyses should be considered as a useful tool for the assessment of antiallergic drugs in clinical practice.

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Poster Discussion Session 3 – T Cells, Cytokines and Antibody Regulation

Poster Discussion Session 3 T Cells, Cytokines and Antibody Regulation 201 Suppression of IgE isotype switch in murine B cells by dendritic cells can be reversed by diesel particles Braun, A1; Bewersdorff, M1; Buters, J1; Jakob, T2; Behrendt, H1; Mempel, M1 1 Division of Environmental Dermatology and Allergy – Helmholtz Zentrum Mu¨nchen, ZAUM – Center for Allergy and Environment, TUM, Munich, Germany, 2University of Freiburg, Allergy Research Group, University Medical Center, Freiburg, Germany

Background: Epidemiological studies demonstrate that air pollution such as diesel exhaust particles (DEP) play a role in the increased incidence of respiratory allergic diseases. Studies in vitro and in vivo have shown that DEP may enhance allergic antibody (IgE) expression. B cells are the key players in the IgE production, and in a defined environment in vitro B cells class switch to IgE. Aim of the study: We first studied whether DEP affect the maturation of murine dendritic cells (DC) and then whether these cells are able to modulate this IgE isotype switching in B cells. Methods: Murine bone marrow-derived DC (BM-DC) were activated with increasing concentrations of the diesel standard reference material SRM1650a in the presence or absence of lipopolysaccharide (LPS). DC maturation was evaluated by FACS analysis of cell surface markers CD40, CD80, CD86 and MHC class II. Splenic B cells sorted by MACS were 495% B220 1 . B cells were co-cultured with/without pre-treated BMDC in the presence of anti-CD40 mAb and IL-4 for various times. B cells and supernatants were analysed for expression and secretion of the immunoglobulin isotypes IgE, IgG, IgM by means of FACS and ELISA. Results: Murine naı¨ ve B cells could be activated in vitro with anti-CD40 mAb and IL-4 to undergo class switch recombination to IgE. The IgE levels in supernatants were increased. When adding BM-DC to this culture, this IgE isotype switch could be reversed. Interestingly, immature BM-DC pre-treated with high concentrations of SRM1650a particles lost the ability to inhibit the IgE isotype switch, although Diesel pre-treated BM-DC show no alteration neither in the expression of DC activation markers or MHC class II molecules. Conclusion: We conclude that particles can modulate immature DC to influence IgE isotype switch of B cells. As CD23, the low-

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affinity receptor for IgE (FceRII), has been proposed as an important regulator of IgE synthesis and is expressed on myeloid cells, it represents a promising candidate for this effect.

202 Increased FOXP3 expression in CD4CD25 regulatory T cells in severe asthma Ghraı¨ri, H1; Berraı¨s, A1; Hamzaoui, K2; Hamzaoui, A3 Tahar Maamouri Hospital Nabeul, Respiratory Diseases, Nabeul, Tunisia, 2Medecine University of Tunis, Respiratory Diseases, Ariana, Tunisia, 3A. Mami Hospital, Respiratory Diseases, Ariana, Tunisia

1

Naturally occurring CD4 1 CD25 1 regulatory T cells are shown to be engaged in the maintenance of self-tolerance and down regulation of autoimmune responses. Objective: The aim of this study was to investigate the Foxp3 expression by CD4CD25 regulatory T cells in severe asthmatics in comparison with mild asthmatics and a healthy control group. Methods: Purified CD4 1 T cells isolated from induced sputum were stained for surface antigen CD25. Two subpopulations were distinguished: CD4 1 CD25 high (with the highest level of CD25) and CD4 1 CD25low. We also analyzed by PCR Foxp3 mRNA which is specifically expressed on CD4 1 CD25 1 high regulatory T cells. Results: Induced sputum CD4 1 CD25 1 T cells were found to be more expanded in severe asthmatics. Foxp3, which is the most specific marker of CD4 1 CD25 high, was expressed at significantly higher levels in severe asthmatics than mild asthmatics and healthy controls. Conclusion: Severe asthma is characterized by expansion of CD4 1 CD25 1 T regulatory cells which may aim to reduce the inflammatory process.

203 Th17 in sensitised human lymphocytes ex vivo

ance corresponding to DNA-promoter gene interaction compared to normal controls. As IL-17E (IL-25) has currently been identified as mediator of tissue inflammation and allergy the aim of this paper was to investigate in how far this factor could be identified in circulating human lymphocytes ex vivo. Methods: Human PBMC from a group of 12 atopic/non-atopic volunteers (IgE 4500 IU, normal age matched- controls:o 50 IU) were stimulated by PHA and antiCD3 in 3-day cultures. Allergic patients were suffering from seasonal hay fever and chronic atopic eczema. Histamine was added 4 h post-plated. Cytokines were measured by the ELISA, STATs by westernblotting, and STAT-DNA-interaction by the electrophoretic mobility shift assay (EMSA) technique respectively. For EMSA the oligonucleotides STAT3 5´ GAT CCT TCT GGG AAT TCC TAG ATC 3´ (LI-COR Nebraska USA) were applied. PBMC differentiation was identified using cytoflowmetric/FACS-analyses. Results: In cells with increased CCR3-expression, STAT6 as well as phosphorylated (activated) aSTAT6 were stimulated after 1 day culture time in atopic patients inducing IL-4-expression and secretion. Western blots showed delayed STAT3 increase. No significant differences could be observed between the atopic and the non-atopic group. (P40.05, n 5 4) The STAT3 concentrations were in a good correlation to IL-17E. In the presence of histamine STAT3 was suppressed. In contrast, IL-17E did not respond to different histamine concentration added to both PHA and anti-CD3 stimulated sensitised cell cultures. However, histamine could influence STAT3 interaction with the lymphocyte promoter gene as it was proved using the EMSA technique. Conclusions: It can be concluded that Th17 showed downstream regulation which was specific in atopic pathology. Whether IL17E is a key cytokine in allergy disease must be elucidated in future work.

Kuzmenko, N1; Rosner, J2; Krieg, C2; Michel, I2; Schulz, N2; Weisser, H3; Khanferyan, R4; Diel, F2 1 2nd Krasnodar Hospital, Allergology, Krasnodar, Russian Federation, 2IUG and University of Applied Sciences HS Fulda, Biochemistry, Fulda, Germany, 3 Klinikum Fulda, Institut fu¨r Labormedizin, Fulda, Germany, 4Medical State University, Institute of Allergy and Asthma, Krasnodar, Russian Federation

Background: Human sensitised T-lymphocytes reveal increased STAT6/STAT1-bal-

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204 Systemic Th1- and Th2-associated chemokines during and after pregnancy in relation to maternal allergic disease Sandberg, M1; Ernerudh, J2; Berg, G3; Matthiesen, L3; Ekerfelt, C2; Nilsson, L1; Jenmalm, M1 1 Institution of Clinical and Experimental Medicine, Division of Pediatrics, Linko¨ping University, Linko¨ping, Sweden, 2Institution of Clinical and Experimental Medicine, Division of Clinical Immunology, Linko¨ping University, Linko¨ping, Sweden, 3Institution of Clinical and Experimental Medicine, Division of Obstetrics and Gynecology, Linko¨ping University, Linko¨ping, Sweden

Background: Allergic diseases and pregnancy are characterized by a Th2-skewed immunity. Prenatal exposure to a strong Th2-like environment could favour the development of Th2-like immune responses in the offspring. We have previously observed increased total IgE levels at gestational week 10 compared to 1 year after delivery in allergic, but not non-allergic, women, indicating that allergy is associated with a more pronounced Th2 deviation during pregnancy. The aim of our study is to characterize the systemic Th1- and Th2associated chemokine levels during pregnancy and after delivery among allergic and non-allergic women. Methods: The chemokine levels were determined by a Luminex assay at five occasions during pregnancy (at gestational week 10– 12, 15–16, 25, 35 and 39), and 2 and 12 months after delivery. Thirteen sensitised women with allergic symptoms and 30 nonsensitised women without allergic symptoms were included. Results: The levels of the Th2-associated chemokines CCL11 (eotaxin), CCL17 (thymus- and activation-regulated chemokine, TARC) and CCL22 (macrophage-derived chemokine, MDC) changed over the course of pregnancy with a similar pattern for the allergic and the non-allergic women. The CCL11 (eotaxin), CCL17 (TARC) and CCL22 (MDC) levels, with the lowest levels found before delivery, decreased during the second and third trimester and then increased postpartum. In contrast, increased levels of the Th1-associated chemokine CXCL10 (interferon-g inducible protein 10, IP-10) were noted at gestational week 39 compared to the first and second trimester. The CXCL10 (IP10) levels were higher in the first than in the second trimester in nonallergic, but not allergic, women. Conlusions: The decrease in the levels of the Th2-associated chemokines CCL11 (eotaxin), CCL17 (TARC) and CCL22 (MDC) and the increase of the Th1-associated chemokine CXCL10 (IP10) at gestational week 39 reflects the normal course of pregnancy, with an increased proinflammatory response close to delivery, and are not influenced by the allergic status of the mother. The absence of increased CXCL10 r 2008 The Authors Journal Compilation

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(IP10) levels at gestational week 10 versus 25 in the allergic women might be associated with the increased IgE responses we previously observed in the first trimester in this group.

205 Recent thymic emigrant peripheral T cells (CD45RO-CD31 1 ), peripheral memory effector T cells (CD45RO 1 CD31-) and regulatory T cells (CD25hiFoxP3 1 ) in 8 year old children Weber-Chrysochoou, C1; Britton, W2; Kemp, A3; Marks, G4; Fazekas de St Groth, B5 1 Woolcock Institute of Medical Research, Allergy Department, Sydney, Australia, 2Centenary Institute, Immunolgy Department, Sydney, Australia, 3Westmead Children’s Hospital, Allergy Department, Sydney, Australia, 4Woolcock Institute of Medical Research, Epidemiology Department, Sydney, Australia, 5 Centenary Institute, Immunology Department, Sydney, Australia

Background: CD4 1 CD25highFoxP3 1 T regulatory cells may have a role in the development of atopic diseases. However, information about these cells in 8 year old children is limited. Here we examine these sub-populations of CD4 1 cells in a selected birth cohort at age eight years. Methods: Subjects were members of the Childhood Asthma Prevention Study cohort, all of whom had a family history of asthma. We performed skin prick tests to inhaled and ingested allergens (SPT) and a clinical assessment at age 8 years. At the same time we collected peripheral blood samples from 144 members of the cohort at age 8 years and identified naı¨ ve and antigen experienced cells by using flow cytometry to detect CD45RO and CD31status. In addition CD4 1 CD25highFoxP3 1 T regulatory cells were identified using intracytoplasmic staining. The distribution of the proportion of these cells was positively skewed and the proportion was log-transformed for analysis. Atopy was defined as any allergen SPT 3 mm and current asthma was defined as doctor diagnosed asthma and wheeze in the past 12 months. Results: Among CD4 1 T cells there was a bimodal distribution of CD31 and CD45RO staining, with subjects clustered as either CD31 1 CD45RO- (naı¨ ve cells) or CD31CD45RO 1 (antigen-experienced cells). Fortyfive per cent of subjects had a pattern of r25% CD31-CD45RO 1 and 440% CD31 1 CD45RO- cells, which was considered antigen naı¨ ve, and 46% had 440% CD31CD45RO 1 cells and r25% CD31 1 CD45RO-, which was considered antigenexperienced. The remaining subjects were in an indeterminate group. The proportion of males and females did not differ between the naı¨ ve and antigen-experienced groups (P 5 0.9). The geometric mean proportion


of CD4 1 cells expressing CD25highFoxP3 1 was 3.68% (95% CI 3.38 to 4.00). Males had a higher proportion of regulatory T cells (4.2%), than females (3.2%, P 5 0.001). The proportion of regulatory T cells was not influenced by atopic (P 5 0.2) or current asthma status (P 5 0.9). Conclusion: We describe the patterns of naı¨ ve, antigen-experienced and regulatory cell populations in CD4 1 T cells of children at high risk for asthma. There were no differences in T reg populations between atopic and non atopic children, or asthmatic and non asthmatic children.

206 Effect on the number of bone marrow T regulatory cells in the absence of CD8 1 T cells in a mouse model of allergic inflammation Malmha¨ll, C; Lu, Y; Lo¨tvall, J; Bossios, A Go¨teborg University, Lung Pharmacology Group, Go¨teborg, Sweden

Background: CD8 1 T lymphocytes have recently been shown to be required for activation of T regulatory cells in the lung. Furthermore, they are also intricately involved in the regulation of bone marrow (BM) eosinophilopoiesis as lack of CD8 1 cells result in reduced number of eosinophils both in BM and in lung during allergen challenge in mice. However, the effect of CD8 1 cells on T regulatory cells in the BM has not yet been shown. The aim of this study was to determine whether a difference in the number of T regulatory cells occurs in the CD8 / mice compared to wild-type (WT) mice in the bone marrow after sensitisation or during allergen exposure. Method: CD8 / and WT mice were sensitised to ovalbumin (OVA) followed by exposure to OVA or PBS. During allergen or vehicle exposure the mice received bromodeoxyuridine (BrdU) which is incorporated to DNA during mitosis. Bone marrow was sampled 24 h after the final exposure. Leukocytes (CD45 1 ), T cells (CD3 1 , CD4 1 ), T regulatory cells (CD4 1 CD25 1 Foxp3 1 ) and newly produced Treg cells i.e. proliferating cells (CD4 1 CD25 1 Foxp3 1 BrdU 1 ) were determined by flow cytometric analysis. Results: No significant difference was found in the total number of BM cells in any group between CD8 / and WT mice and not in the number of CD45 1 or CD3 1 cells as per cent of total BM cells. The CD8 / have increased numbers of CD4 1 cells (per cent of total BM cells) compared to WT mice in the naı¨ ve (0.62 7 0.11 vs. 0.40 7 0.03; P 5 0.042) and OVA/PBS (0.46 7 0.08 vs. 0.20 7 0.03; P 5 0.014) group. Additionally, an increased number of CD4 1 CD25 1 Foxp3 1 cells (per cent of total BM cells) were found only in the OVA/ 91


PBS group (0.025 1 0.002 vs. 0.014 7 0.003; P 5 0.037). The allergen exposure did not induce any significant differences between CD8 / and WT mice. Finally, almost all T regulatory cells in the BM after exposure were newly produced. Conclusion: CD8 / mice have an increased number of T regulatory cells compared to WT mice after sensitisation but not during allergen exposure. This indicates that CD8 1 cells might play a role in the sensitisation phase in the bone marrow.

207 Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells Kimber, I1; Cumberbatch, M1; Maxwell, G2; Westmoreland, C2; Dearman, R1 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom, 2Unilever, Safety and Environment Assurance Centre, Bedford, United Kingdom

Background: Dendritic cells (DC) are required for the initiation of primary immune responses. Healthy skin contains two resident DC subsets: Langerhans’ cells (LC) in the epidermis and dermal DC. We have examined here the characteristics of immature DC derived from murine bone marrow (BMDC) as in vitro surrogates of LC. Responses to a panel of ligands for Tolllike receptors (TLR), pattern recognition receptors that evolved to detect conserved molecules derived from bacteria, viruses and other pathogenic organisms, have been investigated. Methods: BMDC cultured for 6 days in the presence of granulocyte/macrophage colonystimulating factor were stimulated for 24 h with ligands to TLR1-2 (Pam3Cys-Ser-(Lys)4 [PAM]), TLR2-6 (macrophage-activating lipopeptide-2 [MALP-2]; zymosan or peptidoglycan [PGN]), TLR3 (polyinosinic-polycytidylic acid [poly-(IC)]), TLR4 (lipopolysaccharide R515 [LPS]), TLR5 (flagellin), TLR7 (polyuridylic acid [poly-U]) and TLR9 (CpG ODN2395 [CpG]). DC activation was monitored by flow cytometric analysis of membrane marker expression and by analysis of culture supernatants for cytokine/chemokine release by protein bead array. Results: Ligands to TLR3 and TLR7 failed to activate BMDC. All other TLR ligands caused comparable elevations in expression of membrane markers (costimulatory molecules CD40 and CD86). PAM, MALP-2 and LPS induced high level expression of the proinflammatory cytokines interleukin (IL)1a and b, IL-6 and tumour necrosis factor a (TNF-a) and the proinflammatory chemokines macrophage inflammatory protein (MIP)-1a and b. Treatment with CpG was

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associated with a preferential type 1 cytokine and chemokine profile (IL-12p40 and p70 and interferon-g [IFN-g] inducible protein [IP-10] and monokine induced by IFN-g [MIG]). Interestingly, both zymosan and PGN stimulated the production of proinflammatory cytokines and chemokines and also very vigorous secretion of IL-10, but failed to provoke expression of type 1 chemokines IP-10 or MIG. In contrast, flagellin did not cause marked secretion by BMDC of cytokines or chemokines. Conclusions: These data are largely consistent with the reported TLR repertoire of freshly isolated murine LC. In addition, murine BMDC show selective responses to TLR-ligands with respect to general activation, with differentiated cytokine and chemokine patterns suggestive of potential priming for divergent immune responses.

208 IgE-associated allergic disease during infancy is associated with low levels of C-reactive protein at one year of age Abrahamsson, T1; Bjorksten, B2; Vaarala, O3; Jenmalm, M4 1 Department of clinical and experimental medicine, Division of Pediatrics, Linko¨ping, 2Karolinska Institut, Institute of Environmental Medicine, Stockholm, Sweden, 3National Public Health Institute, Department of Viral Diseases and Immunology, Helsinki, Finland, 4 Department of Clinical and Experimental Medicine, Division of Pediatrics, Linko¨ping, Sweden

Background: Stimulation of the innate immune system in the gut has been suggested to be important for the development of tolerance against allergens. C-reactive protein (CRP) may be regarded as a surrogate marker for the activity of innate immune cells. The aim of the study was relate CRP levels in blood with allergic disease and sensitisation in infancy, and to evaluate how CRP levels are affected by factors influencing the gut microflora. Methods: CRP levels were assessed by an ultrasensitive ELISA in plasma or serum samples from the umbilical cord (n 5 97) and at 6 (n 5 106), 12 (n 5 115) and 24 months (n 5 122) in infants completing a double-blind placebo-controlled allergy prevention trial with a daily intake of Lactobacillus reuteri (108 CFU/day) during the last month of gestation and through the first year of life. The infants were followed regarding development of allergic disease and sensitisation (a positive skin prick test and/or circulating IgE to allergens until two years of life). Sensitised infants with symptoms of any allergic disease were regarded to have an IgE-associated allergic disease. The prevalence of Lactobacillus reuteri in stool and breast milk samples was assessed employing conventional bacterial cultivation. Journal Compilation

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Results: Infants developing IgE-associated allergic disease in infancy had lower CRP levels than healthy infants at 12 months of age (P 5 0.04). Low CRP levels at 12 months of age were associated to eczema during infancy (P 5 0.03) and sensitisation at 24 months of age (P 5 0.007). Treatment with Lactobacillus reuteri did not affect CRP levels, although infants to mothers with Lactobacillus reuteri in colostrum and stool perinatally had higher CRP levels at birth (P 5 0.03) and at 24 months of age (P 5 0.002), respectively. Antibiotic supplementation during the first year of life was associated with lower CRP at 12 months (P 5 0.03), attendance to day care with higher CRP at 24 months (Po0.001), and breastfeeding at 12 months with lower CRP at 24 months of age (0.03). Conclusion: Low levels of CRP, a potential marker for innate immune activation, are associated with IgE-associated allergic disease. The CRP levels are influenced by factors which also modulate the gut microflora, but a direct relationship was not confirmed between the two.

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Moved to 393b.

210 Potentially harmful high IgE levels are restricted by a limited humoral IgE immunological memory Achatz-Straussberger, G1; Lamers, M2; Crameri, R3; Achatz, G1 1 University of Salzburg, Molecular Biology, Salzburg, Austria, 2MPI, Immunobiology, Freiburg, Germany, 3 SIAF, Davos, Switzerland

IgE is best known for its strong, unwanted effector functions in allergic diseases, ranging from mild local symptoms, to lifethreatening, systemic reactions. This underlines the potential hazard of high systemic IgE titers and the necessity of a tight biological control of IgE responses. Previously we described the limited expression of IgE as a membrane-bound, antigenreceptor-type molecule, finally restricting the recruitment of IgE-producing cells in the immune response. Here we present mouse strain KN1, carrying a chimeric a˚gene, consisting of the extracellular domains of the a˚-gene and the transmembrane and cytoplasmic domains of the a˜1-gene. KN1 mice show an increased serum IgE level, resulting from an elevated number of IgEsecreting cells. The secondary IgE response in KN1 mice is far more robust and plasmablasts mature much faster to cyclophosphamide-resistant long-lived plasma cells than in wild type mice.Furthermore, IgE-



antibody-secreting-cells with ‘‘a˜1-signaling history’’ migrate more efficiently towards the chemokine CXCL12 gradient guiding plasmablasts to plasma cell niches, than IgE-antibody-secreting- cells with wild type ‘‘a˚-signaling history’’. We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long-lived plasma cell pool and thus to humoral immunologic memory than IgG1 plasmablasts.

211 Individual IgE antibody affinity directly affects T-cell activation mediated by Facilitated Antigen Presentation (FAP) Willumsen, N; Wu¨rtzen, P; Holm, J; Christensen, L; Lund, K ALK-Abello´ A/S, Research, H^rsholm

Background: Activation of mast-cells and basophils responsible for the immediate allergic reaction as well as the activation of allergen-specific T-cells through facilitated antigen presentation (FAP) involve the formation of complexes between IgE, allergen and cell-surface expressed IgE-binding receptors FceRI and CD23, respectively. The influence of IgE antibody-affinity in FAP with respect to complex formation has recently been shown using humanized Derp2-specific recombinant IgE (rIgE). Still, how such differences are reflected by T-cell activation is uncertain. Methods: Complex-formation between rIgE, Derp2 and CD23 on CD23 1 -B-cells was measured directly by flow cytometry (FACS-FAP). The effect of FAP complex formation on T-cell activation was measured in parallel using the complex-bearing B-cells as APC for stimulation of Derp2specific T-cell lines. Complex formation was achieved by use of Derp2 specific rIgE antibodies each specific towards one of three non-overlapping epitopes of Der p 2, and engineered with regard to affinity. Results: In both assays complex formation and T-cell activation, respectively, were most efficient when combining three high-affinity rIgEs. Least efficient was the combination of three low-affinity rIgEs. Stepwise reduction of the avidity of the IgE-allergen complexes by changing individual IgE affinities from high- to medium- to low affinity was more clearly reflected in the T-cell proliferation assay than in FACS-FAP. Conclusion: Manipulating the conditions for FAP complex formation by changing individual IgE affinities is reflected in both FACS-FAP and in FAP-mediated T-cell activation. This strongly indicates that IgE affinity has a marked influence on the efficiency of antigen uptake and subsequent allergen-specific T-cell activation.

212 IL-31 increases serum IgE levels in mice Saito, S; Akiyama, N; Kohno, H; Ohno, Y Jikei Univ. Sch Med, Department of Mol Immunol., Inst. DNA Med, Tokyo, Japan

Background: IL-31 is a cytokine produced by Th2 cells, and overexpression in transgenic mice led to a phenotype closely resembling atopic dermatitis in human patients. Enhanced expression levels of IL31 correlate with IL-4 and IL-13 in atopic and allergic contact dermatitis. In contrast to other IL-31 transgenic (IL-31 Tg) mice, our established IL-31 Tg mice showed increased serum IgE levels, compared with non transgenic (non Tg) mice from littermate. To investigate the association of IL-31 with serum IgE levels, recombinant murine IL-31 (IL-31) was administered into mice. Methods: C57BL/6, BALB/c and IL-4 receptor-deficient BALB/c (IL-4RKO) mice were subcutaneously injected with IL-31 or PBS as a control everyday for 2 weeks. Serum IgE levels of mice were measured by two site capture assay. To investigate the producing activity of IL-4, IL-5, IL-13 and IFN-g, spleen cells from each mice were cultured for 5 days, then culture supernatants were harvested and tested for the cytokine activity by sandwich ELISA. Results: 1) Serum IgE levels in mice injected with IL-31. Serum IgE levels of the IL-31 injected C57BL/6 and BALB/c mice were significantly higher than those of PBS injected mice. On the other hand, no significant increase of serum IgE levels was observed in the IL-31 injected IL-4RKO mice. 2) Cytokine profile in IL-31 injected mice. Significant increase of IL-13 and IL-5 production was found in the IL-31 injected C57BL/6 mice, compared with PBS injected control mice. In contrast, no significant differences on IFN-g producing activity were found between IL-31 and PBS injected mice, whereas concentration of IL-4 was below the detectable level in both group. Conclusion: Increased serum IgE levels and IL-13 production from spleen cells were observed in IL-31 treated mice. These results suggest that IL-31 is involved in the enhancement of serum IgE levels via induction of IL-13 expression.

a bidirectional communication can influence responsiveness of both cell types. Considerable evidence exists that allergic skin inflammation is dependent on functional sensory nerves. In this study, we investigated the role of the receptor for the neuropeptide substance P, the neurokinin-1 receptor (NK1R), in the induction of immediate hypersensitivity responses elicited by local antigen challenge of IgE- or Ig free light chain-sensitised mice. Methods: BALB/c or NK1R – deficient mice (backcrossed to BALB/c; obtained from N. Gerard, Boston) were passively sensitised by an intravenous injection with trinitrophenol-specific IgE or trinitrophenol-specific Ig free light chains. Control mice received PBS. At 30 min after sensitisation, mice were topically challenged with 0.8% picryl chloride in acetone/olive oil and 2 h later the increase in ear thickness was measured with an engineer’s micrometer. NK-1 receptor antagonists (RP 67589 (10 nmoles/mouse) or SR 140333 (40 nmoles/mouse)) were administered intravenously at 60 min before challenge. Results: Topical antigen challenge of IgEor Ig free light chain-sensitised mice resulted in a significant increase in ear swelling at 2 h after challenge. However, in NK1R-deficient animals, this antigen-induced cutaneous edema formation was completely absent in both IgE- and Ig free light chainsensitised animals. Also pretreatment with the specific NK1R antagonists RP67589 or SR140333 completely inhibited the ear swelling response in IgE- or light chainsensitised and hapten challenged BALB/c mice. Conclusion: This study shows that local edema formation by IgE-mediated or Ig free light chain-induced mast cell activation is dependent on the presence of functional neurokinin-1 receptors. Because mast cells and sensory nerves can express both the NK1R and substance P, mast cell activation could induce/influence stimulation of sensory cells and vice versa. In addition, released neuropeptides could stimulate NK1R on endothelial cells to further enhance vascular permeability. Moreover, this study suggests that the mechanism of Ig free light chain-induced skin inflammation shows parallels with the IgE-mediated response.

213 Ig free light chain- and IgE-mediated skin inflammation is neurokinin-1 receptordependent Kool, M; Kraneveld, A; Blokhuis, B; Redegeld, F Faculty of Science, Utrecht University, Pharmacology and Pathophysiology, Utrecht, the Netherlands

Background: Mast cells and sensory nerves are closely associated in various tissues and r 2008 The Authors Journal Compilation

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214 Evaluation of Cytomegalovirus (CMV)specific immunoglobulins as serologic markers for CMV infection in HIV infected patients Mehrkhani, F1; Jabbari, H2; Fattahi, F3; Kourorian, Z3; Jam, S1; Moradmand Badie, B1; Mohraz, M1 1 Iranian Research Center for HIV/AIDS, Medical Sciences/ University of Tehran, Imam Khomeini Hospital, Tehran, Islamic Republic of Iran, 2Center for Environmental Research, Medical Sciences/University of Tehran, Tehran, Islamic Republic of Iran, 3Immunology, Asthma and Allergy Research Institute, Medical Sciences/ University of Tehran, Children Medical Center, Tehran, Islamic Republic of Iran

Background: CMV is an important opportunistic infection in immunocompromised patients. This virus is causative of significant morbidity and mortality among patients with advanced human immunodeficiency virus (HIV) infection. We performed this study to determine the seroprevalence of CMV infection in HIV positive patients in a main referral hospital in Tehran, Iran. Methods: Serum samples from 201 HIV positive patients were collected for antiCMV antibodies (IgG, IgM) between March 2004 and March 2005 by using conventional ELISA kits. An antibody 41.1 Iu/mL was considered positive. Results: The seroprevalence of CMV infection was 94% in HIV positive patients. The maximum prevalence of CMV was seen in patients with unsafe sex and IDUs (100%). The minimum prevalence of CMV was seen in patients with other HIV transmission risk factors like (tattoo, vertical transmission, y) (72.7%) (P 5 0.09). Prevalence of CMV was much higher in patients with low socioeconomic status and low level of education. Mean age of CMV positive patients was 36.5 years (SD 5 9.1 SE 5 0.64) and prevalence of CMV ranged from 50% in (p10) years age group to 100% in (51–60) years age group (P 5 0.001). We assessed the prevalence of CMV in HIV patients by CD4 count 83% of patients with CD4o100 were CMV seropositive. In other groups the prevalence of CMV was approximately the same (P40.05). Conclusion: Our study showed that a significantly high prevalence of CMV in HIV positive patients in Iran. By increasing the level of education and socioeconomic status the prevalence of CMV decrease. And also prevalence of CMV increases by age.

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215 Impaired Th17 immune response in patients with chronic mucocutaneous candidiasis Foerster, S1; Eyerich, K1; Rombold, S2; Hofmann, H2; Ring, J2; Behrendt, H1; Traidl-Hoffmann, C1 1 Technical University Munich, ZAUM-Center for Allergy and Environment, Munich, Germany, 2Technical University Munich, Department of Dermatology and Allergy, Munich, Germany

Background: Chronic mucocutaneous candidiasis (CMC) constitutes a selective inability to clear infection with the yeast Candida albicans resulting in persistent debilitating inflammation of skin, nails, and mucous membranes. To date the underlying defect is unknown. Only recently, Th17 cells have been reported to be involved in clearing Candida infections. The aim of this study was to elucidate possible dysfunctions in cellular immune reactions in patients suffering from chronic Candida infections, focusing on neutrophil functions on the one, and T cell proliferation and cytokine production on the other hand, with a special emphasis on Th17 cells. Methods: Patients with chronic mucocutaneous candidiasis (CMC, n 5 4), patients suffering from chronic Candida paronychia (n 5 2) and healthy volunteers (n 5 7) were enrolled into the study. Neutrophil chemotaxis was assessed by transwell migration assay, MPO release by ELISA. In order to characterise the mechanisms of T cell immune response to Candida, we analysed in vitro T cell proliferation capacity by thymidine incorporation and cytokine secretion into supernatants by ELISA. Results: Neither neutrophil migration nor MPO release differed between CMC patients and healthy controls. The specific proliferation to Candida was heterogenous, but overall higher in CMC patients compared to controls. However, Candida-specific IFN-g production and secretion of the Th17 associated cytokines IL-17 and IL-22 was significantly reduced in CMC patients. Production of IL-17 in CMC patients was also diminished after mitogen stimulation. Interestingly, Candida paronychia patients showed a much higher secretion of these cytokines than both CMC patients and matched controls. Cytokines determining the differentiation of human Th17 cells (IL-1b and IL-6) and another Th17 cytokine, IL-21, were not modified in CMC patients. Conclusion: In summary this study underlines the importance of IL-17 for the clearance of Candida infections. Furthermore our data suggest that an impaired Th17 response seems to be in part responsible for the pathogenesis of CMC.

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216 Detection and characterisation of exosomes of monocyte and human proinflammatory macrophage type I origin Sjo¨strand, M; La¨sser, C; Valadi, H; Lightfoot, A; Malmer, A; Bossios, A; Lo¨tvall, J Go¨teborg University, Internal Medicine, Lung Pharmacology group, Go¨teborg

Background: Exosomes are 40–90 nm membrane vesicles of endocytic origin released by many different cells including dendritic cells, lymphocytes, epithelial cells and mast cells. The function of exosomes is not fully known, but they are proposed to take part in communication between cells through antigen presentation and gene transfer. Recently we have documented that mast cell derived exosomes contain RNA. The aim of this study was to isolate, identify and characterize exosomes from human monocytes and human type I macrophages. Methods: Monocytes from healthy buffy coat were obtained by negative magnetic depletion. The monocytes were cultured for 48 h with and without stimulation with Calcium ionophore 1 mg/mL (CI) and 2 ng/ mL phorbol-12-myristate-13-acetate (PMA). To obtain pro-inflammatory macrophages, the monocytes were cultured for 6 days with 10 ng/mL of human recombinant granulocyte-monocyte colony stimulating factor (GM-CSF) and 24h before harvest half of the macrophage cultures were stimulated with CI/PMA. Flow cytometric (FACS) analysis and cytospin examinations were done for the enrichment and viability of the cells. Exosomes released in the culture media were isolated by repeated centrifugations and filtration steps. Exosomes were detected using FACS analysis by conjugating exosomes to Anti-HLA class II dynabeads followed by additions of the anti-tetraspanins CD9, CD63 and CD81 forming a exosome-microbead complex. Nucleic acids were extracted and detected using spectrophotometry and agarose gel electrophoresis. Results: Eighty to ninety-six per cent of the enriched monocyte population was viable CD14 1 monocytes that were used in the monocyte/macrophage cultures. The amount of protein in secreted exosomes did not varied between stimulated monocytes and macrophages however unstimulated monocytes contained less proteins then macrophages. FACS analysis confirmed the present of the tetraspanins CD9, CD63 and CD81 on exosomes and that stimulation with CI/PMA upregulated the tetraspanins on monocyte exosomes but not on the Type I macrophages. RNA was detected in both stimulated and unstimulated monocyte/ macrophage exosomes.



Conclusion: Both monocytes and type I macrophages can secrete exosomes containing RNA. The results also show that


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proinflammatory mononuclear cells have the ability to upregulate their exosome production. These results can provide in-


formation regarding the communication between cells through exosomes in inflammatory disease.

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Poster Discussion Session 4 – Molecular Allergens Defining a New Approach to Allergy Diagnosis

Poster Discussion Session 4 Molecular Allergens Defining a New Approach to Allergy Diagnosis 217 Native Art v 1: Evaluation of a molecular allergen for mugwort pollen allergy Sen, M1; Huynh, K2; Evangelista, R3; Jaggi, K1; Davoudzadeh, D1; Palazzo, P4; Zennaro, D4; Mari, A4; Hovanec-Burns, D2; Banik, U2 1 Siemens Healthcare Diagnostics, Biochemistry, Los Angeles, United States, 2Siemens Healthcare Diagnostics, Allergy Diagnostics, Los Angeles, United States, 3Siemens Healthcare Diagnostics, Chemistry, Los Angeles, United States, 4Center for Clinical and Experimental Allergology, IDI-IRCCS, Rome, Italy

Rationale: Mugwort (Artemisia vulgaris) pollen is considered in many parts of the world to be the most important cause of polinosis. The molecular allergen Art v 1 from mugwort weed pollen is the major allergen. The posttranslational glycosylation of Art v 1 may be important in the formation of epitopes recognized by IgE antibodies. To help elucidate the variable degrees of cosensitisation to allergens and corecognition by specific IgE (sIgE), we purified native Art v 1 (nArt v 1) for evaluation as a molecular allergen to detect sIgE to weed pollen using the IMMULITEs 2000 3gAllergyt assay. Methods: nArt v 1 was purchased commercially and tested in the IMMULITE 2000 3gAllergy assay. Mugwort extract (W6) was used as a reference. Patient sera, selected on the basis of positive clinical history for mugwort pollen allergy and/or skin test results, were evaluated for immunoreactivity to mugwort extract and nArt v 1 using the IMMULITE 2000 3gAllergy assay. Standardized inhibition studies were performed by preincubating patient serum with the nArt v 1 and mugwort extract. Results: Purified nArt v 1 appeared on SDS-PAGE as a major band (MW 5 B24 kDa). MALD I-TOF spectroscopy confirmed the purity and heterogeneity of nArt v 1. One hundred per cent of patient sera with sIgE to mugwort allergen also demonstrated sIgE to nArt v 1. Competitive inhibition studies with nArt v 1 showed greater than 90% inhibition. Conclusions: Detection of mugwort pollen sensitisation in patients positive for mugwort extract can be achieved using the molecular allergen nArt v 1. The data suggest that nArt v 1 may be a valuable tool to aid diagnosis of mugwort pollen allergy.Under development

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218 Affinity purification of native cockroach allergen Bla g 7 for component-resolved diagnostics

219 Gluten allergy testing – usefulness of specific IgE to gluten and of skin prick test with the extract of gliadins

Everberg, H; Nyga˚rd, K; Unger, E; Ho¨gbom, E; Move´rare, R; Brostedt, P Phadia AB, R&D, Uppsala, Sweden

Bilo, B1; Dyga, W1; Obtulowicz, K1; Waga, J2; Zientarski, J2 1 Jagiellonian University, Medical College, Department of Clinical and Environmental Allergology, Krakow, Poland, 2 Plant Breeding and Acclimatization Institute, Department of Cereals Breeding and Quality Evaluation, Krakow, Poland

Background: Tropomyosin is suggested to be an important cross-reactive allergen among a number of invertebrates such as shrimp, lobster, crab, house dust mite and cockroach. The aim of this study was to use a monoclonal antibody (mAb) developed against shrimp tropomyosin, Pen a 1, for affinity purification of native (n) cockroach tropomyosin, Bla g 7, and to produce an experimental nBla g 7 ImmunoCAPs to be used for component-resolved diagnostics in future applications. Methods: The mAb against recombinant shrimp tropomyosin (a-Pen a 1) was developed and characterized in house. nBla g 7 was purified by a-Pen a 1 affinity chromatography from a heat extracted cockroach protein suspension. Purified nBla g 7 was biochemically characterized by size exclusion chromatography (SEC), SDS-PAGE, immunoblotting, and mass spectrometry (MS). Immunoreactivity was analyzed by coupling of the purified nBla g 7 to ImmunoCAP with subsequent analysis of 68 serum samples from cockroach sensitised individuals. Results: The purity of nBla g 7 was assessed to be 495% from silver-stained SDS-PAGE and SEC. The identity was verified by MSanalysis. A 74% prevalence of IgE reactivity to nBla g 7 was observed by ImmunoCAP analysis which correlated well with IgE immunoblotting. Conclusion: This study demonstrated that a mAb originally developed against the homologous allergen Pen a 1 were successfully used to produce a highly purified preparation of nBla g 7. It was also shown that heat extraction of cockroach proteins provided a source of nBla g 7 which eliminated protease activity and thus rendered affinity purification with mAbs possible. Furthermore, Bla g 7 was concluded to be a major allergen with a high prevalence of IgE reactivity among cockroach sensitised patients as compared to other known cockroach allergens.

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Background: Wheat proteins can cause different types of allergic or immunologic reactions. The main allergens in a typical allergy to wheat flour are albumins and globulins. The allergy to gluten (gliadins) is not frequently diagnosed, which may be due to a limited availability of commercial skin test with gluten. So far the only method for the diagnosis is specific IgE measurement. The aim of the study was to asses the frequency of gluten allergy in patients allergic to wheat flour and in a larger group of screened patients. Methods: Specific IgE was measured by fluoroimmunoenzymatic assay (UniCap100, Phadia). Albumins and globulins were eluted from wheat flour, then the flour underwent alcoholic extraction. This protein extract containing gliadins (GEx) was lyophilized, and its 500 mg/mL solution (0.01 M lactic acid, 20% glycerol) was used for skin prick tests (SPT). In 20 patients with formerly diagnosed wheat flour allergy (symptoms, skin tests and sIgE) sIgE to gluten was measured and in 7 of them SPT with GEx was performed. Further 200 patients diagnosed for suspected allergy (regardless of symptoms) were screened by SPT with GEx. Results: In 9 out of 20 patients with wheat allergy the level of sIgE to gluten was above 0.7 kUA/L. SPT with GEx was positive in 5 patients (sIgE: 0.64–16.7 kUA/L) and negative in 2 (sIgE: o0.35 and 2.12 kUA/L). Only 4 (out of 20) reported the occurrence of symptoms after ingestion of baked wheat (3 skin reactions, 1 anaphylactic – after wholegrain cereals) – all of them had increased levels of sIgE and 3 of them had positive SPT with GEx. SPT was positive in 9 (4,5%) out of 200 patients screened with GEx (mean diameter 3 mm or more) – only 5 of them had also positive skin prick test with standard wheat flour extract (Allergopharma). Four out of these 9 patients reported the occurrence of symptoms after baked flour ingestion (skin, abdominal discomfort,



anaphylactic reaction) and other 4 during the contact with raw flour (asthma, rhinitis). Conclusion: IgE-mediated reaction to gluten appears to be involved in wheat flour allergy in about half of patients. At the same time positive SPT results for gliadins may be observed in subjects without allergy to other flour allergens. Relatively high prevalence of positive SPT in a random sample of allergic patients suggests the need of further research on the epidemiology of gliadin allergy and on the clinical diagnostic usefulness of the extract used in the study.

220 Evaluation of native major peach allergen nPru p 3: a lipid transfer protein for allergy diagnosis Maranõń, M1; Huynh, K2; Poladian, M3; Drummond, A3; Lopez, M2; Jaggi, K1; Davoudzadeh, D1; Zaffiro, A4; Quaratino, D4; Mari, A4; Hovanec-Burns, D2; Banik, U2 1 Siemens Healthcare Diagnostics, Biochemistry, Los Angeles, United States, 2Siemens Healthcare Diagnostics, Allergy Diagnostics, Los Angeles, United States, 3Siemens Healthcare Diagnostics, Molecular Biology, Los Angeles, United States, 4Center for Clinical and Experimental Allergology, IDI-IRCCS, Rome, Italy

Background: Pru p 3, the major allergen of peach, a Rosaceae fruit, is known to be a primary sensitizer and elicitor of allergic reactions. Thus, this nonspecific lipid transfer protein (nsLTP) is a relevant allergenic molecule. We purified and characterized native Pru p 3 (nPru p 3) protein from peach extract for evaluation as a tool for LTP-mediated allergy diagnosis at the molecular level. Method: nPru p 3 was isolated from peach (Prunus persica) extract and further purified to homogeneity using chelating chromatography. The purified protein was tested using the IMMULITE 2000 3gAllergyt assay. Samples from patients known to be allergic to Rosaceae fruit were tested in the assay for specific IgE (sIgE) reactivity to peach and Pru p 3. Additionally, patient sera, selected on the basis of positive clinical histories and/or positive skin test results, were also evaluated for nPru p 3 sIgE using the IMMULITE 2000 3gAllergyt assay. Standardized competitive inhibition studies were performed by preincubating the sera with nPru p 3. Results: Purity, identity and homogeneity of nPru p 3 (apparent MW 5 B9 kDa) were characterized by SDS-PAGE, mass spectrometry and Western blot analysis. Sera of patients allergic to Rosaceae fruit were evaluated for sIgE reactivity to nPru p 3 using the IMMULITE 2000 3gAllergyt assay. Results showed 100% agreement with positive samples. Greater than 85% inhibition was achieved using nPru p 3 as inhibitor.


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Conclusions: Results of this study suggest that the major peach allergen nPru p 3 is a highly sensitive diagnostic tool and support its use by physicians for the diagnosis of severe systemic LTP-mediated allergy. Under development

221 Benefit of the basophil activation test in predicting the oral food challenge outcome with cow milk Rubio, A1; Vivinus-Nebot, M2; Bourrier, T3; Saggio, B4; Albertini, M1; Bernard, A2 1 University Hospital of Nice and Medical University of Nice, Pediatric Department, Nice, France, 2University Hospital of Nice and Medical University of Nice, Immunology Departament, Nice, France, 3University Hospital of Nice, Pediatric and Allergy, Nice, France, 4 Lenval Children’s Fondation, Biology, Nice, France

Background: IgE-mediated cow milk allergy requires a strict diet excluding all cow milk proteins. As it usually disappears in early childhood, children must be re-evaluated regularly to detect its resolution. However no biological test can predict with certainty the persistence or resolution of the allergy. The gold standard is the oral provocation test, which must be carefully discussed considering the risk, time and expense it entails. Objective: The purpose of this investigation was to evaluate the benefit of the basophil activation test (BAT), a recent functional technique for the exploration of allergy, in predicting the child’s reaction to the milk challenge. We compared it with the benefit of the IgE levels for the same purpose. Methods: Thirty-seven consecutive children (average age: 3 years 1 month) admitted for a cow milk provocation test were included. Specific IgE levels had been measured in the previous month. A blood sample was taken for the BAT before the challenge. The degree of basophil activation in response to milk proteins was measured by flow cytometry by the increase in the cell surface expression of CD63. The sensitivity, specificity, positive and negative predictive values were calculated for the BAT and the IgE results. A Khi2 test and the correlation factor between the BAT and the oral provocation challenge, and between the IgE values and the oral challenge, were calculated. Results: Eight children had a clinical reaction during the oral challenge. The BAT was interpretable for 89% of the children, a rate very close to those reported in adults, in spite of the young age of the cohort. It was positive for all the children reactive to the milk challenge. We found a 97% concordance with the oral challenge outcome, with a Khi2(1) of 27.764 (Po0.001) and a correlation factor of 0.917. These scores were higher than those obtained with the


specific IgE values, whichever IgE-value cutoff was chosen. The sensitivity of the BAT in this pediatric cohort was 100%, the specificity 96%, the positive predictive value 88% and a negative predictive value 100%. Conclusion: This study enables us to consider the BAT as a very valuable biological test in the management of cow milk proteins allergy in children. It seems to be the most accurate biological tool in helping to determine whether an oral challenge can be undertaken for a child, avoiding both dangerous positive challenges and the exclusion of cow milk proteins from the diet longer than necessary.

222 Can decision points be generated in open food challenges? O’B. Hourihane, J; Lafford, S; Daly, D; DunnGalvin, A Ireland

Background: Decision points or cut off values of skin prick test (SPT) or specific IgE (spIgE) that predict clinical reactivity have been generated in tertiary centres using the gold standard of double blind food challenge (DBFC), using highly selected populations of patients. Objective: To determine if decision points can be generated using open food challenge (OFC). Methods: We reviewed OFCs for egg and peanut performed in Ireland’s only pediatric allergy clinic. Children were selected for challenge if SPT and/or spIgE were below the DBFC-generated decision points in the literature. Results: Fifty-eight challenges (20 peanut; 38 egg) were available for analysis. Although the cohort was small, no statistical assumptions were violated. SPT 4 mm had a positive predictive value of 92% (95% CI, 66%–98%) for OFC with peanut and 89% (95%CI, 75%–99%) with egg. The area under the receiver operating curve (ROC) was 0.84. The addition of age did not improve the model. SpIgE had a markedly poorer predictive ability than SPT. The probability of clinical allergy for a specific SPT wheal diameter is given as: 1/ [e1.61 5 (0.70 wheal diameter) 1 1]. Conclusion: SPT of at least 4 mm has a high predictive value of OFC outcome for peanut and egg, which is comparable to data from DBFC. If validated in prospective comparison with DBFC, this approach may allow the wider use of decision points in a manner that is not currently possible, as many centres do not have the capability or confidence to do DBFCs on every patient.

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223 In vivo reactivity to grass pollen in patients with allergic rhinoconjunctivitis in correlation to specific serum IgE antibody levels Huss-Marp, J1; Darsow, U2; Pfab, F1; Weichenmeier, I1; Petersson, C3; Borres, M3; Ring, J2; Behrendt, H1 1 Helmholtz Center Munich/Technical University Munich, ZAUM – Center for Allergy and Environment, Munich, Germany, 2Technical University, Department of Dermatology and Allergy Biederstein, Munich, Germany, 3 Phadia AB, Uppsala

Background: Dose-response curves between the severity of clinical symptoms and specific serum IgE antibody levels to both indoor and food allergens have been established in allergic individuals in previous investigations. We questioned whether this also holds true for outdoor allergens e.g. grass pollen allergens. Methods: During the grass pollen season in 2006 and 2007 we recruited 100 patients with allergic rhinoconjunctivitis (ARC) and allergy to timothy grass pollen as well as 40 healthy controls. We determined specific serum IgE antibodies to Timothy grass pollen (ImmunoCAPt, Phadia AB, Uppsala) at three time points during one year (June/July; October; December) to investigate the correlation between IgE antibody levels and threshold in vivo reactivity which was assessed by conjunctival and nasal provocation tests with grass pollen extracts at different concentrations (Phleum pratense, HAL Allergy, Duesseldorf, Germany). Furthermore skin prick tests were performed. Additionally, the patients scored their symptoms with visual analog scales and exhaled nitric oxide was analysed as parameter of inflammatory lung reactions. The investigations were paralleled by grass pollen counts using a Burkard trap performed at the University Campus Biederstein, Munich (o50 m distant from the laboratory, 1.80 m above the ground). Results: Preliminary results from 65 patients with ARC revealed a significant relationship between specific IgE concentration in serum and the titrated level of the skin prick test (P 5 0.0034, JonckheereTerpstas trend test). No correlation between specific serum IgE to timothy grass and thresholds of nasal provocation tests were detected. However, for conjunctival provocation a trend towards a correlation was seen in the preliminary data analysis (P 5 0.0602). Conclusion: Measurement of specific serum IgE levels seems to accurately predict the in vivo reactivity with regard to skin prick test in patients with ARC to grass pollen allergen. The preliminary results indicate a good correlation between indicators of sensitisation, but not between serum IgE levels and threshold allergen concentrations

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that induce nasal symptoms. Further data analysis concerning conjunctival and nasal provocation tests and symptom scores in a greater number of patients is in progress.

224 The wheal-size of skin prick tests does not correlate with specific IgE levels in patients with type-I allergy Wohrl, S1; Binder, M1; Stingl, G1; Prinz, M2 1 Medical University of Vienna, Dep Dermatology, Vienna, Austria, 2Medical University of Vienna, Core unit for Medical Statistics and Informatics, Vienna, Austria

Background: The wheal-size of skin prick tests (SPTs) or allergen-specific IgE serum levels (sIgE) are often regarded as surrogate markers for the symptom severity of type-1 allergic diseases. We investigated whether the wheal-sizes of SPTs with allergen were correlated with sIgE levels in patients suffering from allergic rhinoconjunctivitis (ARC) and whether they were influenced by symptom severity. Methods: We re-analysed a data set of 126 patients of whom 120 had been published previously (Wo¨hrl S. Allergy 2006 : 61;633– 9). Briefly, SPTs and measurement of sIgE had been performed in 126 subjects with ARC (83.3%) and/or bronchial asthma (34.9%) and/or atopic dermatitis (5.6%). There were 34 controls and 84 with a history matching a sensitisation to at least 1 of 5 the 5 study-allergens cat, dust mite, birch, grass or mugwort pollen labelled ‘‘allergic’’. Another 8 patients were labelled ‘‘irrelevant positive’’ with a positive SPT but without an allergen-specific history and added to the controls. SPTs had been measured in mm2 with a semi-automated system as described previously (Wo¨hrl S. Exp Dermatol 2006 : 15,119–24). Disease activity in allergic patients had been classified according to the ARIA recommendations.We used SPSS 15.0 for the data-analysis with scatter and box plots and calculated Pearson’s correlation coefficient (R2) for the area of the SPT and the sIgE of each allergen. We assessed the differences of means with the 2-sided T-test. Statistical significance was assumed at Po 0.05. Results: Box plot analyses revealed that the symptom severity did neither influence the outcome of the wheal-sizes nor the sIgE levels (2-sided t-test, P40.05) except for the subgroup of cat-allergic patients (29.8 mm2 vs. 13.1 mm2, Po0.05). The sIgE did not differ in both groups. None of the allergenspecific wheal-sizes were correlated significantly with the sIgE level (R2, P40.05) in the allergic patients except for house dust mite-allergic patients where there was a weak correlation (R2 0.523, Po0.05). Conclusion: The sIgE levels are hardly correlated with the wheal-sizes of SPTs Journal Compilation

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and both are hardly influenced by the symptom-severity in individuals suffering from ARC. Hence, defining specific in vivo or in vitro cut-off levels for clinical tests may be without clinical relevance and should not be used as a surrogate marker for symptom severity in patients with ARC.

225 Are there differences in the allergen contents of birch, mould and house dust mite skin prick test products from different manufactures that could influence allergy diagnosis? Meno, K1; Giselsson, A1; Grosch, K2; Hansen, G1; Jimeno, L3; Larsen, G1; Schou, K4; Ipsen, H1 1 ALK-Abello´, Allergen Chemistry & Biotechnology, H^rsholm, Denmark, 2ALK-Abello´, Analytical Method Development, H^rsholm, Denmark, 3ALK-Abello´, Biochemical Development, Madrid, Spain, 4ALK-Abello´, Aseptic Production, H^rsholm, Denmark

Background: The objective of skin prick testing (SPT) is to perform quick, safe and allergen-specific diagnosing of allergic patients. This requires standardized allergen extracts that provide high sensitivity and specificity, as the reproducibility and efficacy of SPT products depend on their content of major allergens. The purpose of this study was to characterize the allergen content of mould (Alt a), birch (Bet v) and house dust mite (Der p) SPT products used in Europe as well as the content of preservative (phenol) and stabilizing agent (glycerol). Methods: Mould, birch and house dust mite SPT products from six manufactures were included in the study. The total allergen content was characterized using IgE inhibition studies, whereas the major allergen content was quantified using ELISA and single radial immuno-diffusion. Furthermore, the total protein content was visualised using SDS-PAGE. Finally, the concentrations of phenol and glycerol were determined by HPLC and titration, respectively. Results: The total IgE binding potency was shown by IgE inhibition to vary by a factor of 25 between the most and least potent mould products. The corresponding number was only 2 for the birch products. The house dust mite products yielded non-parallel inhibition curves indicating different allergen compositions. On the major allergen level it was shown by ELISA that the concentration of Alt a 1 in the mould products varied by a factor of 44 whereas the Bet v 1 level in the birch products again varied by a factor of only 2. The Der p 1 and Der p 2 concentrations varied by a factor of 12 and 21, respectively, and the Der p 1/Der p 2 ratio was furthermore very different in the different products. All products contained between 46 and 52% glycerol except the products from one manufacturer, which



contained 42% glycerol. The stated phenol concentrations in the products vary by a factor of 2.5, and the experimentally determined concentrations were significantly different from the stated concentrations for several of the products. Conclusion: This study shows that there are significant differences between the allergen contents in both mould, birch and house dust mite SPT products from six different manufactures. This is of major importance since it suggests that the diagnostic results obtained clinically using these SPT products may be influenced by the brand of the SPT used. The birch SPT products had the most similar allergen composition.

226 Allergen content of grass-pollen preparations for skin prick test and for sublingual immunotherapy Sander, I; Fleischer, C; Meurer, U; Bru¨ning, T; RaulfHeimsoth, M BGFA – Research Institute of Occupational Medicine, German Social Accident Insurance, Ruhr University Bochum, Bochum, Germany

Background: The allergen content of diagnostics and immunotherapeutics is crucial for an effective diagnosis and treatment. It was the aim of the study to quantify and compare the allergen content of different grass pollen preparations for skin prick test and sublingual immunotherapy. Methods: Five skin prick test (SPT) solutions and ten sublingual immunotherapeutics were analysed concerning protein and allergen concentration by Bradford assay, inhibition of IgE-binding to Phleum pratense ImmunoCAPs (Phadia, Sweden), and content of the main allergen Phl p 5 by two-sited enzyme immunoassay (Indoor Biotechnologies, UK). In addition, the grass pollen preparations were compared in SDS-polyacrylamid gelelectrophoresis (SDS-PAGE) and immunoblots. Results: Protein concentrations of SPT solutions ranged from 15 to 427 mg/mL, and Phl p 5 concentrations ranged from 0.15 to 18.3 mg/mL. The ranking of SPT solutions concerning Phl p 5 content and IgE inhibition capacity was the same, the ranking of protein and allergen content was closely correlated (r 5 0.9). Protein content of the maintenance doses of immunotheurapeutics ranged from 5 to 153 mg, Phl p 5 content ranged from 0.2 to 21.6 mg. IgE inhibition capacity of the maintainance doses was closely correlated to their Phl p 5 and protein content. SDS-PAGE and immunoblots confirmed the differences in protein and allergen content. Conclusion: Grass pollen preparations for SPT and sublingual immunotherapy varied greatly concerning protein and allergen r 2008 The Authors Journal Compilation

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content. The impact of these differences on diagnosis and therapy has to be examined carefully.

227 IgE profiles of Bermuda grass pollen sensitised patients evaluated by Phleum pratense allergens Phl p 1, 2, 4, 5, 6, 7, 11, 12 Monasterolo, G1; ROSSI, R2 SS Annunziata Hospita, Laboratorio Analisi, Savigliano, Italy, 2Rete di Allergologia Regione Piemonte, Allergy Unit, Cuneo, Italy

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Background: Despite the difference in geographical dominance of certain grasses, a high degree of allergenic similarity or crossreactivity between Bermuda grass pollen (BGP) and timothy grass pollen (TGP) has been previously demonstrated. The aim of the present study was to ascertain the sensitisation to TGP in four-hundred eleven patients known for their reactivity to BGP extracts by analysing their reactivity to crude timothy pollen extract and timothy pollen purified allergens, establishing their specific IgE-profiles. Methods: Using the immunoenzymatic CAP method we evaluated IgE-specific antibodies for BGP- and TGP- extracts and the timothy recombinant (r) and natural (n) allergens rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12. Results: BGP-IgE positive patients (median 5 8.0 kUA/L, 2.8–22.2 kUA/L 25th– 75th percentile) simultaneously had IgE positive results for TGP (100% of subjects) (median 5 48.9 kUA/L, 19.8-4100 kUA/L 25th–75th percentile) and high prevalence of sensitisation to 6/8 Phleum pratense allergens (Phl p 1, 2, 4, 5, 6, 11, markers of genuine sensitisation to TGP) other than profilin and calcium binding protein. More than 72% of BGP allergic patients were cosensitised to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6. A decrease of total and specific IgE with patients’ age was observed. Conclusions: Our data show that all BGPallergic patients simultaneously exhibit higher IgE antibody levels to recombinant and natural P. pratense allergens as well as to crude TGP extract. This suggests that when choosing an immunotherapeutic regimen for BGP-sensitised patients (after establishing their IgE profile via purified TGP-allergens), subcutaneous or sublingual TGP-extract vaccines in appropriate doses, in order to influence T epitope specificity, might be beneficial. Though extremely uncommon, in cases where a patient is exclusively BGP allergen-sensitised, BGP-extract therapy is the appropriate therapeutic response.


228 Cross-reactive carbohydrate determinants and Hymenoptera venom allergy: IgEdetection in conceptually different automated systems, implicating relevant recombinant allergens Jappe, U1; Sander, I2; Hoffmann, M3; Huebsch-Mueller, C3; Enk, A3; Raulf-Heimsoth, M2 1 Paul-Ehrlich-Institut, Division of Allergology, Langen, Germany, 2Ruhr University Bochum, Research Institute of Occupational Medicine, Bochum, Germany, 3 University of Heidelberg, Department of Dermatology and Venereology, Heidelberg, Germany

Background: Cross-reactive carbohydrate determinants (CCD) are the most widely distributed pan-epitopes seemingly affecting the specificity of IgE-detection methods. Methods: Fifty-six patients with insect venom allergy were investigated in two conceptually different systems with bromelain (BRO), horse radish peroxidase (HRP) and natural rubber latex (NRL): 1. CAP FEIA, based on allergens coupled to a solid ImmunoCAP matrix (Phadia, Sweden) and 2. Immulite E 2000 (IML, DPC Biermann, Germany), based on liquid phase technology, including the CCDs MUXF3 (Phadia) and ascorbate oxidase (DPC), respectively. Most sera were additionally investigated with recombinant venom allergens in ADVIA CENTAUR. Results 40.35 kU/L were considered positive. The controls were 10 grass pollen allergics. Results and discussion: In contrast to CAP, where all patients’ sera had IgE to both insect venoms (yellow jacket, YJ; honey bee, HB), in IML only 39/56 had IgE to HB and 52/56 to YJ. Five out of fifty-six had no IgE to any CCD-allergen presented in both systems. Using CAP, 40/51 had BRO-IgE, 45/51 IgE to HRP, 37/51 to NRL, 38/51 to MUXF3, whereas in IML 24/51 had IgE to BRO, 43/51 to HRP, 19/51 to NRL, and 37/ 51 to ascorbate oxidase. In CAP, IgE to BRO and HRP showed a strong Pearson correlation with r 5 0.9994 (Po0.0001). Although IgE to these CCDs differed individually in IML, the Pearson correlation was still good (0.6953; P 5 0.01). The sera of grass pollen allergics showed IgE to HB (CAP: 4/10; IML 1/10), to YJ (CAP: 5/10; IML: 6/10), to NRL: 8/10, BRO: 6/10, HRP: 4/10 in CAP; in IML: 9/10 to NRL, 7/10 to HRP, 8/10 to ascorbate oxidase and 6/10 to BRO without clinical relevance, strengthening the hypothesis that grass sensitisation induces anti-CCD-IgE. In ADVIA, 28/33 had IgE to YJ-, 14/31 to HBextract, 11 to both. 12/34 bound to Api m 1, 26/34 to Ves v 5, 8/34 to both. IML-results not corresponding with CAP showed accordance with ADVIA for 6 HB-IgE negatives being clearly positive for YJ in ADVIA, 5/6 being positive for YJ in both systems (IML, ADVIA). The serum only HRP- and BRO-

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IgE-positive in IML had YJ-IgE in ADVIA. In 21/34, ADVIA confirmed history, in 6 cases there was no concordance, and where the culprit insect remained unidentified (n 5 7), ADVIA clearly pointed to one venom, 5 YJ, 2 HB. The differences in the IgE-binding to BRO and HRP in IML in contrast to CAP might be due to the presentation of the CCD-epitopes in liquid phase. HRP is the favourable CCD-screening allergen in both systems.

229 Cross-reactive carbohydrate determinant (CCD) from bromelain: a new allergy marker (MUXF Glycopeptide) for specific IgE detection Banik, U1; Huynh, K1; Gan, W2; Evangelista, R2; Lopez, M1; Jaggi, K3; Davoudzadeh, D3; Palazzo, P4; Bernardi, M4; Mari, A4; Hovanec-Burns, D1 1 Siemens Healthcare Diagnostics, Allergy Diagnostics, Los Angeles, United States, 2Siemens Healthcare Diagnostics, Chemistry, Los Angeles, United States, 3 Siemens Healthcare Diagnostics, Biochemistry, Los Angeles, United States, 4Center for Clinical and Experimental Allergology, IDI-IRCCS, Rome, Italy

Introduction: Cross-reactive carbohydrate determinants (CCDs) are a group of structurally similar carbohydrate moieties (e.g., N-glycan) present in association with food allergens of plant, arthropod and mollusk origin. Specific IgE reactivity to CCD is generally considered to have no clinical relevance. To understand the complexities of CCD reactivity to patient sera, we characterized and investigated MUXF (a CCD structure from bromelain, a glycoprotein in pineapple stem) using the IMMULITEs 2000 3gAllergyt assay to detect anti-CCD IgE reactivity. Method: MUXF glycopeptide was obtained by enzymatic digestion of bromelain and purified using column chromatography. Patient sera, selected on the basis of a positive IgE test for glycoprotein-containing food allergenic extracts and a negative clinical history, were tested for reactivity to MUXF glycoprotein and to bromelain allergen using the IMMULITE 2000 3gAllergy assay. Inhibition studies were performed after preincubation of patient sera with excess MUXF prior to assaying for IgE reactivity. Results: Mass spectrometry and sugar analysis indicated the presence of glycopeptides containing the MUXF structure isolated from bromelain. Using both bromelain and MUXF glycopeptide as inhibitors, greater than 90% inhibition was obtained. MUXF glycopeptides and bromelain both demonstrated similar reactivity with anti-CCD IgE in multiserum evaluation studies using clinical samples known to have reactivity against glycoprotein. Conclusions: Our data demonstrate that CCD-specific IgE reacted with the MUXF

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glycopeptides from bromelain. Similar reactivity for MUXF glycopeptides and bromelain implies that they share common determinants. MUXF glycopeptides or bromelain allergen, either together or individually, offer the opportunity to analyze discrepancies between skin prick test, clinical history, and in vitro results.Available outside the US.

230 Laboratory evaluation of a qualitative in vitro test for specific IgE in primary care as compared to quantitative measurement using automated ImmunoCAP FEIA processing Forslid, J; Molldeń, P; Lena, S Karolinska University Hospital, Division of Laboratory Medicine, Stockholm, Sweden

Background: Previous reports of a new sandwich immunoassay from Phadia (ImmunoCAP Rapid), which qualitatively measures ten separate allergens in whole blood and is visually read within 20 min, have shown a good correlation between clinical diagnosis and the obtained IgE results in vitro. The purpose of this study was to compare the laboratory results of the manual test against conventional quantitative IgE measurement with the automated ImmunoCAP FEIA system. Methods: Outpatients from three primary care centres with a clinically suspected allergy were tested for specific IgE against ten allergens. Each individual sample from these patients was immediately analysed in a local laboratory facility using the ImmunoCAP Rapid assay and centrally within 3 days using ImmunoCAP FEIA without any of the laboratories having any previous knowledge of the respective results or clinical diagnoses. The selected allergens were the same in both tests, but the Rapid assay employs a yellow conjugate for visual detection within a cut-off area of 1–2 kU/L, while the ImmunoCAP FEIA has a detection limit of 0.35 kU/L with an FITC conjugate that is read off against a standard curve in a photometer. Results: Seventy patients (aged 2–66 years, average 28) were tested using both assays, but in some cases, the obtained serum volume from the patients was not enough for all 10 separate allergens to be analyzed with the FEIA assay, which is why only 684 allergens were run in tandem. Six hundred and sixty-eitht allergen pairs (97.7%) displayed correct matching between the two methods. A few allergen pairs (4/684; 0.6%) came out as visually doubtful with the Rapid assay, but in all these cases, allergen-specific IgE antibodies were detected with the FEIA. In some cases (12/684; 1.8%) a mismatch was seen between the two assays Journal Compilation

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with an even distribution between falsely positive and falsely negative. Conclusion: There was a very good correlation for the detection of allergen-specific IgE in vitro between the qualitative visual test ImmunoCAP Rapid and quantitative measurement with ImmunoCAP FEIA. The qualitative test is well suited in primary care by virtue of its small sample volume, uncomplicated test procedure, rapid results and easy read-off. It is thus a suitable screening test for extended diagnostics or quantitative measurement of specific IgE prior to deciding on treatment.

231 Development of an ELISA method for detection of specific IgE against saffron pollen allergens in human serum Tayyebi, D1; Rahsaz, M2 Islamic Azad University-kazeroun Branch, Biology, Kazeroun, Islamic Republic of Iran, 2Nemazi Hospital, Transplantation Research Center, Shiraz, Islamic Republic of Iran

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Background: Grass pollen is one of the major causes of allergy worldwide. Grass pollen allergens can cause an immediate hypersensitivity (type I- IgE mediated) response in susceptible individual that leads to the symptoms of hay fever and allergic asthma. Saffron (Crocus sativus) is a monocotyledon plant that belongs to Liliales order, Iridaceae family, Crocus genus and Sativa species. Saffron is a native plant of Iran which is cultivated widely in different places. Our recent observations show that allergy to saffron is partially at high level, especially among the people associated with saffron agriculture. This kind of allergy is characterized by continuous sneezing, rhinorrhea, conjunctivitis, throat irritation, shortness of breath and etc. in order to detect the specific IgE to saffron’s allergen in human serum; we have developed an ELISA method. Methods: Microtitre plate coated with total extract of saffron and remaining sites were blocked by BSA 1% w/v. then sample sera were added, followed by addition of a goat anti-human IgE-HRP. TMB is used as substrate to measure HRP activity and optical density of each well was measured at 450 nm. Results: To verify the specificity of this method, the sera from nonsensitive individuals to saffron but sensitive to other allergens with high levels of IgE were tested and no cross reaction were found. Conclusion: We used this ELISA method to detect saffron specific IgE in 23 people with positive skin prick test to total extract of saffron. 18 people had an O.D. value more that cut-off value. So, a correlation of 78% between these two methods was found.


Poster Discussion Session 5 – Indoor and Outdoor Allergens

Poster Discussion Session 5 Indoor and Outdoor Allergens 232 Cat, dog and horse allergens in allergen avoidance and conventional Swedish daycare centers -associations with building characteristics Cai, G1; Zhao, Z1; Bro¨ms, K2; Kim, J1; Sva¨rdsudd, K2; Norba¨ck, D2 1 Uppsala University, Dept. of Medical Science, Uppsala, Sweden, 2Uppsala University, Dept. of Public Health and Caring Sciences, Uppsala, Sweden

Sweden has had specialised ‘‘allergen avoidance day care centres’’ (AADC) since 1979. These day care centres do not allow pet keeping among staff or the children’s family, to avoid secondary allergen contamination. We have previously compared AADC with matched ordinary day-care centres (ODC), and found that they differ with respect to amount of shelves, textiles and pot plants. Dust collection on Petri dishes is a new method that can detect airborne allergens. The aim was to compare levels of airborne allergens in AADC and ODC. Moreover we studied associations between allergen levels and indoor factors. Airborne allergens were collected on Petri dishes kept open for 6–8 days, and analysed by two-side sandwich ELISA for cat (Fel d 1), dog (Can f 1) and horse (Equ c x). Cat and dog allergens were expressed in ng, horse allergen was expressed as units, where 1 unit corresponds to 1 ng of a horse and dander extract used as standard. The sedimentation rate (ng/m2 and day) for the allergens was calculated, as a measure of the airborne allergen concentration. Geometric mean values were calculated (GM). In total, 11 AADC and 11 ODC in southern and western part of Sweden was studied (33 AADC and 37 ODC rooms). The cat allergen level (GM) in AADC and ODC was 0.4 and 2.4 ng/m2 and day, respectively (Po0.001). The dog allergen level (GM) in AADC and ODC was 2.1 and 4.1 ng/m2 and day, respectively (P 5 0.001). The horse allergen level in AADC and ODC was 1.1 and 2.0 ng/m2 and day, respectively (P 5 0.01). Total allergen level in air (sum of cat, dog, horse allergens) was higher in older buildings (Po0.001), and related to amount of carpets on the floor (Po0.001), textile factor (excluding carpets) (Po0.001), number of pot plants (Po0.001), but was not related to ventilation flow (CO2 levels). When analysing AADC and ODC separately, associations with building factors were strongest in ODC, and not significant (except for building age) in AADC. In conclusion, allergen avoidance day care r 2008 The Authors Journal Compilation

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centres have lower levels of furry pet allergens in the air, particularly cat allergen, mainly due to the restriction of pet keeping. The effect of reduction of open shelves and textiles and pot plants in these day care centres, on allergen levels in air, may be of secondary importance. The levels of airborne allergens were lower in both types of day care centers, as compared to allergen levels in Swedish schools, measured by the same Petri dish method.

233 Prevalence of rhinitis among office workers of a naturally ventilated old building: indoor air quality concerns Rios, J1; Boechat, J1; Gottens, A2; Ramos, C3; Aquino Neto, F3; Lapa e Silva, J1 1 Federal University of Rio de Janeiro, Thorax Disease Institute, Rio de Janeiro, Brazil, 2Policlinica Geral do Rio de Janeiro, Servic- o de Alergia, Rio de Janeiro, Brazil, 3 Federal University of Rio de Janeiro, Chemistry Institute, Rio de Janeiro, Brazil

Background: In modern life people spend approximately most of their time indoors. Many factors contribute to indoor air quality (IAQ), such as temperature, humidity, odors, air movement, bioaerosols and volatile organic compounds (VOCs) contamination. The range of complaints attributed to indoor air pollution is labeled sick building syndrome. This abstract aims to determine the prevalence of allergic rhinitis (AR) and rhinitis complains in office workers of an antique building. Method: One hundred and forty from 280 full-time office employers, working for more than 1 year in a naturally ventilated building, located in the downtown area of Rio de Janeiro, were studied. All of them worked from the 7th to 14th floor, out off the street spectrum of air pollution. They answered the ISAAC rhinitis written questionnaire (WQ) and performed medical examination, spirometry and skin prick test (SPT) for Dermatophagoids pteronissinus, Blomia tropicalis, Aspergillus sp, Cladosporium sp, Alternaria alternata and Periplaneta americana. Temperature, humidity, air movement and indoor pollutants were measured in the selected floors. Standardized international methodologies were used to investigate the indoor concentration of CO2, VOCs, endotoxin, temperature, humidity and air movement and fungal colonies count. The criteria to be considered a case of rhinitis was: positive answer to the questions 2 and 6 from the WQ or at least


two of the four rhinitis symptoms in clinical evaluation, plus a positive SPT (43 mm) to at least one antigen. Results: Seventy-one (50,7%) of the employees answered positively the WQ or had AR symptoms. But only 32 (22,8%) had SPT positive and could be considered rhinitis cases. Indoor humidity and air movement and the concentration of CO2, VOCs, fungal colonies and endotoxins did not exceed the national safety limits. Indoor temperature was above the recommended level, as Rio de Janeiro is a hot tropical city and the building did not have air conditioner. Conclusion: Despite the building adequate IAQ parameters, many workers (50,7%) presented rhinitis complains, although only 32 (22,8%) had at least one positive SPT. The prevalence of allergic rhinitis, using this criterion, was similar the prevalence of rhinitis in the general population. The adverse attributes of the building (older then 60 years) and offices (large quantities of old paper), might be contributing to the high prevalence of nasal symptoms, in spite of adequate IAQ parameters.

234 Natural and recombinant allergens of olive and chenopods in the diagnosis of allergic patients Flores, E1; Cervera, L2; Salinas, M1; Barber, D3; Fernańdez, J4 1 San Juan University Hospital, Biochemistry, San Juan de Alicante, Spain, 2Vistahermosa Hospital, Biochemistry, Alicante, Spain, 3ALK-Abello-Spain, I 1 D 1 I, Madrid, Spain, 4Elche University Hospital. UMH, Allergy Section, Elche, Spain

Background: Olive and Chenopods (Chenopodiaceae) pollens are an important cause of allergy in Mediterranean countries. Up to date, many allergens of these pollens(Ole e1 to Ole e10, Che a1 to Che a3 and Sal k1) have been isolated and characterized. We have studied the sensitisation to these allergens in a sample of patients suffering from pollinosis and asthma in the east of Spain. Methods: We select 112 consecutive patients (36.1%male) with pollinosis and/or asthma by history, skin prick tests and CAP (Phadia) positive. Sera from these patients were tested against both recombinant and natural allergens of olive and chenopods (nOle e1, nOle e7, nOle e9, rChe a1, rChe a3 and nSal k1), using an ELISA AdviaCentaur system (Bayer). All patients were 101


classified by clinical symptoms (Asthma and/or rhinitis). Data management and statistical analyses were implemented in SPSS 11.0 for windows. Results: The mean age of the sample (112 patients) was 31.45 7 9.7 years. 80 patients (71.4%) were allergy to Olive. In these patients nOle e 1 was the main allergens in all of them (Mean value of nOle e1 15.73 kU/ L against 0.44 kU/L of nOle e9. Meanwhile, from the 83 patients (74,1%) who were allergic to Chenopods, 9 subjects did not had positive nSal k1. Besides, Olive allergic patients with asthma had a significant (Po0.01) higher nOle e1 than patients with only rhinitis (17.22 kU/L vs. 7.52 kU/L). Conclusions: Ole e1 may be an important marker of severity in olive allergy patients in our area. The use of natural and recombinant allergens from different pollens in the ADVIA-Centaur system allows to light up the sensitivity and even severity of pollen allergic patients.

235 Polycyclic aromatic hydrocarbons (PAHs) are proallergic diesel exhaust particle chemicals: they generate oxidative stress in epithelial cells and drive IL-4 secretion from basophils in an allergen independent fashion Schober, W1; Lange, J1; Gebauer, G1; Lubitz, S1; Matuschek, G2; Lintelmann, J2; Behrendt, H1; Buters, J1 1 Division of Environmental Dermatology and Allergy, Helmholtz Zentrum Mu¨nchen/TUM, ZAUM – Center for Allergy and Environment, Munich, Germany, 2Helmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health, Institute of Ecological Chemistry, Neuherberg, Germany

Background: Exposure to diesel exhaust particles (DEPs) is associated with a variety of adverse health effects, including exacerbation of allergic rhinoconjunctivitis and asthma. It is increasingly being recognized that generation of reactive oxygen species (ROS) plays an important role in DEPinduced allergic inflammation. High levels of oxidative stress can trigger NFkappaB and MAP kinase signaling pathways that are responsible for transcriptional activation of Th2-polarizing cytokine genes. In addition to the particles themselves, adsorbed redox-cycling organic compounds, such as PAHs, may highly contribute to the proallergic effects of DEPs by generating oxidative stress. We tested PAH containing airborne extracts and phenanthrene (Phe), a major DEP-PAH, for their ROS inducing activity in lung epithelial cells and explored the PAH-induced cytokine secretion from human basophils. Methods: A549 cells were exposed for 3 h to organic 1 day-extracts of PM2.5 (AERex) collected at a highly frequented six-lane road and Phe alone or after pretreatment 102

with 100 mM buthionine sulfoximine (BSO), which inhibits glutathione (GSH) synthesis. ROS levels were detected by dichlorofluorescein fluorescence and multiparameter flow cytometry. Additionally, the decrease of intracellular GSH upon PAH exposure was measured by colorimetric analysis. Moreover, purified basophils from birch pollen allergic donors were incubated in the presence of 1 mM Phe for 165 min alone or in combination with rBet v 1 over the last 45 min of treatment. Supernatants were assayed for IL-4 and IL-8 by means of ELISA. Results: Strongest ROS inducing activity of PM extracts and Phe was seen in BSO pretreated A549 cells (in % of control): AERex#1 12% (total Phe content: 2.3 mM)4Phe 10%4AERex#2 8% (2.7 mM)4AERex#3 6% (1.2 mM). Oxidative stress occurred dose dependently and was found significant for Phe, AERex#1 and #3. In addition, Phe significantly induced IL-8 and IL-4 secretion from purified sensitised basophils in the absence of antigen suggesting an adjuvant role of DEP-PAHs in allergic sensitisation. No relevant further increase in IL-4 secretion was seen when rBet v 1 was added. Conclusion: Our results provide further evidence that PAHs from diesel emissions drive proallergic processes in biologic systems through generation of oxidative stress in airway epithelial cells and initiate a Th2 dominated immune response via direct action of single PAHs on sensitised basophils.

236 Short-term effect of ozone and grass pollen exposure on antiasthmatic drug consumption Mur Gimeno, P1; Feo Brito, F2; Martin Iglesias, A1; Guerra Pasadas, F3; Lara de la Rosa, P2; Castro Jimenez, A2 1 Hospital Santa Barbara, Allergy Unit, Puertollano, Spain, 2General Hospital Ciudad Real, Allergy Section, Ciudad Real, Spain, 3Hospital Reina Sofia, Department of Allergy and Pathology, Cordoba, Spain

Background: The prevalence of respiratory allergic reactions induced by pollens in Europe has been on the increase in the past decades. Westernised lifestyle is correlated with the increasing frequency of polleninduced asthma, specially in urban areas. Air pollution is detected in industrial areas although in urban cities of the Mediterranean area there are frequently high concentrations of ozone (O3) favoured by sunny days and ultraviolet rediations. Our objective was to analyze the short-term association between pollen exposure and antiasthmatic drug consumption in the industrial city of Puertollano compared to the tertiary sector city of Ciudad Real,

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taking into account the potentially confounding effect of air pollution. Methods: We studied a cohort of 137 patients from Puertollano and Ciudad Real during two pollen seasons (2000–2001) and analysed the relationship between air pollutant and pollen levels and the medication used. We used Student’s t-test to study the relationship between terbutaline consumption (1 point per 500 mg dose) in Ciudad Real and Puertollano as well as the mean terbutaline consumption (T) on normal days versus the days on which the health threshold was exceeded. Results: The total annual grass pollen collected in the year 2000 reached 4863 grains/ m3 an in the year 2001 were 26% lower (3701 grains/m3).The (T) in Ciudad Real was 0.56 doses versus 0.79 in Puertollano (P 5 0.001). Over 2000 and 2001 years, the (T) on normal days (with no O3 exceedances) was 0,78 doses in Puertollano, versus 0.85 on the days that the O3 exceeded the health threshold (P 5 0.506). The influence of the pollutants on the patients in Puertollano also differed depending on the year of the study. In the year 2000, the (T) was 0.83 doses versus 0.92 on the days that exceeded the O3 health threshold (P 5 0.738). However in the year 2001 the consumption of terbutaline on the normal days was 0.64 doses per day, versus 0.85 doses on the days with O3 exceedances (P 5 0.004). Conclusion: 1- An increase in the use of rescue medication is a clear sign of deterioration in asthma symptoms. 2- The use of rescue medication in Puertollano was significantly related to the O3 exceedances in the year 2001 but not in the year 2000. 3- In years of high pollination (2000), the effect of the pollens can mask the additional deterioration caused by environmental pollution. 4- In the year 2001 with lower pollen levels, it was easier to detect the clinical effects of the pollutants.

237 The development of the algorithm for allergic pollen forecast Oh, J1; Lee, H2; Kim, H3; Kang, I4; Kim, S5; Kim, B6; Kook, M7 Hanyang University Guri Hospital, Pediatrics, Guri, Republic of Korea, 2Hanyang University Guri Hospital, Pediatrics, Seoul, Republic of Korea, 3Republic of Korea, 4 Daegu Fatima Hospital, Department of Pediatrics, Daegu, Republic of Korea, 5Busan St. Mari Hospital, Department of Pediatrics, Busan, Republic of Korea, 6 Kangneung Asan Hospital, Department of Pediatrics, Kangneung, Republic of Korea, 7Kwangju Veterans Hospital, Department of Pediatrics, Kwangju, Republic of Korea

1

Background: A growing number of people are recently contracting allergic diseases caused by pollen because of climate change



in the world. Since 1997 research for pollen forecast has conducted in Korea. Method: There are eight pollen observation stations, which are operated by the Korean Academy of Pediatric Allergy and Respiratory Diseases through Korea. Pollen grains are counted and reported and stored in a pollen database station for over 10 years. We have focused to develop the equations for the pollen forecast like the weather forecast. Daily fluctuation in the amount of pollen have to do with a variety of meteorological factors. According to multiple regression equations for the meteorological factors and the concentration of pollen, different meteorological elements affect the concentration of pollens each year. Ten weather elements to affect the concentration of pollen are used to develop algoirthm for the pollen forecast. Forecast algorithm for each variety of allergic pollen as alder and ragweed and for each month as April and September have been developed based on multivariate regression analysis. Results: The Algorithm for forecast tree pollen in April and May showed accuacies of 72.7% and 42.9% for pine pollen respectively and 77.3% and 57.1% for the other tree pollens such as alder, birch and oak. The forecast algorithm for weed pollen in September and October achieved an accuracy of 74.2% and 46.7% respectively. Conclusion: The algorithm may be useful for allergic pollen forecast in future. In other hands, in order to enhance the accuracy of the pollen forecast algorithm, it will necessary to evaluate the curren index for pollen allergies for suitability to allegic patients based on experimental data.

238 Spread of ragweed plants and ragweed sensitisation in the Netherlands de Weger, L1; van der Linden, A1; Terreehorst, I1; van der Slikke, W2; van Vliet, A3; Hiemstra, P1 1 Leiden Unversity Medical Center, Department of Pulmonology, Leiden, the Netherlands, 2FLORON Foundation, Leiden, the Netherlands, 3Wageningen University, Environmental Systems Analysis Group, Wageningen, the Netherlands

During the last decades the importance of ragweed (Ambrosia) pollen for allergic patients has increased in Europe. The aim of the present study was to investigate the current spread of ragweed plants and pollen in the Netherlands, as well as the degree of allergic sensitisation to ragweed. Data on the spread of ragweed were obtained from two different sources: (i) floristic observations by the FLORON foundation; and (ii) a web-based entry of ragweed observations by the public following a media campaign in 2006 and 2007. The first observations of ragweed in the Netherlands originate from the first part of the twentieth century. In the r 2008 The Authors Journal Compilation

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past decade an increase in the number of observations was documented. The recent increase in ragweed plants was mainly observed in private gardens where the plants were most likely introduced by means of bird-seed. In 5% of the observations in 2007 it concerned locations of more than 100 plants, suggesting local establishment of the plant. In contrast, the counts of ragweed pollen at the Leiden pollen counting station did not show an increase in the past decades, and the daily pollen concentrations did not exceed 10 pollen/m3. The sera of 250 patients that were submitted for allergy diagnosis and collected in 2004, 2005 and 2006, were analyzed for the presence of specific IgE directed against a panel of inhaled allergens (Phadiatop) and against ragweed pollen. Within the group of patients with a positive Phadiatop, 17% also had IgE directed against ragweed. Ragweed-specific IgE was often present in those patients with IgE directed against mugwort pollen and those with IgE to a range of other inhaled allergens. Based on these findings, we conclude that despite the increased observations of ragweed plants, at present both the degree of ragweed sensitisation and the number of ragweed pollen in ambient air in the Netherlands are relatively low. Careful monitoring will be required to determine whether ragweed develops into a major cause of hay fever symptoms in late summer in the Netherlands.

239 Clinical and epidemiological findings in patients monosensitised to Artemisia pollen in Ciudad Real (Spain) Lara de la Rosa, P; Borja Segade, J; Feo Brito, F; Galindo Bonilla, P; Go´mez Torrijos, E; Alonso Llamazares, A; Garcıá Rodrı´guez, R General Hospital, Allergy Section, Ciudad Real, Spain

Introduction: Artemisia gender is distributed elsewhere. Artemisia spp flowers in late summer (august-september) with regional differences and pollen accounts registered are usually low due to high pollen weight. In our zone (Spanish center) mean annual pollen concentration between 1.996 and 2004 was very low: 13 g/m3 (range 4–26 g/m3). In Europe, 3–10% of pollen sensitised patients are sensitised to Artemisia vulgaris, usually associated to grass sensitisation. In Spain prevalence of sensitizacion is between 9–38% in pollen sensitised patients. Clinical relevance of this pollen, specially in our monosensitised patients is unclear. Objective: The aim of the study was to determine the clinical and epidemiological findings in our patients monosensitised to artemisia pollen.


Material and methods: We performed a retrospective search from our outpatients database (41.508 patients in a 10 years period 1997–2007). We selected all patients monosensitised to Artemisia vulgaris pollen without food allergy and collected the following data: sex, age, clinical picture, symptomatic period, wheal area of ART skin test and total IGE. All patients had been tested at least in two occasions with an aeroallergen battery (ALK-Abello) which included mite, dander, pollen (grass, olea, cupressus, platanus, quenopodium, plantago, salsola and Artemisia vulgaris) and molds. Specific IGE was not routinely determined. Results: Twenty (0.05%) patients fulfilled the selection criteria: 15 male, 5 female. Mean age: 34.4 years (range 21–67). Mean age when clinical picture began: 24,3 years (range 11–60). Clinical picture: 10 patients (50%) suffered from rinoconjunctivitis (RC) and asthma (A), 4 RC, 4 Rhinitis, 1 A and 1 conjunctivitis. Symptomatic period: 12 perennial, 8 seasonal (4 spring, 4 springautomn). Mean wheal area (mm2): 50.5 (range 9–240); Mean total serum IGE: 76.9 kU/L (range 9.8–126). Conclusions: Patients monosensitised to Artemisia vulgaris pollen without food allergy are predominantly male, whose symptoms began after the second decade. Fifty per cent of patients were submitted with perennial symptons of RC and A, not related to pollen season. Mean total serum IGE was lower than usually found in atopic patients sensitised to grass pollen. Only 4 patients experimented seasonal symptoms during pollen season although low pollen levels do not probably explain this clinical picture.

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Abstract withdrawn.

241 Airborne allergen (Ole e 1) distribution associated with various size fractions De Linares, C; Nieto, D; Alba, F; Dıáz de la Guardia, C University of Granada, Botany Department, Granada, Spain

Background: The knowledge of airborne particles is fundamental for Aerobiology. In the Mediterranean area, Olea europaea pollen causes one of the major health problems. The symptoms are mainly asthma with or without rhinoconjunctivitis. The particles larger than 20 mm usually do not enter the lower respiratory tract. However, if the Olea pollen reaches the human respiratory tract, the allergens released could

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induce this allergopathy. Nevertheless, the air contains allergenic material of smaller size, which might be the causes of bothersome symptoms for allergic persons. However, little is known about the prevalence of these particles in the atmosphere, their temporal variations or their detailed chemical composition. The present research was finance for a project of the Ministry of Science & Technology (PROYECT I 1 D 1 I: CGL2006-1648-CO3-02) and offers a study of Ole e 1 distribution associated with various size fractions and a comparative analysis between this allergenic activity and the dynamics of airborne Olea europaea pollen. In this way, we can establish whether the allergy symptoms could be related to the distribution of allergens in the atmosphere. Methods: The air was sampled during the pollination period of Olea europaea, from 2005 and 2006, using a cascade impactor (Andersen Samplers) collector and a volumetric collector (Burkard Spore Trap) situated in the Science Faculty of the University of Granada (southern Spain). The indirect ELISA was used to detect the allergenic activity whereas the counting method was that recommended by Spanish Aerobiological Network. Results and conclusion: This research demonstrates that the dynamics of allergenic activity and pollen particles are coincides with the main Olea pollen season. However, the study of the distribution of the allergenic particles reveals that the highest concentrations were localized below 3.3 mm. Therefore, the allergens are detected in the respirable fraction, and thus could be the cause of the significative percentage of the asthmatic symptoms in the population.

242 Profile of allergen pollen reactivity in patients sensitised to Olea pollen Corominas, M1; Lleonart, R1; Martıń, C1; De la Torre, F2 Hospital Universitari de Bellvitge, Allergology, L’Hospitalet de Llobregat, Spain, 2Research and development, ALK-Abello, Madrid, Spain

1

Background: Previous studies performed in our area (with low levels of Olea pollen) showed that 38% of patients with pollinosis were sensitised to Olea, although only 7% of them are monosensitised. The aim of this study is to evaluate sensitisation to olive pollen as a consequence of cross reactivity with other pollen allergens. We studied the sensitisation pollen profile of patients with positive skin prick test (SPT) to Olea and the cross reactivity with other pollen allergens. Patients and methods: Fourteen adult patients (8 males and 6 females) whith a positive SPT to Olea pollen were included. We analyzed their clinical history and SPT to common airborne allergens and allergens 104

with a high LTP, profilin or polcalcin content. Specific serum IgE against to recombinant allergens Ole e1, Ole e 9, Salk1, Par j 1, Phl p 1, Phl p 5, Art v 1, Cup s 1, Bet v 1, Pru p 3, Mal d 4, Che a 3 (ALK-Abello) were quantified in the ADVIA Centaur platform. Results: Ten patients presented rhinitis and four asthma and rhinitis. According to SPT, 78% were positive to grasses, 64% to Parietaria, 57% to Salsola, 57% to Plantago, 50% to Platanus, 43% to Artemisia, 36% to Cupressus, 36% to peach, 21% to polcalcin, 14% to profilin and 14% to Betula. When we analyzed specific IgE against recombinant allergens, we found that 11 of the 14 patients (78%) were reactive to Ole e1 and none to Ole e 9. Only one patient had specific IgE to Che a 3 (polcalcin), and another to Che a 3 and to Mal d 4 (profilin). Ole e 1 specific IgE levels correlated with IgE levels of Phl p 1, Phl p 5, Pru p 3, Cup s 1, Che a 3, Mal d4, Pla l 1 and Bet v 1 (Po0.002). Conclusions: Most of the patients sensitised to Olea had specific IgE to Ole e 1 allergen. Profilin and polcalcin allergens are not responsible for polysensitisation present in these patients. In this group, sensitisation to Olea pollen corresponds to the great number of sensitisation present, but it is not a result of cross reactivity.

243 Comparison of the characteristics and allergenicity of fresh and commercialised Phleum pratense pollen Abou Chakra, O1; Rogerieux, F2; Senechal, H3; Peltre, G4; Lacroix, G2 1 INERIS, Experimental Toxicology Department, Verneuil en Halatee, France, 2INERIS, Experimental Toxicology Department, Verneuil-en-Halatte, France, 3ESPCI – INSERM, LECA, Paris, France, 4ESPCI – CNRS, LECA, Paris, France

Background: Grass pollen is quantitatively one of the most important aeroallergen, which can induce allergic diseases (asthma and rhinitis). Most studies on the pollen allergenicity were carried out with commercialised pollen which has been often collected 2 or 3 years ago and which has undergone a conservative treatment. A few works have pointed out that pollen evolved in time (for example in their germination capacity or in their resistance to an osmotic shock). The aim of our study was to compare physical characteristics and allergenicity of pollen of different ages, with or without post-harvest treatment. Methods: Two pollen collected in 2003 and 2005 with a post-treatment (Allergon), and one pollen harvested in 2006 without treatment were used. Phleum pratense pollen was suspended in water. Damaged pollen and intracytoplasmic granules (CGP) released by Journal Compilation

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pollen in contact with water were counted by a Z2t COULTER COUNTERs. Brown Norway rats were sensitised (day 0) and challenged (day 21) intratracheally with 0.15 mg of pollen. Negative control (NC) received saline. At day 25, blood and bronchial lymph node were collected. The humoral response was assessed by the determination of IgE and IgG1 in sera. The cell-mediated response in the lymph node cells was determined by measuring the uptake of [3 H] thymidine in a proliferation assay. Results: After an osmotic shock, the number of damaged pollen collected in 2003 (1.5 7 0.05%) was significantly lower than the 2 other harvests (B2 7 0.1%). In the same way, the release of GCP by the pollen collected in 2005 and 2006 was more pronounced than the release by the pollen harvested in 2003. For the three collects, specific IgE and IgG1 were present in the sera, comparing with NC. The IgG1 level in sera was higher in the rats sensitised in pollen without post-harvest treatment (1.5) than rats sensitised in pollen of 2003 and 2005 harvest (0.4 and 0.3, respectively). Cultured lymph node cells of pollensensitised rats proliferated more than the NC but no significant difference was observed between the 3 different pollen grains. Conclusions: We confirm here that the ageing of pollen induces a better resistance to water and thus lead to less GCP release. The post-harvest treatment of pollen appears to weaken the pollen structure. The three different Phleum pratense pollen grains induced humoral and cellular responses in our animal model, with a higher sera IgG1 level for the pollen without treatment.

244 Foreign allergenic pollen. Some cases observed in Catalonia (NE Spain) and Tenerife (Canary islands) Belmonte, J1; Alarcoń, M2; A´vila, A3; Izquierdo, R3; Cuevas, E4 1 Universitat Auto`noma de Barcelona, Unitat de Bota`nica/ ICTA, Bellaterra (Cerdanyola del Valle`s), Spain, 2 Universitat Polite`cnica de Catalunya, Fı´sica i Enginyeria Nuclear, Vilanova i la Geltru´, Spain, 3Universitat Auto`noma de Barcelona, CREAF, Bellaterra (Cerdanyola del Valle`s), Spain, 4Instituto Nacional de Meteorologıá, Observatorio Atmosfe´rico de Izanã, Santa Cruz de Tenerife, Spain

Background: Pollen dispersal, a crucial process in the life cycle of wind-pollinated plants, depends on the atmospheric dynamics, and can vary from a few meters to thousands of kilometres. Several studies have shown the occurrence of long-range pollen transport; e.g. the transcontinental transport from Africa to North America or to Europe, or from the boreal/austral forests to the poles. Pollen transport and dispersion



can have important consequences, such as the expansion of the bio-geographical range of different organisms, the transport of pathogens and effects on the human health due to allergenic pollen. Methods: Singular aerobiological Hirst pollen records (appearance of unexpected pollen types or detection of simultaneous pollen peaks at different sites) were analyzed together with the back trajectories and the meteorological setting during the peak days. Dust transport model maps, satellite images and source-receptor models were also used for the analysis. Results: In Catalonia, NE of Spain, significant concentrations of Ambrosia pollen (species scarcely present in Catalonia) were collected in September1996 and its appearance were attributed to long range transport from the Lyon region in France, where the species is abundant. In the same way, Fagus pollen peaks appeared simultaneously at different sites across the Catalan geography in 2004 and 2006, indicating a broad-scale transport phenomenon. The analysis of back trajectories and the meteorological setting during the peak days indicated a consistent European provenance. The application of a source-receptor model showed regions in Central Europe as the probable areas of Fagus pollen emissions. Also, Chenopodiaceae/Amaranthaceae, Artemisia and Casuarina pollen collected in Santa Cruz de Tenerife and Izanã (Canary Islands) has been clearly related to an African provenance and associated with dust storms. Conclusion: The aerobiological pollen spectra are occasionally influenced by pollen and


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spores of far provenance transported by synoptic movements of the air masses. Sporadic episodes of allergy may be associated with these situations.

245 Season variability in the relationship between grass pollen counts and ICTbased daily hay fever symptom scores in patients with allergic rhinitis: implications for hay fever forecasts de Weger, L1; Beerthuizen, T2; Gast-Strookman, J1; van der Plas, D1; Hiemstra, P1; Terreehorst, I1; Sont, J2 1 Leiden Medical University Center, Department of Pulmonology, Leiden, the Netherlands, 2Leiden Medical University Center, Department of Medical Decision Making, Leiden, the Netherlands

Rationale: A multi-day hay fever forecast based on grass pollen counts may allow patients with allergic rhinitis to timely adjust medications and daily activities in order to reduce symptoms. A previous study (Spieksma, pers. communication) suggested that symptom severity for a given grass pollen concentration is variable during the grass pollen season. The aim of this study was to delineate the relationship between hay fever symptoms and grass pollen concentration. In addition, we assessed whether there was a difference in symptom scores at the start of the season as compared to the end of the season. Methods: Daily grass pollen counts were obtained after sampling the air by a Burkard sampler. After screening, 80 non-asthmatic patients that were positive in a skin-prick test for grass pollen were enrolled into the


study. They were asked to enter their hay fever symptoms (runny nose, sneezing, blocked nose, post nasal drip, eye symptoms) daily on a scale from 0 to 3 to the study center either by short message service (SMS) or by internet from May–July 2007. Start period and end period of the grass pollen season were defined before and after June 12th. This date was preceded by a peak period with high grass pollen concentrations ( 7 100 pollen/m3). Results: Among the hay fever symptoms, the mean daily eye symptom score (average [range]: 0.61 [0.28–1.29]) and sneezing score (0.74 [0.29–1.49]) showed the strongest positive relationship with grass pollen counts (r 5 0.66 and r 5 0.64, respectively). At similar grass pollen concentrations, eye symptoms and sneezing scores were lower at the end of the season as compared to the start of the season (b 5 0.30 and 0.35, respectively (ANCOVA, Po0.001). Conclusion: We conclude that hay fever symptoms are lower at the end of the grass pollen season as compared to the start of the season. The milder symptoms at the end might be related to the preceding experience of a grass pollen peak period. This suggests that the date within the grass pollen season and/or the occurrence of recent pollen peaks should be included in the multi-day hay fever forecast.Supported by a grant from the Netherlands Asthma Foundation.

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Poster Discussion Session 6 – Drug Allergy – From Diagnostics to Management

Poster Discussion Session 6 Drug Allergy – From Diagnostics to Management 246 The negative predictive value of negative drug challenge test involving antibiotics

247 Allergy to blactam antibiotics: from diagnosis to patient compliance

Botelho, C; Silva, R; Rodrigues, J; Castel-Branco, M Hospital Saõ Joaõ E.P.E, Allergy and Clinical Immunology Division, Porto, Portugal

Campina Costa, S1; Costa Silva, I2; Chambel, M2; Romeira, A2; Martins, P2; Leria Pinto, P2 1 Pulido Valente Hospital, Immunoallergy Unit, Lisbon, Portugal, 2Dona Estefaˆnia Hospital, Immunoallergy Department, Lisbon, Portugal

Background: Drug challenge tests (DCT) are considered the gold standard to establish or exclude the diagnosis of drug hypersensitivity reactions (HR). They consist on the controlled administration of the drug, under medical monitoring, either with diagnostic purpose or to determine a safe alternative drug. Aim: To evaluate if patients, in which we excluded the diagnosis of HR to antibiotics with a negative DCT, had been re-exposed to the suspected drug and determine if they tolerated it. Methods: From all patients who performed DCT to antibiotics since January 2000 until June 2007, the authors selected those with negative results to the suspected antibiotic (HR excluded). These patients were contacted by phone or letter and answered a questionnaire, in order to know if they took the culprit antibiotic after the study and evaluate its tolerance. Results: Seventy two DCT involving antibiotics were carried with diagnostic purpose, with negative results. Fifty four patients (mean age of 37 years 7 18.7, 67% female) answered the questionnaire. These patients were referred to our clinic for the study of 76 reactions (70% involving one antibiotic (100% beta-lactams); 95% of reactions had cutaneous involvement and 63% were delayed). From the 54 patients who answered the questionnaire, only 20 (37%) took the drug (or another from the same family) previously indicated as safe. The reasons for re-exposure were: treatment of tonsillitis, H. pylori, dental and upper respiratory tract infections. Four out of 20 patients (20%) had HR after re-exposure, with the same pattern as before (delayed and with cutaneous involvement). Comments: The negative predictive value of DCT in this sample was 80%. The authors speculate about the reasons of recurrence of HR after negative DCT: 1) coexistence of infection, or other factors, at the time of the reaction. 2) possibility of desensitisation/tolerance induction during the provocation procedure.

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Background: The diagnosis of allergy to beta-lactams (bL) includes clinical history, and both in vivo and in vitro methods. Finding an alternative bL is of great importance, especially in children, when re-exposure to bL is frequently needed. Aim: To evaluate the prevalence of allergy to bL in a population of patients (Pt) referred to an allergy outpatient clinic; to assess the tolerability to the alternative bL on re-exposure; to determine the diagnosis relevance for the Pt. Methods: We retrospectively analysed the charts of Pt with adverse reaction to bL. We reviewed data concerning: clinical history, specific IgE, skin tests and drug provocation test with the suspicious or an alternative drug. In order to evaluate post test avoidance or intake of bL, follow-up phone calls were made. Results: Fifty-six Pt were enrolled, median age 12,5 years (P25-75 : 8–28), 55% were women. Suspected drugs were: amoxicillin and clavulanic acid (27 Pt); amoxicillin (15 Pt); penicillin (10 Pt); and cephalosporins (4 Pt). The majority (71%) reported cutaneous symptoms, and 14% reported anaphylaxis. The median time between the suspected drug reaction and the investigation was 1 year (P25–75 : 0.7–4). The median length of follow-up was 8 months (P25–75 : 4–12). Twenty-three per cent were considered allergic to at least one bL (Group bL) and 77% concluded their investigation with negative results (Group Neg). Group bL (n 5 13), median age 12 years (P25–75 : 11– 43), 69% women, 69% described cutaneous symptoms and 23% reported anaphylaxis. An alternative bL was recommended to 77% of Group bL, and 60% of them had been re-exposed to the alternative drug, with 100% tolerability. Group Neg (n 5 43), median age 13 years (P25–75 : 7–23), 51% women, 74% described cutaneous symptoms. Sixty per cent of Group Neg had been re-exposed to bL, with 100% tolerability. On both groups the main reasons pointed for bL avoidance were: no need for antibiotherapy (50%); fear from adverse reactions (33%) and medical advice (17%). Journal Compilation

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Conclusion: Cutaneous manifestations dominate the clinical spectrum of bL adverse reactions. The diagnosis of bL allergy was established in 23% of the population, and safety of the recommended alternative bL on re-exposure was demonstrated. A considerable number of Pt unnecessarily avoided re-exposure to bL, even when allergy to bL was not confirmed. Follow-up of Pt with adverse reactions to bL is needed in order to reinforce avoidance or to reassure the safety on re-exposure, for a better Pt compliance.

248 Comparison of two immunoassays for measuring specific IgE antibodies to betalactams Mayorga, C1; Torres, M2; Blanca-Lopez, N3; RodriguezBada, J1; Perez-Inestrosa, E4; Montan˜ez, M4; Blanca, M2 1 Carlos Haya Hospital-Fundacioń IMABIS, Research Laboratory, Ma´laga, Spain, 2Carlos Haya Hospital, Allergy Service, Ma´laga, Spain, 312 de Octubre Hospital, Allergy Service, Madrid, Spain, 4Ma´laga University, Organic Chemistry Department, Ma´laga, Spain

Background: In vitro quantification of IgE to penicillins can be done by either radioallergosorbent test (RAST) or sepharose radioimmunoassay (SEPH). The aim of the study was to compare sensitivity and specificity of both methods. Methods: Patients with immediate allergic reactions to penicillins diagnosed by skin testing or drug provocation tests were classified in group A (n 5 32, RAST( 1 ) to benzylpenicillin and/or amoxicillin) and group B (n 5 25, RAST( ) to benzylpenicillin and amoxicillin). Two control groups with good tolerance to penicillin with either high (Control 1: n 5 10, mean 2475, 1 UI/L) or low (Control 2:n 5 19, mean 85, 3 UI/L) total IgE were selected. RAST was done using benzylpenicillin or amoxicillin at 10 mg/mL conjugated to Poly-L-Lysine and SEPH with benzylpenicillin or amoxicillin at 10, 100 and 500 mg/mL (SEPH10, SEPH100 and SEPH500 respectively) conjugated to sepharose epoxy-activated-6B. Results: Regarding benzylpenicillin, Group A showed 64,5% of positivities with RAST, being lower with SEPH (SEPH10 : 16.1%, SEPH100:29,4% SEPH500:12.9%). In Group B, RAST and SEPH were negative; Interestingly, 50% of positivities were detected with SEPH10 in the high IgE group (control 1). Regarding amoxicillin, Group A showed 67,7% of positivities with RAST. SEPH



showed different results depending on concentration used (SEPH10 : 29%, SEPH100 : 88,2%, SEPH500 : 74,2%). However, in Group B we only detected positive results with SEPH (SEPH100 : 26,1%, SEPH500 : 30,4%). SEPH with the highest concentrations of penicillin showed a high number of false positives in the high IgE group (Control 1, SEPH100 : 90% SEPH500 : 100%). Conclusions: RAST showed higher sensitivity and specificity than SEPH in patients with immediate reactions to betalactams. SEPH with amoxicillin had the lowest specificity due to non-specific binding.

249 Delayed-type hypersensitivity reactions to heparins: Improvement of lymphocyte transformation test using dendritic cells Torres, M1; Blanca-Lopez, N2; Lopez, S3; Mayorga, C3; Canto, G2; Chaves, P3; Rondon, C1; Bravo, I4; Blanca, M1 1 Carlos Haya Hospital, Allergy Service, Ma´laga, Spain, 2 12 de Octubre Hospital, Allergy Service, Madrid, Spain, 3 Carlos Haya Hospital-Fundacioń IMABIS, Research Laboratory, Ma´laga, Spain, 4Carlos Haya Hospital, Emergency Unit, Ma´laga, Spain

Background: In vitro tests are not useful for diagnosing delayed type hypersensitivity (DTH) reactions to heparins since the lymphocyte transformation test (LTT) may give false negatives. The aim of this study was to improve the LTT with heparins by using dendritic cells (DC) as antigenpresenting cells (APC). Methods: Patients with DTH reaction to heparins (N 5 7) and controls (N 5 9) were studied. For the LTT, either monocytes and B cells (classical LTT) or immature DC (obtained from peripheral monocytes after incubation with rhGM-CSF and rhIL-4) were used as APC. The heparins tested were: Sodium Heparin, Dalteparin, Bemiparin, Nadroparin, Tinzaparin, Enoxaparin, Sulodexide, Fondaparinux and Lepirudin. TTL results were expressed as stimulation index (SI) and were considered positive when 43. LTT was repeated in 3 patients one year after the reaction. Results: Patients were diagnosed by skin testing (N 5 6) or drug provocation tests (N 5 1). In six patients, including the one who was skin test negative, LTT was positive to most of the heparins tested with the two types of APC, monocytes/B cells or DC, but showed a higher SI when DC was used. In those patients where LTT were repeated after one year, proliferation to the culprit drug was only detected when DC were used as APC. All LTT were negative in controls. Conclusions: Autologous monocyte-derived dendritic cells used as APC may improve the in vitro lymphocyte responses in patients with delayed allergic reactions to heparins, increasing and prolonging the specific r 2008 The Authors Journal Compilation

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proliferative responses, especially to the culprit heparin.

250 Influence of age, anaesthesia and surgery on serum tryptase Garvey, L1; Bech, B2; Mosbech, H3; Kroigaard, M1; Rasmussen, L4; Poulsen, L3 1 Danish Anaesthesia Allergy Centre, Rigshospitalet, Department of Anaesthesia, Copenhagen, Denmark, 2 Bispebjerg University Hospital, Department of Anaesthesia, Copenhagen, Denmark, 3Rigshospitalet, Allergy Clinic, Copenhagen, Denmark, 4Rigshospitalet, Department of Anaesthesia, Copenhagen, Denmark

Background: Mast cell tryptase is released from mast cells and is a clinically useful marker of anaphylaxis during anaesthesia, although little is known about the effect of anaesthesia and surgery on serum levels. Data from the Danish Anaesthesia Allergy Centre (DAAC) has suggested an increase in basal level of tryptase with increasing age. Our aim was to investigate the effect of general anaesthesia (GA) and surgery, and patient age, on tryptase levels in patients undergoing elective surgery. Additionally, a possible age effect was investigated retrospectively in patients referred to DAAC after suspected anaphylaxis during anaesthesia. Methods: The study included 120 patients (median age 54 years (range 19–94)) undergoing elective orthopaedic surgery in GA. Exclusion criteria were allergic reactions during this or previous anaesthesia, mastocytosis or steroid treatment. Blood samples for tryptase analysis (ImmunoCAPs, Phadia, Uppsala, Sweden) were drawn shortly before anaesthesia and after anaesthesia/ surgery. A difference in tryptase 43 mg/L before/after surgery or between age groups was considered clinically relevant. DAAC patients included 221 patients (median age 51 yrs (range 18–86)) referred to DAAC 1999–2007, who had tryptase basal levels measured as part of standard investigations. Results: Median duration of anaesthesia was 105 min (range 45–263) with a median interval between blood samples of 139 min (range 39–370). Mean tryptase before surgery was 5.0 mg/L vs. 4.5 mg/L after surgery, giving a mean difference of 0.5 mg/L (Po 0.01, 95% CI 0.3–0.8). This difference could be partly explained by dilution, as 0.2–2 L of fluid was given. For elective patients 75/120 (62.5%) were aged o60 years vs.150/221 (67.9%) of DAAC patients. Mean tryptase in elective patients o60 years was 4.9 mg/L vs. 5,2 mg/L in patients 460 years, giving a mean difference of 0.3 mg/L (P 5 0.7, 95% CI 2.0–1.4). Mean tryptase for DAAC patients o60 years was 6.0 mg/L vs. 8.3 mg/L 460 years, mean difference 2.3 mg/L (P 5 0.02, 95% CI 4.1– 0.4).


Conclusion: General anaesthesia and surgery did not increase tryptase basal levels. In contrast a small decrease was seen, probably due to dilution by iv fluid. No significant difference in tryptase basal levels was found between elective patients aged over and under 60 years. However, when comparing same agegroups in DAAC patients, referred for allergy testing after suspected anaphylaxis during anaesthesia, a statistically significant difference was found.

251 Coetaneous drug hypersensitivity reactions in database of the centre for adverse drug reactions monitoring in Cracow Porebski, G1; Woron, J2; Czarnobilska, E1; Rembiasz, M1; Antoszczyk, G1; Obtulowicz, K1 1 Jagiellonian University, Dep. of Clinical and Environmental Allergology, Cracow, Poland, 2 Jagiellonian University, University Centre for ADRs Monitoring, Cracow, Poland

Background: Skin hypersensitivity drug reactions are very often in clinical practice. Cutaneous symptoms are one of the most common types of adverse reactions to drug therapy. Almost any medicine can induce skin reactions, and certain drug classes, such as NSAID, antibiotics and antiepileptics, have drug eruption rates approaching 1–5%. Methods: We perform an analysis of spontaneous reports received to University Centre for Adverse Drug Reactions Monitoring and Investigation in Cracow between August 1st, 2006 and August 1st, 2007 to identify the case reports containing data on skin hypersensitivity drug reactions. Results: Analysis revealed 224 spontaneous reports. In 73 cases reactions occurred as the result of the use of NSAIDs, in 41 cases in the course of treatment with metamizole, in 56 cases during antibiotic therapy, in 14 cases during antiepileptic treatment, in 14 cases in patients using benzodiazepines, in 11 cases during treatment with anticonceptive pills, in 9 cases due to the use of antihistamines, in 6 cases skin hypersensitivity drug reactions were reported during treatment with tramadol. Most often symptoms due to hypersensitivity drug reactions were urticaria, angioedema and maculopapular exanthema, however there were some cases of sever anaphylactic reactions including anaphylactic shock, as well. Conclusion: The skin hypersensitivity drug reactions are important problem in clinical practice, because they are caused by popular groups of drugs. Although most druginduced skin eruptions are not serious, some of them are severe and potentially lifethreatening.

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252 Alternative algorithm for l-asparaginase allergy in acute lymphoblastic leukemia Uysal Soyer, O1; Aytac, S2; Tuncer, A1; Gumruk, F2; Cetin, M1; Sekerel, B1 1 Hacettepe University School of Medicine, Pediatric Allergy and Asthma Unit, Ankara, Turkey, 2Hacettepe University School of Medicine, Pediatric Hematology Unit, Ankara, Turkey

Background: Though L-asparaginase (Lasp) is a critical drug in the treatment of acute lymphoblastic leukemia (ALL), its use may be limited by hypersensitivity reactions where substitution with Erwinia or PEGasparaginase (PEG-asp) is preferred approach. However, in many parts of the world, their availability is limited and premedication and/or desensitisation protocols may be an alternative practice. Aim: To evaluate the effectiveness of the treatment protocol used in children with ALL presented with allergic reactions to L-asp. Method: The children with ALL, treated according to Modified St Jude Total XIII protocol and who experienced allergic reactions with L-asp (Leunases) were included. In case of any allergic reaction to L-asp, PEG-asp (Oncospars) was preferred if could be provided. If PEG-asp could not be provided, L-asp was administered with premedication plus desensitisation in patients with anaphylaxis and alone with premedication in mild allergic reactions. Desensitisation was begun with 1 IU of L-asp and dose was doubled in every 10 min until cumulative dose reached total dose. If allergic reaction repeated with L-asp after premedication and desensitisation, PEG-asp was mandatory for subsequent doses. Results: Nineteen patients (8.8 7 5.4 years old) experienced allergic reactions to L-asp during induction (n 5 7), reinduction (n 5 1) and maintenance (n 5 11) treatments. Mild allergic reactions involving skin and/or gastrointestinal systems were seen mainly during induction (n 5 7) whereas anaphylaxis was observed during maintenance (n 5 9) (P 5 0.002) of which 6 cases were life threatening episodes. Only 3 patients could be treated with PEG-asp as first line. Ten children who experienced anaphylaxis underwent premedication and desensitisation and any subsequent clinical hypersensitivity reaction were not observed in 8 of them. The other 2 non-tolerant patients received PEG-asp successfully. Six patients who had mild allergic reactions were premedicated and only two experienced allergic reactions. In summary, a total of 12 patients were treated with L-asp after an initial reaction with L-asp. Conclusion: L-asp could be administered to more than half of the patients who had a hypersensitivity reaction with L-asp before. 108

In countries with shortages of alternative asparaginase preparations, this approach may be a suitable option.

253 A computer alert system to prevent allergic reactions to drugs due to administration errors in the hospital Gastaminza, G1; Idoate, A2; Goikoetxea, M1; Ferrer, M1 Clıńica Universitaria de Navarra, Alergologıá e Inmunologıá Clıńica, Pamplona, Spain, 2Servicio de Farmacia, Spain

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Introduction: Drug allergic reactions are a frequent cause of morbimortality in in- and out-patients. Sometimes these reactions are due to the mistaken administration of the drug to a patient already diagnosed of allergy. Methods: A computer system has been designed that, when the prescription is being prepared, authomatically checks the information about drug allergy included in the computarized medical history. If a patient is being prescribed a drug to which he/she has already been diagnosed or relates an allergy, the system emits an alert to the doctor, who must decide whether the drug should be substituted by another one. The results of the system have been analysed 6 months after its implementation (July-December 2007): number of attempts stopped or not, causes, most frequent drugs, system errors (wrong alert). Results: In these 6 months, a total of 535 attempts to prescribe a drug to which the patient is presumably allergic were recorded. Out of these, 445 attempts were stopped in 224 different patients; 41% of the drugs were NSAIDs, 38% antibiotics and 21% other drugs. In 52% of the cases, the drug prescribed was directly forbidden, in 25% it belonged to a family related to the patient’s allergy and in 17% the patients were intolerant to NSAIDs. Only 2% were considered errors of the system. In 90 attempts in 75 different patients, the doctor decided to continue with the administration of the drug (31% antibiotics, 29% NSAIDs, 12% anaesthetics and opiates, 11% iodinated contrast media and 17% other drugs). The reasons to continue the administration were: good previous tolerance (35%), ‘‘medical criteria’’ (28%), allergological study (24%), error in the history documents (7%), error in the administration (2%), other reasons (4%). Conclusions: By means of a simple computer system, the mistaken administration of a drug was avoided in an important number of patients allergic to that drug and therefore it could prevent the appearance of a sizeable number of allergic reactions to drugs. The number of system errors is small.

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254 Aspirin desensitisation on patients with aspirin intolerance and nasal polyps: a new therapeutic approach by the intravenous route in comparison to oral application Pfaar, O; Klimek, L University Clinic Mannheim, Center for Rhinology and Allergology, Wiesbaden, Germany

Background: Intolerance reactions to acetylsalicylacids (ASA) and to other nonsteroidal anti-inflammatory drugs (NSAIDs) show a classic triad of symptoms (Samters triad): chronic rhinosinusitis with nasal polyps, bronchial asthma and hypersensitivity to ASA, respectively Aspirin. In recent years, clear evidence has been found that ASA intolerance is likely to be mediated by a deviation of the arachidonic acid metabolic pathway toward excessive leukotriene (LT) production. ASA desensitisation represents the only specific treatment that can be offered to ASA-intolerant patients. In classic ASA desensitisation, gradually ascending doses of NSAIDs are given by oral application. This study was initiated to evaluate the possible side effects by using the intravenous route (i.v.) for the ascending doses in ASA desensitisation via infusions in comparison to the (conventional) oral route. Methods: We hospitalized 90 patients (49 females, 41 males, mean age of 35,7 years) with a clear positive history of ASA-intolerance, (recurrent) sinunasal-polyps and a positive test result in the ‘‘Analgetics Intolerance’’in-vitro-test and randomised them in three groups: 30 patients were given oral-ASA in ascending doses (to a maximum of 500 mg at day of discharge) whereas 60 patients received ASA via an infusion through the i.v-route (29 patients once daily and 31 patients twice daily to the maximum dose of 500 mg, respectively 900 mg at day of discharge). Results: Systemic side effects in the two i.vgroups were lower compared to the peroral route (in this group 21% of patients). Furthermore, the rate of systemic reactions were even lower in the group receiving i.vASA twice daily (4.1%) compared to a single daily infusion (9.4%). Conclusion: Together, our study clearly revealed that the i.v. route for the ascending phase of ASA desensitisation is a safe procedure without severe complications. To our knowledge this is the first study showing the methodical feasibility of the intravenous route for the dose-increase phase of ASA desensitisation and comparing these results with the (conventional) peroral route. A clinical benefit of this procedure might be the opportunity of interrupting the ASA application by stopping the infusion therapy in case of (beginning) systemic reactions. Furthermore, high



cumulative doses of ASA could be safely reached with the i.v.-route in this study.

255 Rapid and semi-rapid desensitisation protocols for patients with acute coronary artery disease and aspirin hypersensitivity Labrador-Horrillo, M1; Bartra, J2; Guilarte, M1; MunozCano, R2; Sala-Cunill, A1; Rueda-Garcia, M1; Luengo, O1; Valero, A2; Cardona, V1; Picado, C2 1 Hospital Vall d’Hebron, Allergy Unit, Barcelona, Spain, 2 Hospital Clinic, Pneumology Department, Allergy Unit, Barcelona, Spain

Background: Aspirin and other nonsteroidal anti-inflammatory drugs are among the most common causes of adverse drug reactions. Some patients with cardiologic indications for combination therapy with aspirin and clopidogrel (stent placement and/or acute coronary syndrome) have a history of aspirin hypersensitivity. Aim: to apply a rapid or semi-rapid aspirin desensitisation protocol in a group of patients with an acute coronary event and a history of aspirin intolerance. Patients and methods: From October 2006 to December 2007, 17 patients (14 men), median age 64 years old (range 27–77), with an acute coronary artery disease and a history of aspirin hypersensitivity who had imperative indications for combination therapy with aspirin and clopidogrel antiaggregation for a stent placement were included. These patients were desensitised with oral aspirin (or endovenous lysine-aspirin equivalent) in a rapid (final dosis 100 mg) or semirapid (final dosis 300 mg) procedure depending on theirs characteristics. Three different desensitisation protocols were applied: Protocol A (5 patients): 0.1, 0.3, 10, 30, 40, 81, 100 mg each 20 min. Protocol B (7 patients): 5, 10, 20, 40, 75 and 100 mg each 30 min. Protocol C (6 patients): 30 and 60 mg with a 90 min period on the first day and 150 and 300 mg each 90 min on the second day. All patients were monitored in the coronary care unit during and after the 24h procedure. Results: In all 17 patients, reintroduction was successful. Three patients developed aspirin related reactions. One patient (protocol C), 3h after 60 mg of oral aspirin presented palpebral angioedema. On the following day, protocol A was applied and 5 min after the 100 mg dosis he developed a mild anaphylaxis with good response to treatment. Twenty-four hour later, the same patient tolerated the last two doses of protocol A at 40 min intervals. A second patient (protocol A), developed a mild anaphylaxis with the 40 mg dosis and presented a good response to treatment. However, the next day he tolerated this protocol starting with the 30 mg dosis. A third patient (protocol B), presented facial urticaria 90 min after the last dosis which resolved r 2008 The Authors Journal Compilation

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successfully with treatment. On the next day he tolerated 100 mg aspirin. Conclusion: This aspirin desensitisation procedures may allow us the introduction of this drug at antiplatelet doses in aspirin allergic or intolerant patients with imperative cardiologic indications for double antiplatelet therapy. 256 Drug provocation tests with cyclooxygenase-2 inhibitors: perception of the results and clinical utility Pereira da Silva, S; Lopes da Silva, S; Mascarenhas, I; Lopes, A; Spıńola Santos, A; Branco Ferreira, M; Pedro, E; Pereira Barbosa, M Hospital Santa Maria, Immunoallergology, Lisbon, Portugal

Background: Drug provocation test (DPT) is widely considered as the gold-standard to establish or exclude drug hypersensitivity (HS) and it occupies an important proportion of our outpatient clinic activity. Written information on which drugs patients (pts) can use and wich they must avoid should be made available after DPT, based on the immediate result of this procedure. Aim of the study: 1-Evaluate the level of awareness on safe alternative drugs of pts previously submitted to DPT with cyclooxygenase (COX)-2 inhibitors and negative immediate result; 2- access the clinical utility of the test. Methods: We selected 94 pts from our database with at least one negative DPT with COX2 inhibitors between Jan 2005 and Jan 2007. Each patient was submitted to a questionnaireguided personal interview. Pts were enquired about the results of DPT performed, whether they kept related written information, if they had ever since taken the safe alternative they were given and if it had been well tolerated. Results: Pts were divided in two groups: A – Clinical history highly suggestive of nonsteroidal anti-inflammatory drugs (NSAIDs) HS (F-67%; mean age: 47 7 15 years-old); B – NSAID HS less probable (F-93%; mean age: 47 7 14 years-old). Three DPT were reclassified as late positive and were excluded from analysis. Results are summarized in the following table. (legend: T-Tolerated; NNNo need; F-Fear)

Independently of clinical history, only approximately one third of the patients used the alternative drug, corresponding to the patients that recalled the name of the tested drug. Pts that did not tolerate presented similar symptoms to those that motivated the DPT, 12 h after consecutive intake of the drug. Reasons not to use the alternative drug were different between the two groups. Most of the patients in group A stated they didn’t need the drug while in group B the main reason not to use it was fear from a reaction. Conclusion: DPT procedures consume a huge proportion of time and human work. Our results alert for the need to reinforce the information to our patients about the drugs they can use after the DPT, to optimize the utility of the DPT procedure.

257 Work up of patients with a history of betalactam hypersensitivity Silva, R; Cruz, L; Botelho, C; Cadinha, S; Rodrigues, J; Castel-Branco, G Hospital S. Joaõ, EPE, Allergy Division, Porto, Portugal

Introduction: Beta-lactam (BL) antibiotics, such as penicillin, amoxicillin and cephalosporins are the most frequent cause of antibiotic hypersensitivity reactions (HR), which may be immediate or take several days to present. Most of them are maculopapular or urticarial rashes, but severe reactions such as anaphylaxis also occur. Objective: To evaluate the demographics, clinical manifestations, and management options of patients with suspected BL HR. Material and methods: A review of clinical files of all patients with suspected BL HR who were studied in our Department in the last 30 months. Results: Sixty-three patients (52 F, 11 M), with a mean age of 36.1 7 19.0 years (4–78 years) were studied for suspected BL HR. The most frequent self-reported antibiotics were penicillin in 23 cases (36.5%), amoxicillin and amoxicillin/clavulanic acid in 28 (44.4%), cephalosporins in 10 (15.9%) and flucloxacillin in 2 (3.2%). The clinical

Table 1 for abstract 256. Posterior use of the alternative drug

Posterior use of the alternative drug

Yes

Yes

No

No

Recall the name of the drug used in the DPT

Recall the result of the DPT

Written information

TOTAL (n 5 31)

T

TOTAL (n 5 60)

NN

F

A (n 5 61) B (n 5 30) TOTAL (n 5 91)

37% 40% 37%

79% 80% 79%

51% 47% 49%

31% 40% 34%

84% 92% 87%

69% 60% 66%

69% 44% 65%


No

31% 56% 35%

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manifestations were cutaneous in 41 patients (65.1%), respiratory in 3 (4.8%), gastrointestinal in 2 (3.2%), cutaneous 1 respiratory in 7 (11.1%), cutaneous 1 gastrointestinal in 6 (9.5%) and others in 4 (6.3%). Skin prick tests (SPT) and intradermal tests (ID) with PPL, MDM, penicillin G, amoxicillin, ampicillin and the suspected cephalosporin were performed, as well as specific IgE determination. In non-immediate HR, epicutaneous testing was also done. If all were negative, an oral drug challenge (ODC) was performed in most cases. SPT were positive in 3 patients (4.8%), ID were positive in 3 patients (4.8%) and specific IgE was positive in 9 patients (14.3%), with values between 0.40 and 2.30 KU/L. Only one patient with positive specific IgE had positive skin testing. Of the remaining 49 cases, 32 were submitted to ODC with the culprit antibiotic, with a positive reaction to amoxicillin in 2 cases (6.3%). In all positive cases and when an ODC with the suspected antibiotic was not indicated, an ODC with an alternative drug was done, all with negative results (27 patients). Comments: Of the 63 studied cases with history of BL HR, 16 (25.4%) were confirmed after testing. A combination of skin testing, specific IgE determination and ODC is necessary, since none has enough sensitivity to be used alone. In cases of confirmed BL HR, an ODC with an alternative antibiotic is indicated, in order to determine a safe alternative.

258 Hypersensitivity to gelatin used as excipient for soft capsules of Ibuprofen Heffler, E; Guida, G; Badiu, I; Pizzimenti, S; Bergia, R; Bommarito, L; Nebiolo, F; Rolla, G University of Torino – ASO Ordine Mauriziano ‘‘Umberto I’’, Allergy and Clinical Immunology, Torino, Italy

Background: Allergic reactions to non-steroidal anti-inflammatory drugs (NSAIDs) are common. Ibuprofen is frequently involved in hypersensitivity to NSAIDs. Hypersensitivity to excipients contained in NSAIDs are less frequently reported. In the last years soft capsules has been developed in order to speed up the absorption of the drug by the oral mucosa; these capsules may contain ‘‘gelatin’’ as excipient. Patient and Methods: A female 49 y.o. patient referred to our Allergy Outpatients’ Clinic for several episodes of labial angioedema about 180 min after having taken a soft capsule of Ibuprofen. The patient did not have any reaction after ingestion of other pharmaceutical formulations of Ibuprofen, nor with other NSAIDs. She underwent to skin prick tests (SPT) for common inhalant and food allergens, including 110

bovine and swine meat, patch test with gelatin diluted with physiological solution at the rate of 1/1 and, 7 days after, to a prickby-prick with gelatine diluted in the same way. Five atopic and five non atopic volunteers underwent to the same skin tests for gelatin. Results: SPT with inhalant and food commercial extracts and patch test with gelatin were negative. Prick-by-prick with gelatin resulted positive after about 30 min, and after about 180 min the patient had urticaria on the left harm starting from the point in which prick-by-prick was performed and labial angioedema. Controls subject had all negative tests for gelatin. Conclusions: We here describe a case of a patient with an allergic reaction to an excipient of soft capsules: gelatin. Allergy to gelatin is well known as a major problem for anti-viral vaccines, while it is probably underdiagnosed for other drugs. In conclusione, allergy to excipients should be investigated in selected cases of suspected drug allergy.

259 Sitagliptin-induced rhinorrhea, cough, headache and fatigue Baraniuk, J1; Jamieson, M2 Georgetown University, Rheumatology, Immunology and Allergy, Washington, DC, United States, 2 McMinnville, Tennesse, United States

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Sitagliptin is a dipeptidyl peptidase-4 (DPPIV) selective inhibitor indicated for Type II diabetes. DPP-IV cleaves the N-terminal dipeptide from glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), RANTES, neuropeptide Y, substance P and other airway peptides, and causes their inactivation. Blocking DPP-IV increases tissue levels of these peptide hormones. In diabetes, insulin secretion is increased while glucacon and glucose are decreased. Case Report: A fifty five year old, atopic and hypothyroid white female developed Type II DM. She had hypothyroidism, atopic asthma, and history of ACE inhibitor-induced cough. Several weeks after starting sitagliptin 100 mg po daily, she developed nasal congestion, post-nasal drip, and a throat-clearing cough. PEFR was 450 when the drug was stopped. The next day her headache and congestion cleared. The cough ceased 3 days later. PEFR rose to 620. Sitagliptin (50 mg) was restarted. Symptoms returned over the next 3 days. Her lowest PEFR was 430 after 2 weeks. She scored congestion severity, post-nasal drip, throat clearing and tiredness/decreased energy at 5 to 8 out of 10, and headache as 5 to 7/10. Cough was intermittent in these 2 weeks. All symptoms disappearred and Journal Compilation

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PEFR returned to normal after stopping the drug. Case Series: Five Type II diabetics on sitagliptin were assessed. Three had no adverse events, including one atopic asthmatic. One atopic diabetic developed copius rhinorrhea refractory to fexofenadine, followed by cough and then fatigue. A nonallergic woman with an history of ACE inhibitor cough developed severe rhinorrhea and cough that did not respond to inhaled mometasone furoate. Conclusions: We anticipated that DPP-IV inhibitors may promote cough, especially in subjects with ACE inhibitor-induced coughing. Surprises were the copious anterior and posterior rhinorrhea, nasal congestion and headache, and potential central nervous system symptom of fatigue. Analysis of the secretions may reveal the glandular or vascular source of the discharge and the nature of the offending peptide in this new syndrome. Unknown substrate(s) of DPPIV may induce fatigue. These substrates may be novel targets for future drug development. Sitagliptin and possibly other DPP-IV inhibitors now join the list of other drugs that cause nonallergic rhinitis symptoms.

260 Adverse drug reactions prevalence in south Italy Manfredi, G Religious Gen. Hospital ‘‘F. Miulli’’, Allergy and Clinical Immunology Unit, Acquaviva delle Fonti (BA), Italy

Background: It’s well known that the higher the adverse drug reaction (ADR) database size, the bigger the powerful to detect drug safety is. The goal of this study is to evaluate ADR prevalence in our country, in order to improve the drug safety. Methods: We analyzed the prevalence of ADR occurred to our observation in the last 2 years. We subdivided ADR into reactions to NSAIDs (Non Steroid Antinflammatory Drugs), Antibiotics and Other Drugs (myorelaxants, local anaesthetics, H1 antihistamine, H2 antihistamine, antithrombotics, leukotriene antagonists, xanthine oxidase inhibitors). Results: Out of 1317 guys observed, 564 (42,8%) had ADR; 302 (53,5%) to NSAIDs, 235 (41,6%) to antibiotics, 28 (4,9%) to other drugs. Among NSAIDs, acetylsalicylic acid/pyrazolones caused 99 ADR (32,7%), arylpropionics 59 (19,5%), nimesulide 47 (15,5%), arylacetic acid derivatives 41 (13,5%), paracetamol 20 (6,6%), oxicam 15 (4,9%), niflumic acid 10 (3,3%), glucosamine 4 (1,3%), tramadol 3 (0,9%), coxib 3 (0,9%), nabumetone 1 (0,3%). Among antibiotics, beta-lactams caused 112 ADR (47,6%), cephalosporins 32 (13,6%), macrolides and lincosamides 23 (9,7%), cotrimoxazol and



sulfamydes 14 (5,9%), aminoglycosides 5 (2,1%), tetracyclins 5 (2,1%), amphenicols 4 (1,7%), unknown 3 (1,2%), glycopeptides 2 (0,8%), other antibiotics 5 (2,1%). Among Other Drugs, myorelaxants caused 15 ADR (2,6%)(joscine 7, thiocolchicoside 5, dipyrone 1, domperidone 1, pridinol 1), local anaesthetics 4 (0,7%)(mepivacaine 2, lidocaine 1, articaine 1), H1 antihistamines 3 (0,53%) (levodropropizine 1, ebastine 1, oxatomide 1), H2 antihistamines 1 (0,17%)(ranitidine),


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antithrombotics 3 (0,53%)(heparin 2, ticlopidine 1), leukotriene antagonists 1 (0,17%) (montelukast), xanthine oxidase inhibitors 1 (0,17%)(allopurinol). Conclusions: This study shows the lowest ADR prevalence among NSAIDs belongs to nabumetone, coxib, tramadol; among antibiotics to rifaximin, metronidazole, clofoctol, ketolides, neomycin, glycopeptides; among myorelaxants to pridinol, dipyrone, domperidone; among local anaesthetics to


lidocaine, articaine. Among H1 antihistamines none ADR occurred to desloratadine, levocetirizine, acrivastine, mizolastine, fexofenadine; among antithrombotics none to sulodexide nor vitamine K antagonists. Leukotriene antagonists and xanthine oxidase inhibitors showed a very low ADR prevalence. It’s possible to improve the knowledge regarding drug safety by comparing these data with drug use in Italy.

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Poster Discussion Session 7 – Basic Mechanisms of Asthma From Mouse Models to Humans

Poster Discussion Session 7 Basic Mechanisms of Asthma From Mouse Models to Humans 261 Teaching old protocols new tricks: adjuvant is not required for asthma induction in mice

262 A role of sialidase in the hyaluronic acid binding ability of CD44-bearing CD4 1 T cells in a murine model of allergic asthma

263 Phosphatidylinositol 3-kinaseg is involved in the pathogenesis of OVA-induced chronic mouse asthma model

Conrad, M; Garn, H; Renz, H Philips University, Biomedical Research Centre, Marburg, Germany

Katoh, S1; Maeda, S2; Fukuoka, H2; Wada, T3; Yamaguchi, K3; Miyagi, T3 1 Faculty of Medicine, Kagawa University, Department of Cell Regulation, Kagawa, Japan, 2CREST, Japan Science and Technology Agency, Saitama, Japan, 3Miyagi Cancer Center Research Institute, Division of Biochemistry, Miyagi, Japan

Takeda, M1; Ito, W1; Ueki, S1; Yamaguchi, K1; Kayaba, H1; Sasaki, T2; Chihara, J1 1 Akita University School of Medicine, Department of Clinical and Laboratory Medicine, Akita, Japan, 2Akita University School of Medicine, Department of Pathology and Immunology, Akita, Japan

Background: The murine model of asthma is an essential tool for in vivo analysis. As the asthma phenotype observed in mice is a product of the method used, protocol selection is paramount. The standard asthma induction model used today involves intraperitoneal (IP) sensitisation with the antigen ovalbumin (OVA) and the adjuvant aluminum hydroxide (alum). As it is unknown how alum stimulates the immune response, this compound is not desirable for use in studies pertaining to the discovery of mechanism. The purpose of this study is to develop a simple, adjuvant free murine asthma induction protocol. Methods: Open field behavioural tests were performed in this study to demonstrate the effect of adjuvant on mice. Several asthma induction protocols were tested varying in; use of adjuvant, mode of antigen delivery and length of aerosol challenge (minutes or days). The resulting asthma phenotype was determined by measurements of airway reactivity, OVA specific antibody levels, lung histology, bronchoalveolar lavage (BAL) cell counts and BAL and spleen cytokine measurements. Results: Subcutaneous (SC) OVA injection without alum produces increased OVA specific IgE titres, airway reactivity, increased eosinophils, lung inflammation and increased BAL IL-5. This model resembles the standard OVA IP 1 alum model with the exception that the SC model produces less IgG1. Behavioral data indicate that mice injected with the adjuvant alum undergo significantly higher stress/discomfort levels than individuals treated without adjuvant therefore, an adjuvant free protocol is preferable. Conclusion: Through comparison of several different asthma induction methods we have developed a simple, SC OVA, adjuvant free protocol that induces a phenotype comparable to the benchmark adjuvant protocol used in the literature. The use of an adjuvant free protocol is significantly less stressful for mice and avoids the added complication of adjuvants that have no known mode of action.

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Background: CD44 is a widely expressed cell adhesion molecule, which is involved in lymphocyte infiltration into inflammatory tissues. We have demonstrated that CD44 plays an important role in accumulation of Th2 cells into the lung in a murine model of allergic asthma. Although CD4 1 T cells express CD44 on their surface, few of the cells use the molecule for recognition of the ligand hyaluronic acid (HA). We previously reported that an inducible sialidase might influence the glycosylation and receptor activity of CD44 on monocytes. Now we evaluate the role of sialidase in the HA binding ability of CD44 expressed on CD4 1 T cells using a murine model of allergic asthma. Methods: We developed a murine model of allergic asthma sensitised by intraperitoneal injection of mite allergen with alum, followed by transnasal administration of same allergen. HA binding ability of splenic CD4 1 T cells in this asthmatic model was examined by flow cytometry. Expression of sialidase (Neu-1, Neu-2, and Neu-3) in the spleen of this model was evaluated by RTPCR. Results: A small but significant number of CD4 1 T cells in the spleen of this asthmatic model was able to bind HA in a CD44 dependent manner. This binding was upregulated by treatment with bacterial sialidase in vitro. These CD4 1 T cells displayed increased receptor activity of CD44 after culture with antigen in vitro, along with characteristic paralleled induction of sialidase (Neu-1 and Neu-3) expression. This induction of HA binding ability was significantly suppressed by the sialidase inhibitor 2-deoxy2, 3-dehydro-N-acetylneuraminic acid. Conclusion: CD4 1 T cells may be capable of enzymatic remodeling cell surface CD44, altering their ability to interact with the extracellular matrix. Our observation suggests that endogenous sialidase could influence Th2-related airway inflammation of asthma.

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Bronchial asthma is a syndrome associated with persistent functional and structural changes within the airways through chronic airway inflammation. Airway mucosal inflammation is characterized by an influx of inflammatory cells such as activated eosinophils and T lymphocytes. These cells play critical roles in orchestrating the allergic inflammatory response leading to airway hyperresponsiveness (AHR). Furthermore, persistent inflammation leads to airway remodeling which includes subepithelial fibrosis, hyperplasia of mucus glands, and myofibroblast and smooth muscle proliferation. Phosphatidylinositol 3-kinase (PI3 K), which is a family of conserved lipid kinase, plays a prominent role in various inflammatory cells by regulating fundamental cellular responses, including proliferation, differentiation, cell survival, cytokine production, cell migration, and adhesion through its downstream components. Recently, it has reported that the inhibitor of PI3Ks attenuates chemokine-induced eosinophil migration in vitro. However, few studies has reported that PI3K is involved in the pathogenesis of asthma in vivo, especially airway remodeling. In this study, we investigated a role of PI3Kg in allergen-induced airway inflammation and airway remodeling using PI3Kg deficient mice. Following ovalbumin (OVA) sensitisation and challenge, wild type (WT) and PI3Kg deficient mice were exposed to aerosolized OVA on 3 days/week for 5 weeks. In OVA-sensitised and challenged WT mice, AHR to inhaled methacholine, the number of lung inflammatory cells as well as Th2 cytokines and growth factors in bronchoalveolar lavage fluid were significantly increased compared with negative control mice. In addition, the features of airway remodeling such as goblet cell hyperplasia, smooth muscle hyperplasia, and subepithelial fibrosis were similarly increased compared with negative control mice. On the other hand, these responses were not found in OVA-sensitised and challenged PI3Kg deficient mice. These



results indicate that PI3Kg is involved in the pathogenesis of OVA-induced chronic asthma model, especially airway remodeling.

264 EP receptor agonists induce upregulation of lung cyclooxygenase-2 mRNA expression in vivo in non-sensitive and house dust mite-sensitive mice Herrerias, A1; Torres, R2; Marco, A3; Picado, C4; de Mora, F1 1 Universitat Auto`noma de Barcelona, Department of Pharmacology, Barcelona, Spain, 2IDIBAPS, Clinical experimental respiratory immunoallergy, Barcelona, Spain, 3Universitat Auto`noma de Barcelona, Department of Animal Pathology, Barcelona, Spain, 4Hospital Clıńic, IDIBAPS, Universitat de Barcelona, Department of Pneumology and Respiratory Allergy, Barcelona, Spain

Background: Both, cyclooxygenase (COX)1 and 2 are responsible for the synthesis of prostaglandins (PG), but the inducible COX-2 form has classically been viewed as a pro-inflammatory enzyme. In spite of this, the COX-2 product PGE2 has been suggested to exert an anti-inflammatory effect in the respiratory system by means of its EP receptors. This discrepancy could partly be attributable to PGE2 downregulation of COX-2 mRNA expression as previously shown in vitro in macrophages, but experiments with fibroblasts do exhibit opposing data. In order to elucidate the effect of EP receptors on the enzyme’s regulation in vivo, we investigated the impact of exogenous EP agonists on airway COX-2 production in the lungs of non-sensitive and HDM-sensitised mice. Methods: We assessed the effects of exogenous PGE2 (0.5 mg/kg, sc) and of the EP1/ EP3 agonist sulprostone (80 mg/kg, sc) on COX-2 mRNA expression (real time PCR) in the lungs of mice exposed intranasally to either house dust mite (HDM) allergens (n 5 8–10/group) or saline (n 5 5/group). In parallel, we looked at the effect of PGE2 and sulprostone on the mice’s airways inflammation. Results: In non-sensitive animals both PGE2 and sulprostone induced respectively a 76% and 141% increase of COX-2 mRNA expression above baseline lung levels. Exposure to HDM significantly upregulated (262%) baseline COX-2 mRNA expression in untreated animals. Such allergen-induced enhanced enzyme’s production was further increased (156%) by sulprostone pretreatment of the mice but not by PGE2. Interestingly, the EP receptors-induced fluctuations of COX-2 mRNA expression were not correlated with changes in the counts of the inflammatory cells infiltrating the lungs. Conclusion: EP receptors do modulate lung COX-2 production in vivo in mice with a predominating positive feedback possibly by means of an EP3/EP1-mediated mechanism. r 2008 The Authors Journal Compilation

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Such modulation is observable both in allergen-sensitive and non-sensitised animals. Surprisingly, this work furthers into the complex EP-COX-inflammation network through the demonstration of EP agonists-induced upregulation of lung COX-2 in vivo with no impact on airway inflammation.

265 Altered expression of TRESK in a mouse model of bronchial asthma Jeong, Y Republic of Korea

Introduction: TRESK, a member of twopore domain K1 channel family, is strongly expressed in the spleen and thymus as judged by Northern blot analysis. TRESK is modulated by intracellular Ca2 1 , calcineurin, arachidonic acid, immunosuppressant (FK506), and GPCR agonists such as acetylcholine and histamine. Based on these properties, this study was carried out to investigate whether the expression of TRESK is changed in animal models for asthma. Methods: To generate an asthma mouse model, 6-week-old BALB/c WT mice were sensitised intraperitoneally twice with 75 fı` g of ovalbumin (OVA) plus 2 mg of alum on days 1 and 7, and then challenged intranasally with 50 fı` g of OVA on days 14, 15, 21, and 22. As a negative control group, sameaged WT mice were sensitised intraperitoneally and then challenged intranasally with phosphate-buffered saline (PBS) on the same days. The expression of TRESK was analyzed using real-time PCR and Western blot analysis. The cDNA and proteins were prepared from lungs of mouse models. Results: Protein levels were up-regulated by B30-fold compared to control group and TRESK mRNA was also markedly increased compared to control group. Conclusion: TRESK might contribute to the background K1 conductance in lung cells and be a good target for therapeutic potential for the treatment of asthma.


266 Modulation of the antigen dose during allergic sensitisation reveals a key role for CD8 1 T cells in reducing allergic airway inflammation Aguilar-Pimentel, J1; Huster, K2; Alessandrini, F3; Jakob, T4; Behrendt, H5; Ring, J1; Busch, D2; Mempel, M4; Ollert, M4 1 Klinikum rechts der Isar der TU Mu¨nchen, Dermatology and Allergy, Biederstein, Munchen, Germany, 2Klinikum rechts der Isar der TU Mu¨nchen, Institute of Microbiology, Immunology, and Hygiene, Munich, Germany, 3Klinikum rechts der Isar der TU Mu¨nchen/ Helmholtz Zentrum Mu¨nchen, Division of Environmental Dermatology and Allergy, Munchen, Germany, 4Klinikum rechts der Isar der TU Mu¨nchen, Dermatology and Allergy, Biederstein, Munich, Germany, 5Klinikum rechts der Isar der TU Mu¨nchen, Division of Environmental Dermatology and Allergy, Munchen, Germany

In murine allergy models the dose of the sensitizing antigen has previously been shown to affect the phenotype of IgEmediated airway inflammation. Application of abundant antigen during the sensitisation phase lead to a significant reduction of the inflammatory response in contrast to a low dose sensitisation. Studies in both, low and high dose sensitisation protocols indicated that in addition to CD41 T cells, CD81 T cells also have an important role in mediating and regulating allergic inflammation. In this study we used different markers of CD41 T cells (e.g. CD154) and CD81 T cells (e.g. MHC class I multimer technology) to analyze the induction, the natural distribution and the phenotype of allergenspecific CD41 and CD81 T cells in a murine C57BL/6 model of alum-ovalbumin (OVA)-induced IgE-mediated allergy. Responses in our allergy model were typically characterized by the induction of OVAspecific IgE and IgG1 and airway eosinophilia following OVA allergen aerosol exposure. Conditions of antigenic overload, which resemble specific immunotherapy and antigen presentation in the context of bacterial signals, were favourable for inducing a protective and cytotoxic OVA257-264-specific CD81 T cell response. In parallel we observed a dramatic reduction in the number of antigen-specific CD41 T cell and significantly reduced airway eosinophilia and total cell number in lung and BAL. Thus, our data suggest a protective role for allergen-specific effector CD81 T cells in the regulation of airway inflammation, which may be due to their specific cytokine and migration pattern, and most importantly to the context of antigen priming.

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267 Increased expression of Lipocalin 2 suppresses allergen-induced airway disease in mice

268 A. fumigatus induced allergic asthma in a mouse model of sensitisation to self HSP70 protein

Meyer, H1; Krokowski, M1; Dittrich, A2; Cowland, J3; Hamelmann, E1 1 Charite, Department of Pediatric Pneumology and Immunology, Berlin, Germany, 2Hannover Medical School, Junior Research Group SFB 587, Hannover, Germany, 3University of Copenhagen, Department of Hematology, Copenhagen

Shevchenko, M; Shekhovtsova, E; Sapozhnikov, A Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Immunology, Moscow, Russian Federation

Background: Allergen-induced bronchial asthma is a chronic airway disease in which the interplay of various endogenous genes with environmental factors regulates inflammatory responses in the lungs. The single factors mediating this process are yet not fully described. The aim of the project was to identify and validate the biological significance of new genes/proteins involved in the development of experimental allergic airway disease in a murine asthma model. Methods: RNA microarray analysis of expression profiles from lung tissues of two mouse strains with different allergic susceptibility (BALB/c vs. C57/Bl6) was used to identify common genes regulated upon allergen sensitisation and airway challenges. Target regulation and in situ localization were confirmed by western blot of bronchoalveolar lavage fluid and immunohistochemistry of lung tissues, respectively. Requirements for induction of target genes and its effects on cell survival were assessed in epithelial cell cultures via RT-PCR and TUNEL-assay, respectively. Finally, functional relevance of the discovered new target genes was assessed utilizing knock-out mice. Results: Among various genes, Lipocalin2 was found to be strongly upregulated in lung tissues of sensitised and challenged mice compared to control animals. The expression of lipocalin2 in lung tissue increased in both strains after induction of airway inflammation on RNA and protein level. Functional analysis revealed induction of Lcn2 in epithelial cells via different proinflammatory cytokines and LPS and suggested a pro-apoptic effect of Lipocalin2. Allergen sensitisation and airway challenges of lipocalin2 knock-out mice resulted in vastly elevated airway inflammation and hyperresponsiveness, associated with decreased levels of apoptosis in lung tissue. Conclusion: These data suggested a protective role for Lcn2 in allergic airway disease with the pro-apoptic effect as the underlying mechanism. Lcn2 thus is a newly discovered mediator between innate and adaptive immune responses with a potential protective role for this molecule in allergic airway disease.

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Autosensitisation was shown to be associated with severe manifestation of asthma or atopic dermatitis. This phenomenon is explained as a cross-reactivity of antibodies, initially produced in response to foreign antigens, to structurally homological self proteins. Elongation of aerosol challenge phase, mimicking the chronic allergen exposure, in a standard mouse model of OVAinduced asthma, was shown to increase IgG autoreactivity. The aim of the present study is to investigate the effect of sensitisation to self HSP70 protein on A. fumigatus (Af) induced allergic asthma in a mouse model. Mouse HSP70 (mHSP70) for mice sensitisation was yelled from liver and kidney homogenate of syngeneic mice by chromatography on ATP-agarose column. Control groups of mice were immunized with HSP70, obtained from sonicated culture of Af, (AfHSP70) or OVA respectively. BALB/ c mice received correspondent antigen i.p. in PBS, in dose 10ug/mouse/injection, 10 times during two weeks – short protocol, or 3 short protocol courses with a month interval after each course – long term sensitisation. A week after the last injection mice were i.n. challenged with Af crude extract (Af crude) 125ug/mouse/time, 3time/day, 3days/week. Serum specific IgG production was estimated by ELISA. Lung inflammation was characterized by total and differential BAL cell count. No serum mHSP70-specific IgG production were found after both short and long term immunization of mice with mHSP70. Af crude challenge didn’t influence anti-mHSP70 production as well. OVA- and AfHSP70-specific IgG production in respective groups started a week after the first injection with the maximum on the third week. Induced by Af crude challenge lung inflammation was significantly enhanced in mice, underwent long term immunization with mHSP70 (17 7 0,7 mln cells/mouse BAL) compare to mice, sensitised with AfHSP70 or OVA (1 7 0,3 mln cells/mouse BAL). Bronchoalveolar infiltrates in mice, immunized with self HSP70 were dominantly represented by neutrophils, number of which was more then 10 fold increased compare to mice, immunized with OVA or Af HSP70. Mice, received mHSP70 according to the short protocol, followed by Af crude challenge, didn’t show pronounced lung inflammation, similarly to challenged with Af crude normal mice. Sensitisation to self HSP70 protein is characterized with

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latent specific antigen production, but causes severe lung inflammation, induced by exposure to inhaled Af allergens.

269 Selective nasal allergen provocation rapidly induces bronchial hyperresponsiveness via substance P release by pulmonary nerves Callebaut, I1; Hens, G1; Raap, U2; Vanoirbeek, J3; Nemery, B3; Bullens, D1; Ceuppens, J1; Hellings, P1 1 Katholieke Universiteit Leuven, Division of Clinical Immunology, Leuven, Belgium, 2Hannover Medical University, Department of Dermatology and Allergology, Hannover, Germany, 3Katholieke Universiteit Leuven, Division of Lung Toxicology, Leuven, Belgium

Background: Allergic rhinitis patients have an increased prevalence of bronchial hyperresponsiveness (BHR). The mechanism by which nasal allergen deposition causes BHR is unknown. This study explores the role of substance P in the induction of BHR after nasal allergen deposition. Methods: In mice sensitised to ovalbumin (OVA), selective nasal OVA (1 mg in 10 mL saline) provocations were performed following complete anatomical separation of upper and lower airways by means of a tracheotomy. Control mice received saline (10 mL). Four hours after nasal allergen provocation, bronchial responsiveness to metacholine was measured using a computer-controlled small animal ventilator. Endobronchial substance P was measured by ELISA and the cellular source of substance P was investigated by immunohistochemistry. In separate groups of mice, the substance P receptor antagonist L-732138 was administered before the nasal provocation. Finally, we studied the effect of endobronchial substance P administration on the bronchial responsiveness to metacholine. Results: Four hours after selective nasal OVA deposition, a clear BHR was found as compared to control mice. Simultaneously, an induction of substance P in the bronchial lumen was demonstrated. Substance P was produced by pulmonary nerves, as demonstrated by immunohistochemistry. Treatment with a substance P receptor antagonist abolished the allergeninduced BHR completely. Moreover, endobronchial administration of substance P dose-dependently increased bronchial responsiveness in OVA-sensitised mice. Conclusion: We show a rapid induction of BHR after selective nasal allergen deposition. This is mediated via substance P release by pulmonary nerves, suggestive of a neural reflex pathway between nose and bronchi after nasal allergen contact.



270 Transcriptional regulation of type I interferon-b and type III interferon-k in bronchial epithelial cells. Common role of the transcription factor IRF-3 Edwards, M; Slater, L; Haas, J; Kotenko, S; Bartlett, N; Johnston, S Imperial College London, Respiratory Medicine, London, United Kingdom

Asthmatic bronchial epithelial cells cultured ex vivo and infected with rhinovirus show a deficiency in interferon-b and interferon-l mRNA and protein compared to nonasthmatic controls, this deficiency may contribute to the pathogenesis of viral induced asthma exacerbations. Using siRNA studies, and reporter gene experiments to investigate the transcriptional regulation of interferon-b and interferon-l genes. Deletion mutants and point mutations of the interferon-b promoter ( 125 bp fragment) and interferon-l1 promoter ( 1kB fragment) were constructed, and used to transfect primary bronchial epithelial cells and BEAS-2B cells. We observed that the PRDII (NF-kB), PRDI, III (IRF) and PRDIV (ATF2/cJun) sites were all required for maximal interferon-b promoter activation at 24 h using rhinovirus infection, or by transfection with constitutively active RNA helicase RIG-I (DRIG-I) or TLR3 adaptor, TRIF (DTRIF) compared to control cells (Po 0.01, n 5 3–4 experiments) Using the interferon-l1 promoter, and human rhinovirus infection or A¨RIG-I transfected cells, maximal activation was observed with several deleted constructs, however promoter activation was markedly reduced using a construct lacking a IRF/ISRE site at position 766 bp from the transcription start site (Po0.01 vs. full length contruct, n 5 3 experiments). Using siRNA studies in nonsmoking, non-asthmatic primary bronchial epithelial cells, siRNA targeting ATF2, IRF-3, IRF-1, c-Jun but not IRF-7 or NF-kB p65 reduced rhinovirus induced interferon-b mRNA levels compared to cells transfected with control, non-specific siRNA at 24h post infection (Po0.01, n 5 4–5 experiments). In similar experiments focusing on interferon-l1 and interferon-l2/3 mRNA, siRNA targeting IRF-3, but not IRF-1, IRF-7, ATF2, c-Jun or NF-kB p65 reduced rhinovirus induced mRNA levels compared to cells transfected with control siRNA at 24h (Po0.01, n 5 4–5 experiments). All siRNA reduced their respective target mRNA by at least 75%, and also reduced endogeneous protein levels, and did not induced interferon mRNA without rhinovirus infection. We conclude that IRF-3 is a common transcription factor required for rhinovirus induced interferon-b and interferon-l gene expression in bronchial epithelial cells, and suggests that r 2008 The Authors Journal Compilation

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deficient IRF-3 mediated signalling events or altered IRF-3 signalling in the asthmatic phenotype could be implicated in deficient interferon-b and interferon-l expression observed in asthmatic bronchial epithelial cells.

271 Activation of NFkB and induction of T-cell recruiting chemokines by rhinovirus infection in vitro and in vivo Bartlett, N1; Walton, R1; Slater, L2; Message, S2; Clarke, D3; Edwards, M1; Johnston, S1 1 Imperial College London, Respiratory Medicine, London, United Kingdom, 2Imperial College London, Respiratory Medicine, London, United Kingdom, 3 Imperial College London, Respiratory Pharmocology, London, United Kingdom

Respiratory virus infections, most commonly rhinovirus (RV), are the most frequent cause of acute asthma exacerbations. Recruitment of lymphocytes to the airways is a characteristic feature of asthma exacerbations. In vitro RV-infected primary human bronchial epithelial cells (HBECs) produce T cell-recruiting chemokines IP10, RANTES and I-TAC. Knock down of the NFkB P65 subunit expression by siRNA in cultured HBECs resulted in almost complete inhibition of RV-induced IP-10 and RANTES gene expression demonstrating a central role for this transcription factor in T cell-chemokine production. Using a mouse-RV infection model we tested the hypothesis that RV infection in vivo induces lymphocytic airways inflammation preceded by activation of NFkB and production of T cell recruiting chemokines. EMSA experiments demonstrated binding of lung nuclear protein from RV-infected mice to an NFkBspecific nucleotide probe. Analysis of BAL cells from RV-infected mice demonstrated lymphocytic airways inflammation (peaking days 4 to 7 after infection) following production of the T cell recruiting chemokines IP-10 (750 pg/mL), RANTES (275 pg/ mL) and I-TAC (800 pg/mL) peaking on days 1 to 2. These responses were replication dependent as they were not observed in mice treated with UV-inactivated RV. We have also developed a human-RV experimental infection model and hypothesized that production of T cell recruiting chemokines would be enhanced in RV-infected asthmatics. Normal (n 5 12) and asthmatic (n 5 7) subjects were experimentally infected with RV and bronchial alveoloar lavage (BAL) performed prior to- (baseline) and four days post-infection. RV infection induced a small but non-significant increase in I-TAC and IP-10 over baseline in normal subjects. In contrast I-TAC (Po0.01) and IP-10 (Po0.05) in BAL from asthmatics were significantly increased compared to baseline asthmatic- and day 4 normal-levels.


We show for the first time that chemokine production is significantly up-regulated in asthma following experimental RV infection of humans whilst mouse and cell-culture based infection models have demonstrated the potentially important role of NFkB in the pathogenesis RV-induced asthma exacerbations.

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Abstract moved to 898b.

273 Intranasal prostaglandin E2 reduces airway inflammation in a house dust miteinduced mouse model of allergic asthma Herrerias, A1; Torres, R2; Marco, A3; Picado, C4; de Mora, F1 Universitat Auto`noma de Barcelona, Department of Pharmacology, Barcelona, Spain, 2IDIBAPS, Clinical experimental respiratory immunoallergy, Barcelona, Spain, 3Universitat Auto`noma de Barcelona, Department of Animal Pathology, Barcelona, Spain, 4Hospital Clıńic, IDIBAPS, Universitat de Barcelona, Department of Pneumology and Respiratory Allergy, Barcelona, Spain

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Background: Clinical and experimental data suggest that prostaglandin (PG) E2 could play a defensive role in the lungs. Accordingly, previous reports showed that PGE2 prevents allergen-induced airway hyperresponsiveness (AHR), and airway eosinophilia in asthma patients. In order to establish a murine system in which to further into the anti-inflammatory mechanisms of PGE2 in asthma, we assessed the effect of this PG in a house dust mite (HDM)-sensitive mouse model, the features of which, namely daily delivery exclusively through the airways of a natural aeroallergen known to cause human asthma, provide ideal conditions to mimic the human disease. Methods: PGE2 was administered intranasally to mice either non-exposed or exposed to allergens through the airway for 10 consecutive days (n 5 6 mice/group). Four therapeutic conditions were established by combining 2 doses of PGE2 (0.3 or 0.15 mg/ kg) with 2 delivery schedules; either one day (10) or two days (5 and 10) administration of the PG along the exposure to HDM allergens. We then analyzed the in vivo impact of the local administration of PGE2 on the AHR to methacholine, the number of bronchoalveolar lavage (BAL) inflammatory cells, and the production of IL-13 protein in the lungs. Results: HDM allergens induced a strong inflammatory process in the airways paralleled by changes of airway reactivity to methacholine. Although AHR was not affected by the local administration of PGE2 under any of the established therapeutic conditions as measured by whole body plethysmography, inflammation was 115

Poster Discussion Session 8 – Food Allergy – New Findings and Clinical Responses

significantly reduced under a dose-dependent pattern only when PGE2 was given twice (days 5 and 10) along the course of the induction phase. Inflammatory cells in BAL were diminished by 43% at the dose of 0.15 mg/mL and by 48% at the dose of 0.3 mg/mL. In parallel, PGE2 led to a dosedependent reduction of airway IL-13 production of 26% and 41% at 0.15 mg/kg and 0.3 mg/kg respectively. Conclusion: As for reported data from mild asthmatics, intranasal PGE2 administered twice during the 10-day allergen exposure did partly prevent airway inflammation in HDM-sensitive mice. PGE2 had therefore a beneficial effect on the airway reaction despite the mild therapeutic protocol applied in relation to a sustained allergenic stimulation. We thus have established an in vivo system to investigate the antiasthmatic effect of PGE2, in which we have shown that fluctuating lung IL-13 levels might be a relevant mediating mechanism.

274 Mast cells can mediate vascular permeability through regulation of PI3KHIF-1a-VEGF axis Moon, H; Park, S; Kim, S; Lee, K; Choe, Y; Lee, Y Chonbuk National University Medical School, Department of Internal Medicine, Jeonju, Republic of Korea

Background: Mast cells release a number of mediators that act directly on the vasculature, resulting in vasodilatation, increased permeability, and subsequent plasma protein extravasation. Vascular endothelial growth factor (VEGF) has been implicated to contribute to asthmatic tissue edema through its effect on vascular permeability. However, the effects of mast cells on VEGF-

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mediated signaling in allergic airway disease are not clearly understood. Methods: We have used genetically mast cell-deficient WBB6F1-KitW/KitW v(W/Wv) mice and the congenic normal WBB6F1 7 / 1 ( 1 / 1 ) mouse model for allergic airway disease to determine the role of mast cell in allergic airway disease, more specifically in vascular permeability. Results: We found that levels of VEGF and plasma exudation in W/Wv mice after ovalbumin (OVA) inhalation were significantly lower than the levels in 1 / 1 mice. Moreover, mast cell-reconstituted W/Wv mice restored vascular permeability and VEGF levels similar to those of the 1 / 1 mice. Our data also showed that VEGF expression was regulated by hypoxia-inducible factor-1a (HIF-1a) activation through phosphatidylinositol 3-kinase (PI3K) pathway in OVA-induced allergic airway disease. Conclusion: These results suggest that mast cells modulate vascular permeability by the regulation of PI3K-HIF-1a-VEGF axis. Thus, these findings suggest one of the molecular mechanism(s) by which mast cells induce vascular permeability and provide a therapeutic strategy for allergic airway disease through modulation of signal transduction.

275 Thrombocytopoiesis in severe asthma

processes at the same time, that cause increased airway obstruction. Objective: The aim of the present study was to evaluate the platelet count, the percentage of the reticulated platelets and the concentration of thrombopoietin as well as interleukin 6 (IL- 6) and IL-3 in plasma of bronchial asthma patients. Patients and Methods: The study was performed on fifteen patients with chronic severe allergic asthma and fifteen patients with chronic severe nonallergic asthma. Patients were treated according GINA 2006 criteria for chronic severe asthma. Eighteen healthy persons served as negative control. Blood was collected in the morning on CTAD. Results: The mature platelets and reticulated platelets count was statistically higher in allergic asthma patients as compared to nonallergic and healthy controls. The concentration of IL-6 was significantly greater in allergic asthma patients as compared with nonallergic and healthy subjects. The concentration of IL-3 and thrombopoietin was slightly elevated in allergic as compared to nonallergic asthma patients but not significantly. There were no differences between asthma patients and control group. Conclusions: In our study we demonstrated higher reticulated platelets count in allergic asthma patients which may indicate increased megakariocytopoesis. Platelets may participate in allergic inflammation and play significant role in remodeling of the airways.

Kemona-Cheˆtnik, I1; Bodzenta-£ukaszyk, A1; Kucharewicz, I1; Dymnicka-Piekarska, V2; Kemona, H2 1 Medical University of Bialystok, Dept. of Allergology and Internal Medicine, Bialystok, Poland, 2Medical University of Bialystok, Dept. of Clinical Laboratory Diagnostic, Bialystok, Poland

Background: Allergic asthma is characterized by bronchial inflammation and repair

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Food Allergy – New Findings and Clinical Responses

Poster Discussion Session 8 Food Allergy – New Findings and Clinical Responses 276 High risk of immediate type reactions to soy drinks in 50 patients with birch pollinosis Treudler, R1; Werner, M2; Thiery, J2; Kramer, S1; Gebhardt, C1; Averbeck, M1; Su¨X, A1; Simon, J1 1 Universita¨tsklinikum Leipzig A.o¨.R., Dermatology, Venerology and Allergology, Leipzig, Germany, 2 Universita¨tsklinikum Leipzig A.o¨.R., Lab. Medicine, Clin. Chemistry and Mol. Diag., Leipzig, Germany

Background: Patients with birch pollinosis are at special risk for pollen associated allergy to soy, and may show severe immediate type reactions already at first consumption. This allergy is due to the substantial similarity of peptides (80%) and proteins (63%) between Gly m 4 and Bet v 1. We aimed at investigating the frequency of sensitisation to soy allergen Gly m 4 and the clinical relevance of this sensitisation in patients with birch pollen allergy in Leipzig. Methods: Fifty consecutive patients (30 female, 20 male, mean age 35 years) with confirmed type-1 allergy to birch pollen (positive skin prick test/SPT and/or specific IgE for birch 4CAP1) were included. All patients were asked for former consumption and tolerance of any soy products and in n 5 32 we were able to perform skin prick test (SPT) with a commercially available soy-drink product. In vitro tests included total IgE, specific IgE to soy bean, birch, rBet v1, rBet v 2, rBet v 4, rGly m4, tryptase (Phadia, Germany). Values 4CAP 1 were assessed as positive. Results: Mean total IgE was 318 kU/L, specific IgE values were found positive in 46 (92%) for rBet v 1, 7/50 (14%) for rBet v 2, 2/50 (1%) for rBet v 4, 36/50 (72%) for Gly m 4, 5/50 (10%) for soy. Tryptase values were within normal range. SPT was positive in 31/32 (94%). 28/50 patients (56%) reported former consumption of any soy product (most frequently soy drinks n 5 15 and tofu n 5 7). In this group, 26/28 were positive for rBet v 1, 4/28 for rBet v 2, none for rBet v 4, 21/28 for rGly m 4, 3/28 for soy. SPT was positive in 19/20. 8/28 had experienced an immediate type reaction at consumption of soy drinks (n 5 2 oral discomfort, n 5 3 anaphylaxis grade 1, n 5 3 grade 2 according to Ring and Messmer), 7 of whom were positive for rBet v 1 (all CAP class 43) and rGly m 4; one of them was positive for soy, and showed CAP class 1 for rGly m 4, while Bet v 1 was negative. r 2008 The Authors Journal Compilation

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Comment: Sensitisation to soy allergens due to Gly m4 was documented in 72% of our 50 patients with birch pollen allergy. 29% (8/28) of those patients who had consumed soy products previously, reported to have suffered from immediate type reactions due to soy drinks. We recommend to advise all patients with birch pollen allergy to be reluctant with consumption of any soy products.

277 Specific allergy to P. monodon (seawater shrimp) or M. rosenbergii (freshwater shrimp) in shrimp-allergic children Piboonpocanun, O; Sripramong, C; Pacharn, P; Udompunturak, S; Chinratanapisit, S; Piboonpocanun, S; Visitsunthorn, N; Vichyanond, P Thailand

Background: Allergy to specific shrimp species has not been systematically studied by oral challenges. The concordance between skin test reactivity from commercial and crude shrimp extracts as well as from prick to prick (PTP) test has never been studied. The objective of this study is to identify cases of specific allergy to seawater shrimp, Penaeus monodon (Pm) or freshwater shrimp, Macrobrachium rosenbergii (Mr) among shrimp-allergic children. Comparison of skin tests using commercial and crude shrimp extracts plus PTP method was investigated. Methods: Sixty-eight children with history of shrimp allergy were oral challenged to both shrimp species. Skin prick tests using extracts of Pm (PmSPT), Mr (MrSPT), commercial shrimp (ComSPT), and PTP method (PmPTP, MrPTP) were performed. Results: Food challenges identified specific allergy to Pm and Mr in 17.65% and 23.53% of subjects, respectively. Positive and negative challenges to both shrimp species were found in 47.06% and 11.76% of subjects, respectively. Correlations between mean wheal diameter (MWD) from ComSPT-PmSPT, ComSPT-PmPTP, ComSPT-MrPTP, PmSPT-PmPTP and MrSPT-MrPTP were observed. In Pm allergy group, PmSPT with MWD of 30 mm provided 80% predictive probability for positive challenges. PmPTP and ComSPT with MWD of 22.5 and 20 mm provided 95% predictive probability, respectively. In Mr allergy group, MrSPT with MWD of 30 mm provided 95% predictive probability.


Conclusion: Specific allergy to Pm or Mr was confirmed by food challenges. Predictive probability of SPT is helpful where food challenge is not feasible.

278 A search for peach Pru P 3-homologous lipid transfer proteins in pollens Tordesillas, L1; Sanchez-Monge, R2; Cuesta-Herranz, J3; Compes, E3; Garcia-Carrasco, B2; Lombardero, M4; Barber, D5; Salcedo, G2; Diaz-Perales, A2 1 ETS Ingenieros Agronomos, Biotecnologia, Madrid, Spain, 2ETS Ingenieros Agronomos, Unidad Bioquimica. Dept. Biotecnologia, Madrid, Spain, 3Fundacioń Jimenez Diaz, Alergia, Madrid, Spain, 4ALK-Abello´, Departamento de I 1 D, Madrid, Spain, 5ALK-Abello, Alergia, Madrid, Spain

Background: Two different patterns of sensitisation to Rosaceae fruits have been described in the Centre/North (Bet v 1-like as main allergens) and South (Lipid Transfer Proteins, LTPs, as major allergens) of Europe. Sensitisation to distinctive pollens have been claimed as a putative source of these differential profiles. Objetive: To explore the presence of putative Pru p 3-like allergens in Ambrosia artemissifolia (ragweed), Artemisia vulgaris (mugwort), Betula verrucosa (birch), Dactylis glomerata (barnyard grass), Helianthus annus (sunflower), Parietaria judaica (pellitory), Phleum pratensis (timothy), Plantago lanceolata (plantain) and Platanus occidentalis (plane tree) pollens. Methods: PBS extracts from the different pollens were separated by SDS-PAGE and immunodetected with anti-Pru p 3 polyclonal antibodies or IgE of sera from peach allergic patients (with associated pollinosis). ELISA-inhibition assays using purified Pru p3 and mugwort Art v 3 allergens as inhibitors were also performed. Pollen RNA samples and a cDNA encoding Pru p 3 probe were used in Northern analysis. Results: Pru p 3-like proteins were detected only in mugwort, but not in the other pollens analyzed, using either anti-Pru p 3 antibodies or sera from allergic patients. The cDNA-Pru p 3 probe hybridized with specific RNA bands from mugwort and plane tree pollens, but no band was found in the rest of pollens. Conclusion: Pru p 3-like LTPs were present in mugwort and plane tree pollens, as previously reported, but were not detected in ragweed, mugwort, birch, barnyard grass, sunflower, pellitory, timothy, plantain and plane tree pollens. A limited structural 117


similarity with Pru p 3 and or even null levels of putative homologous allergens could explain the lack of detection of Pru p 3-like LTPs in most pollen analyzed.

279 Peanut lipid transfer protein (Ara h 9): expression, characterisation and its biological activity in comparison to the peach LTP, Pru p 3 Lauer, I1; Dueringer, N1; Pokoj, S1; Foetisch, K1; Reese, G1; San Miguel-Moncin, M2; Malet, A3; Cistero-Bahima, A4; Enrique, E5; Vieths, S1; Scheurer, S1 1 Paul-Ehrlich-Institut, Division Allergology, Langen, Germany, 2Pius Hospital de Valls, Xarxa Santa Tecla, Allergy Department, Tarragona, Spain, 3Alergo Centre, Barcelona, Spain, 4Institut Universitari Dexeus, Allergy Department, Barcelona, Spain, 5Hospital General de Castellon, Allergy Division, Castellon, Spain

Background: Peanut (Arachis hypogaea) is the most frequent cause of fatal food allergic reactions. Peanut allergy is also reported in the Mediterranean area where patients are frequently sensitised to plant non-specific lipid transfer proteins (nsLTP). So far a homologous allergen in peanut has not been described in detail. The aim of this study was to identify and to clone a homologous nsLTP from peanut, to investigate its IgEantibody reactivity and biological activity in comparison to the most important food nsLTP, Pru p 3, from peach. Methods: Full length cDNA was obtained by PCR strategy based on known EST cDNA sequences of peanut nsLTP. The coding sequence was cloned into pPICZalphaA for expression in P. pastoris. Recombinant non-fusion protein was purified by IEC/preparative SEC chromatography. Three different groups of peanut allergic patients were recruited: 35 patients from Spain, 6 patients from United States and 35 patients from Germany. Inclusion criteria were a positive clinical history and either a positive response to SPT with peanut extract or specific IgE levels to peanut of CAP class 1 or higher. For analysis N-terminal sequencing, CD-spectroscopy, immunoblotting, inhibition tests and in vitro basophil histamine release assays were carried out. Results: A 9 kDa protein was expressed and purified from the supernatant at high level (45 mg/L). The pure non-fusion protein was identified by N-terminal amino acid sequencing and nsLTP specific antibodies as nsLTP (rAra h 9). Recombinant Ara h 9 (96 aa, 9.56 kDa, pI 8.9) showed 59.6% amino acid sequence identity with Pru p 3. CD-spectroscopy revealed the typical ahelical secondary structure similar to Pru p 3. Frequency of IgE antibody-reactivity to rAra h 9 was 25.7% in peanut allergic patients from Spain, 16.7% in USA and 5.7% in Germany. IgE inhibition experi-

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ments showed a strong cross-reactivity to Pru p 3 and no cross-reactivity to the hazelnut nsLTP (Cor a 8). In peanut allergic patients with concomitant peach allergy, rAra h 9 showed a weaker allergenic potency in comparison to Pru p 3 in histamine release assays using four individual patient sera from Spain. Conclusions: Recombinant nsLTP from peanut was characterised as minor allergen in different patient groups even in peanut allergic patients from the Mediterranean area. Recombinant Ara h 9 is capable of inducing histamine release from basophils but to a lesser extent than Pru p 3.

280 Double blind trial of a fermented infantile formula in cow’s milk allergy prevention 1

2

3

Morisset, M ; Soulaines, P ; Aubert-Jacquin, C ; Codreanu, F1; Maamri, N2; Hatahet, R1; Kalindjian, A2; Cuny, J1; Gueńard, L1; Babchia, H2; Frentz, P1; Bouillot, F4; Florac, L2; Cordebar, V1; Beaudouin, E1; Kanny, G1; Moneret-Vautrin, D1; Dupont, C2 1 Nancy University Hospital, Internal Medicine, Clinical Immunology and Allergy, Nancy, France, 2Hoˆpital Cochin-St Vincent de Paul, Pe´diatrie-Neónatalogie, Unite´ de Gastroente´rologie, Paris, France, 3Bledina SA, Villefranche-sur-Saoˆne, France, 4Clinique d’Essey-le`sNancy, Pediatric department, Essey-le`s-Nancy, France

Background: Previous studies have shown that probiotics supplementation during infancy decrease atopic manifestations. Infant formulas with afterwards heat killed ferments (FIF) have not yet been evaluated. An infant formula (IF) with cow’s milk proteins (CMP) and with added heat-killed ferments (Calismas technology, Bledina SA, France) was compared with a standard formula (SIF) in cow’s milk allergy (CMA) prevention. Methods: A prospective double-blind randomised trial was conducted with 2 groups of infants at high risk of atopy, fed with a FIF or a SIF, given from birth -or weaningto 12 month of age. During breastfeeding, mothers ingested the same IF as their child. Every event was collected during follow-up. At 4th and 12th month, clinical exams and prick-tests to CM were performed. In case of CMA suspicion, atopy patch tests and IgE to CM were carried out and after CMP avoidance, CM oral challenges were performed at 6 month of age. Results: One hundred and fifteen out of one hundred and twenty-nine children were followed during 12 months: 59 FIF, and 56 SIF. The percentage of digestive and respiratory events was significantly lower in the FIF group. A significant lower rate of IgE-dependent sensitisation to CM was observed in the FIF group: 1.7% versus 12.5%. A persistent CMA was observed in respectively 10.2% and 16.1% of children ingesting FIF and SIF respectively.

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Conclusions: CMA seems common in children at high risk of atopy but most cases, early and limited, disappear before the first oral challenge, leading to underestimation. The benefit of fermented formula needs confirmation with a larger number of infants.

281 Identification and characterisation of novel crustacean allergens from the north sea shrimp Crangon crangon Bauermeister, K1; Ballmer-Weber, B2; Falk, S1; Wangorsch, A1; Conti, A3; Holzhauser, T1; Vieths, S1; Reese, G1 1 Paul-Ehrlich-Institut, Division of Allergology, Langen, Germany, 2University Hospital Zurich, Department of Dermatology, Zurich, Switzerland, 3CNR-ISPA, Proteome Lab, Colleretto Giacosa, Italy

Background: So far only two Crustacean allergenic proteins are described, tropomyosin and arginine kinase. However, there are shrimp-allergic subjects without specific IgE to these known allergens. Therefore, we sought to identify, clone and characterize further Crustacean allergens to improve diagnostic reagents for Crustacean allergy. The North Sea shrimp Crangon crangon, a species frequently consumed in Europe, was used. Methods: Twelve patients with a positive double blind, placebo-controlled food challenge (DBPCFC) with shrimp, and 5 patients with anaphylactic reactions to shrimp were included in the study. By immunoblot analysis of cooked North Sea shrimp extract with sera that do not contain tropomyosin-specific IgE, prominent IgE reactive proteins were identified. Their identity was determined by means of mass spectrometry. Synthetic genes of homologous proteins from other Crustacean species were expressed and the IgE reactivities of recombinant proteins proven by immunoblot analysis. Homology search with published sequence data of different Crustacean species permitted cloning of the full-length cDNA sequences of the homologous C. crangon proteins by PCR strategy. The IgE binding properties of the expressed C. crangon proteins were investigated. Results: Four of five recombinant Crustacean proteins with molecular masses between 14 and 30 kDa bound IgE by immunoblot analysis: A sarcoplasmic Cabinding protein, troponin C, myosin light chain and triosephosphate-isomerase. Additionally, the full-length cDNA sequences encoding these four proteins were cloned from C. crangon and their IgE reactivities were verified. Conclusion: With a combination of proteomic and molecular biological methods we identified four novel IgE binding proteins from various Crustacean species including C. crangon. In addition to tropomyosin and



arginine kinase the novel C. crangon allergens will be included in component-resolved diagnosis to improve the sensitivity of the in vitro diagnosis of shrimp allergy.

282 Pomegranate LTP isoforms identified by a new proteomic approach show different immunological properties Zoccatelli, G1; Consolini, M1; Fusi, M1; Dalla Pellegrina, C1; Chignola, R1; Peruffo, A1; Marsano, F2; San MiguelMoncin, M3; Scheurer, S4; Rizzi, C1 1 University of Verona, Dept. of Science and Technology, Verona, Italy, 2University of Piemonte Orientale, Dept. of Environmental & Life Sciences (DISAV), Alessandria, Italy, 3Pius Hospital de Valls, Dept. of allergy, Tarragona, Spain, 4Paul-Ehrlich-Institut, Div. Allergology, Section of Recombinant Allergens, Langen, Germany

Background: Plant Lipid Transfer Proteins (LTPs) belong to a class of high conserved proteins extensively studied in the last decade. These proteins are recognized as food allergens capable of eliciting severe systemic symptoms, especially among the Mediterranean population. Several polypeptides belonging to this family have been characterized so far. For some LTPs different isoforms have been identified in a given plant. The sequence information was mainly derived from the cDNA, by contrast, the investigation of isoforms diversity is frequently not addressed. Allergen isoforms often exhibit different IgE binding capacities, hence contributing differently to the whole allergenic potencies (e.g. Bet v 1 and Mal d 1). Recently two different LTP isoforms have been isolated from pomegranate (PG) and characterized by mass spectrometry (MS). Here we applied a new proteomic approach based on acid-urea electrophoresis (AU-PAGE) followed by SDS-PAGE for the separation of LTP isoforms from PG and peach. Methods: Proteins from PG juice and peach peel were separated first by the AU-PAGE. The lanes were cut, equilibrated in SDS containing buffer, and subjected to 15% SDS-PAGE. The separated proteins were transferred to PVDF membrane and assayed with a polyclonal antibody raised against Pru p 3 (PAB). IgE immunoblotting assays were carried out using sera from two groups of patients: 1) allergic to pomegranate, 2) allergic to peach (sensitised to Pru p 3). For identification the proteins were subjected to MS. Results: By this approach up to 10 spots with an apparent MW of 9–12 kDa (depending on the redox conditions) were separated for PG. Five of these proteins were recognized by the PAB and by the patient’s sera. IgE immunoblotting experiments clearly showed that PG LTP isoforms are differentially recognized. In contrast, 2D analysis of peach revealed only a single predominant r 2008 The Authors Journal Compilation

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9kDa spot recognized by the PAB and by the patient’s sera. Conclusions: Up to 5 LTP isoforms have been separated from PG by the proteomic approach. The power of this technique is demonstrated by the identification of a large number of IgE reactive LTP isoforms never described for other organisms. The immunological study of the identified isoforms indicates that they possess different IgE epitopes which might contribute differently to the whole allergenic potency of pomegranate.

283 Introduction of baked egg in the diet of egg allergic children may accelerate the induction of oral tolerance to egg proteins Giavi, S; Vassilopoulou, E; Konstantinou, G; Chatziioannou, A; Lagara, K; Douladiris, N; Manoussakis, M; Saxoni-Papageorgiou, P; Papadopoulos, N NK University of Athens, 2nd Pediatric Clinic, Allergy Department, Athens, Greece

Introduction: Hen’s egg allergy is one of the most frequent allergies in children. Although most children have outgrown their allergy by the age of five, the fact that hen’s egg can be found in a wide range of foods increases the risk for accidental allergen exposure and allergic reaction until then. To date, the only management of egg allergy is strict avoidance of egg proteins in any form and anaphylaxis action plan, although studies have shown that some children with egg allergy can tolerate heat treated egg. The purpose of this report was to describe the natural history of egg allergy in egg-allergic children that have been eating foods containing heat-treated egg. Method: We retrospectively evaluated histories of 25 children with IgE-mediated egg allergy, who were challenged with baked egg in cake, at a median age of 30 months. Tolerant patients were instructed to receive baked egg regularly and were re-challenged with egg at an average of 6 months later. Results: Twenty two out of the 25 challenges (88%) to cake were negative and foods containing heat treated egg proteins were introduced regularly in the children’s diet. Thirteen of these children were challenged to cooked egg after an average of 6 months (median age: 36 months). Ten out of the 13 children (77%) were by then able to tolerate egg. Conclusion: A considerable proportion of children with egg allergy can tolerate baked egg; this can be quite important in designing their diets and other aspects of their daily life. Based on the existing literature, approximately 30% of children with egg allergy may tolerate egg at the age of 3 years and up to 66% at the age of 5 years. Although the number of patients in this


report is small, the proportion of children who outgrew their allergy is quite high, suggesting that introduction of baked egg might have contributed to the induction of oral tolerance. A prospective controlled study is currently underway to evaluate this hypothesis.

284 Tolerance of wines fined with allergenic proteins in allergic individuals- doubleblind, placebo controlled food challenge study Belloni, B; Kirschner, S; Kugler, C; Ring, J; Brockow, K Klinik fu¨r Dermatologie und Allergologie am Biederstein, Technical University Munich, Department of Dermatology and Allergy, Munich, Germany

Background: Labelling of allergenic ingredients present in foods has been instituted by the EU to protect allergic consumers. However, it is unknown if egg-, milk-, and fish-derived products used as processing aids in wine making, which may remain in the finished product could elicit clinical reactions in patients with IgE-mediated food allergy. Method: Fourteen adults with egg, milk or fish allergy were included (12 female, two male, median age and range 47 years, 26–71 years) in the study. German wines were fined with an excessive dosage of egg albumin, egg lysozyme, milk casein or fish isinglass or gelatine followed by routine filtering. Skin prick tests were performed with fining agents as well as with fined and unfined wines. All patients underwent double-blind placebo-controlled food challenges with fined and unfined wines up to a cumulative dose of 200 mL in female and 300 mL in male. Results: Skin prick tests were positive to egg albumin, lysozyme and milk casein, but not to isinglass or fish gelatine. Few positive skin prick tests to wines were detected, however, there was no difference between fined and unfined ones. In the provocation test no reaction to German fined wines or placebo occured. Conclusion: The concentrated albumin-, lysozyme- and casein-containing, but not fish protein-containing fining agents or diluted fining agents in present wine were allergenic in the skin prick test, however, no patient reacted adversely in the provocation to fined or unfined filtered wine. Wines fined with these agents appear not to present a risk to allergic individuals after filtration.

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285 Food allergy, asthma and atopic dermatitis: the atopic march Pecora, V; Nucera, E; Decinti, M; Aruanno, A; Lombardo, C; Colagiovanni, A; Rizzi, A; Buonomo, A; Pollastrini, E; Musumeci, S; Domenico, S; Patriarca, G Catholic University, Department of Allergology, Rome, Italy

Food allergy and asthma are both atopic diseases and therefore frequently co-exist. Four to eight per cent of pediatric population with asthma have also food allergy, and more than a third of children with food allergy have also asthma. In the literature, there are few studies that analyse the association between food allergy, asthma and atopic dermatitis.We studied 148 patients (F/M 5 68/80; age: 2–64 years) with food allergy, 50 (33.8%) of these were affected by asthma and 50 (33.8%) by atopic dermatitis. All patients underwent a complete allergological evaluation: clinical history, physical examination (score of atopic dermatitis), skin prick tests performed with extracts of common food and respiratory allergens and with fresh foods (prick-by-prick method), in vitro laboratory tests (specific IgE and IgG4, total IgE and eosinophilic cationic protein in serum) and double-blind placebo-controlled food challenge. 80 patients underwent a food oral specific desensitising treatment according to standardized protocols; since some patients were allergic to more than one food, 92 treatments were performed. In our study the most common allergenic foods are cow milk, hen egg, cod fish and peanut; we didn’t notice essential differences between patients with and without asthma. Regarding 50 patients with asthma, the allergological tests performed with respiratory allergens were positive in 37 (74%) and negative in 13 (26%). Seventeen patients showed asthma only after the ingestion of allergenic food. Regarding 50 patients with atopic dermatitis, we analysed the atopic march and we noticed that in the most of cases (80%) after the clinical appearance of the cutaneous lesions the second step is food allergy and the third one respiratory diseases (rhinitis and/or asthma). Analysing the influence of atopic dermatitis and asthma on the results of 92 food oral specific desensitising treatments, we noticed that the patients affected by asthma show a 10% successful treatment less than the population undergone to desensitisation. This type of treatment doesn’t seem modify the course of asthma. Atopic dermatitis and food desensitising treatment don’t affect each other.

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could further inactivate some heat-resistant potential allergens.

286

Abstract withdrawn.

287 Wheat seed salt-soluble allergens from wheat-derived foodstuffs show lower IgEbinding capacitiy than those from flour De Gregorio, M1; Armentia, A2; A Palacıń1; Quirce, S3; Salcedo, G1; Dıáz-Perales, A1 1 E.T.S.Ingenieros Agrońomos, Biotecnologıá, Madrid, Spain, 2Hospital Universitario Rio Hortega, Seccioń de Alergia, Valladolid, Spain, 3Hospital Universitario Doce de Octubre, Servicio de Alergia, Madrid, Spain

Salt-soluble proteins from wheat flour, such as a´-amylase/trypsin inhibitors and lipid transfer proteins have been described as main allergens associated with both baker’s asthma and food allergy. However, most of their identification studies have used raw flour as starting material, thus not considering potential changes in allergenic properties induced by the heat treatment and other industrial processing to produce wheatderived foodstuffs. Objetive: To investigate the IgE binding capacity of salt soluble proteins from wheat derived foods as well as their stability on simulated gastric digestion. Methods: Salts extracts from different commercial wheat-derived products (bread, pasta, etc) were obtained, and their IgE-binding properties investigated by IgE-immunodetection and ELISA assays. Chromatographic fractions enriched on IgE-binding proteins were obtained and characterized by N-terminal sequencing and fingerprinting. Simulated gastric digestion of these fractions was also accomplished. Results: The IgE-binding capacity of saltsoluble proteins from commercial breads and cooked pastas was reduced around 50% compared with that from raw flour, being the reduction less dramatic in non-cooked pasta and cookies. Most wheat-derived foodstuffs shown major IgE-binding components of 20 and 35 kDa, identified as avenin-like and globulins proteins. These proteins were hydrolyzed when subjected to simulated gastric fluid digestion. IgE binding bands of around 12–15 kDa, corresponding most probably to a´-amylase inhibitor subunits were only detected in non cooked pasta. Tri a 14, the wheat LTP allergen, was located in some kind of breads by using anti-peach Pru p 3 antibodies but it was not recognized by sera from patients with food allergy. Conclusions: Processing of wheat flour to obtain wheat-derived foodstuffs seems to decrease strongly the IgE binding capacity of the major salt-soluble wheat proteins. Moreover, simulated gastric fluid digestion

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288 Clinical and immunological characteristics of patients with food hypersensitivity according to the timing of the reaction Jedrzejczak-Czechowicz, M; Makowska, J; Gwardys, T; Lewandowska-Polak, A; Wysocka, M; Kowalski, M Medical University of Lodz, Department of Immunology, Rheumatology and Allergy, Lodz, Poland

Background: Food hypersensitivity reactions may have various timing (early, late, delayed), clinical manifestations and may involve both immunological and non immunological mechanisms. Aims: The aim of this study was to characterize food hypersensitivity reactions distinguished according to the timing of manifestation. Patients and methods: The group of 372 patients (175 children, mean aged 5 years (0,4–17) and 197 adults; mean age 37 years (18–67) with a history of food hypersensitivity reactions was recruited from the university allergy clinic as a part of the Europrevall study. Patients underwent physical examination and filled standardized Europrevall questionnaire. Skin prick tests with 42 inhaled and food allergens was performed. Results: Out of 351 patients studied in 249 (67%) symptoms started within 2 h after food intake (early reactors – ER) and in 123 (33%) patients symptoms started later (late reactors -LR). The family history of atopy was reported in 36% of ER and 31% of LR. Patients with early food reactions were older – mean age 25 years (0,4–67) vs. mean age 9 years (0,6–62) of patients with late reactions. Patients from both groups reported similar skin symptoms – urticaria, rash, itching and gastrointestinal system but ER reported also bronchial symptoms (dyspnoe, cough, wheezing) and conjunctivitis. The most often reported culprit foods in ER were milk (42%), hazelnut (30%), walnut (30%), apple (29%), peanut (29%) and egg (29%) and in LR–milk (66%), egg (43%), fish (12%), peanut (14%), hazelnut (11%), walnut (11%) and soy (11%). Positive SPTs to at least one food allergens were found in 43% of ER and 13% of LR. In early food reaction patients SPT were positive to hazelnut in 57%, to sunflower in 57%, to celery in 56%, to carrot in 44%, to apple 42%. Among LRs SPT were positive to eggs in 19%, fish in 13% and to milk in 9% of subjects. Conclusions: Patients with early and late food reactions report similar offending foods and similar symptoms pattern but differed with the prevalence of positive skin



prick tests to foods.The study is a part of EuroPrevall.

289 Oral challenge with pasteurized egg white from Gallus domesticus Cuevas-Durań, T; Jurado-Palomo, J; Bobolea, I; FiandorRomań, A; Pascual, C; Quirce, S University Hospital La Paz, Allergy, Madrid, Spain

Background: Gallus domesticus egg’s main allergens are partially thermostable, so tolerance to egg has to be tested by challenge with raw egg white. However, raw egg is potentially dangerous because it can cause digestive toxic-infections. This risk can be avoided by using pasteurized egg white (in the process of pasteurization heat is used). The objective of this study was to assess if pasteurized raw egg white is as reliable as raw egg white in egg allergy diagnosis. Methods: Our study group included 32 eggallergic children (mean age 4.7 7 0.6 years; IQR 3.01–5.13) consecutively challenged with both pasteurized and fresh raw eggwhite during 2007. Open challenge was


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carried out with increasing doses (4, 8 and 16 mL) of pasteurized egg white, administered every 60 min. On the second day, increasing doses of fresh raw egg white (1/ 8, 1/4, 1/2 egg white) were given at 60-min intervals. The challenge was immediately stopped if objective clinical symptoms arose. To avoid the rejection of the food, egg white was mixed with a cold dessert. Natural egg white had to be previously whipped; this was not necessary with the pasteurized egg. Sera of egg-challenged children were grouped in three pools. Pool A: Sera of patients who reacted to cooked egg. Pool B: Sera of patients who reacted to raw egg but tolerated cooked egg. Pool C: Sera of patients who tolerated both cooked and raw egg. SDS-PAGE and IgE-immunoblotting (IgE-IB) with boiled, natural raw and pasteurized raw egg white as well as IgE-IBinhibition and ImmunoCAP-inhibition assays with the different egg-white extracts were performed. Results: Eleven children (34.4%) had positive challenges with pasteurized egg white. The 21 children (65.62%) who tolerated


pasteurized egg white also had a negative challenge with natural egg white. If challenge with pasteurized egg white resulted positive the study was stopped as considered sufficient to detect allergy to raw egg proteins. SDS-PAGE, IgE-IB: the protein profile and IgE-binding capacity of both pasteurized and raw egg white were almost identical. IgE-IB-inhibition and ImmunoCAP-inhibition: both extracts behaved in a similar way. Conclusion: We did not find any in vitro or in vivo allergenic differences between fresh and pasteurized egg white. The use of pasteurized egg on oral allergen challenges may avoid infections (Salmonella, Avian flu). This study supports the use of pasteurized egg white in the diagnosis of allergy to raw egg proteins.

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Abstract withdrawn.

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Poster Discussion Session 9 – Anaphylaxis and Drug Allergies in Children

Poster Discussion Session 9 Anaphylaxis and Drug Allergies in Children 291 An audit of children presenting to a Birmingham hospital with anaphylaxis Ruth, N; Rodgers, H; Makwana, N Sandwell General Hospital, Pediatrics, West Bromwich, United Kingdom

Aims: 1) To identify common presenting features of anaphylaxis in children, and analyse management. 2) To compare current management to new recommendations. Background: Anaphylaxis is a rare but potentially fatal presentation to Emergency Departments across the World. Its management is therefore very important. Studies have suggested that epinephrine (Adrenaline) should be administered immediately to all children with anaphylactic reactions. Management of such children is however variable, with a variety of treatment regimes instigated. Methods: Retrospective case based analysis of all children aged 0–16 years presenting with anaphylaxis in a five-year period. Data was collected about presenting symptoms, trigger factors, previous history and management received. Results: Between January 2002 and December 2006, 62 children were diagnosed with anaphylaxis. Of these, 40 case notes were identified and analysed (the rest being unavailable). Demographics: i) Total patients/year ii) Male:female ratio/year iii) Age at presentation Year 0–2 2–4 4–6 6–8 8–10 10–12 12–14 14–16 Total Male Female 2002 2003 2004 2005 2006 Totals

1 9 2 2 0 4 2 2 6 4 1 0 5 4 1 14 16 13

1 2 0 2 1 6

0 2 0 1 2 5

1 0 0 0 1 2

0 0 0 1 2 3

0 0 0 0 1 1

14 10 12 9 17 62

10 4 7 4 12 37

4 6 5 5 5 25

The incidence of anaphylaxis stayed relatively constant with a slight male dominance. The majority of cases (72%) presented below the age of 6 years. All children presented with rash and angioedema; only 15% had respiratory symptoms, whilst 5% had circulatory collapse recorded). The commonest trigger was peanuts (55%) followed by egg and tomato (10% each); 20% had no identified cause. Ninty five per cent of patients received chlorpheniramine, with only 20% receiving epinephrine. Ten per cent and 7% respectively received salbutamol nebuliser and prednisolone and 50% received IV hydrocortisone. Eighty five 122

per cent of patients were identified as atopic, 15% of whom had had previous anaphylactic reactions. Conclusion: The majority of patients given a diagnosis of anaphylaxis should have had a diagnosis of an allergic reaction. Of those patients with true anaphylaxis, according to the recent definition, only (57%) received epinephrine although all had equally good outcomes. The confusion in management appears to lie in the lack of a clear simple definition of anaphylaxis. To improve therapy of children with anaphylaxis, schematic diagrams detailing management, have been placed in pediatric clinical areas.

292 Referral to allergy unites of patients with anaphylaxis who attended to emergency departments Tejedor, M; Moro, M; Mugica, M; Vila, C; Rosado, A; Gomez-Traseira, C; Lindo, D Fundacion Hospital Alcorcon, Allergy, Alcorcon, Spain

Background: Referral of patients with anaphylaxis episodes from Emergency Units to Allergy Units is low (about 25–50%). We have assessed the clinical reasons which influence referral of patients with anaphylaxis by physicians of the Emergency Unit to Allergy Unit and the reasons why the same patients assist to Outpatients Allergy Unit. Methods: Patients with anaphylaxis episodes were identified, in computerized clinical records of our hospital, among patients who attended the Emergency Unit of our hospital in the years 2004 and 2005, using alpha-numeric sequences which identify acute allergy syndromes. We have noticed that this strategy has 95% of sensitivity in identifying these patients. We showed 433 anaphylaxis episodes. Two models of logistic regression are showed according to the studied binary outcome variable: the referral of patients with anaphylaxis episodes by physicians of the Emergency Unit to Outpatient Allergy Unit and the assistance of patients to the Outpatients Allergy Unit. Results: 58.4% (95% CI 53.6–63.1) of patients with anaphylaxis episodes from Emergency Unit were referred to Allergy Unit, whilst the assistance to Outpatients Allergy Unit of our hospital in the same group of patients was 50.8% (95% CI 45.9– 55.6). The large majority of these patients (74.3%–95% CI 68.5–79.6) were referred by the Physicians of the Emergency Unit of our hospital. Upper respiratory system involveJournal Compilation

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ment favoured referral (O.R. 1.9) and assistance to Allergy Unit (O.R. 1.9). In addition, Idiopathic and anisakis anaphylaxis were associated to a higher assistance to Allergy Unit. On the other hand, ages higher than 15 years had lesser assistance to Allergy Outpatients Unit (several O.R.o1) In the next table we showed the clinical reasons associated to higher referrals to Outpatient Clinic Allergy Odds Referral Allergy ratio Unit

Assistance to Allergy Unit

95% 95% Odds 95% 95% lower upper ratio lower upper limit limit limit limit Age 0–15 years Age 16–40 years Age 41–60 years Age 460 years Mild anaphylaxis Severe anaphylaxis Cardiovascular involvement Upper respiratory system involvement Idiopathic anaphylaxis Food anaphylaxis Drug anaphylaxis Anisakis anaphylaxis Referral by a physician’s Emergency Unit

1 0.53 0.71 0.61 1 0.94 1.44

0.29 0.36 0.28

0.96 1.41 1.34

0.37 0.72

2.42 2.89

1 0.14 0.16 0.28 1 0.50 1.67

1.99 1.14

3.50

0.38 0.24 0.57 0.88

1.00 0.59 1.34 2.51

0.14 0.10 0.24 0.31

0.06 0.07 0.10

0.31 0.37 0.76

0.16 0.71

1.59 3.90

1.97 1.00

3.88

4.55 1.12 1.89 4.39 15.50

1.52 0.43 0.77 1.38 8.98

13.59 2.95 4.64 13.95 26.75

Conclusion: Ages lower than 15 years’ old favored referral and assistance to Allergy Unit from Emergency Unit. Out of them, the principal reason fot assistance to Allergy Unit, among patients with anaphylaxis episodes attended in Emergency Unit, was to be referred by a physicians of Emergency Unit.

293 Anaphylaxis in Brazil – survey of Brazilian Association of Allergy and Immunopathology (ASBAI) Bernd, L1; Fleig, F2; Alves, M3; Bertozzo, R4; Coelho, M5; Correia, J5; Di Gesu, G2; Di Gesu, R2; Geller, M6; Mazzolla, J7; Oliveira, C8; Peixoto, D9; Sarinho, E10; Silva, E11 1 FFFCMPA, ASBAI, Division of Immunology, Porto Alegre – RS, Brazil, 2P. Alegre (RS), Brazil, 3Tubaraõ (SC), Brazil, 4Corumba´ (MT), Brazil, 5B. Horizonte (MG), Brazil, 6 Rio de Janeiro (RJ), Brazil, 7Roca Salles (RS), Brazil, 8 Campinas (SP), Brazil, 9Vitoria (ES), Brazil, 10Recife (PE), Brazil, 11S. Paulo (SP), Brazil

Anaphylaxis is the most serious clinical condition observed at allergologists clinical practice. From September 2005 to October



2007 we conducted a survey among Brazilians allergologists asking them to report new cases who they have examined after an anaphylaxis episode. Method: Allergologist members of ASBAI received a questionnaire to be filled with patient’s characteristics and investigation of clinical cases. Results: We received report of 113 patients; 49 male, 64 female; ranging from 8 months to 83 years old. Dermatologic manifestations (erythema, urticaria and/or angioedema) was observed by 94% of patients; respiratory symptoms (dyspnea, chest tightness, suffocation) was verified by 49% patients; hypotension and other cardiovascular signal were present in 30% of crisis; and gastrointestinal symptoms (abdominal cramps, nausea, vomiting) were observed at 22% of anaphylaxis episodes. It can synthesized that were three major group of precipitating agents: drugs (50/113 patients); insect venoms (20/113); and foods in 18 of 113 reported crisis. This was the same for children, young adults and older people. Trigger agent was not identified in 8,9% of episodes. Ten patients had anaphylaxis induced by more than one trigger. Analgesics and NSAID were drugs which have provoked more number of anaphylaxis episodes. Also important trigger agents were antibiotics and ECA inhibitors. Bee and wasp were frequently associated with anaphylaxis. In children, cow milk and white egg were the most important foods in anaphylaxis. Food allergy in adults was induced principally by crustaceans. Majority of patients have sufferer a two or more anaphylaxis crisis before having a consultation with allergologists. Conclusion: We studied causes of anaphylaxis among patients evaluated by Brazilian allergologists. We found that drugs (analgesics and NSAID), insect venoms (bee and wasp) and foods (milk and egg in children; crustacean in adults) were the major triggers of anaphylaxis in our population. 294 Clinical presentation and causes of anaphylaxis in children of an industrial urban area of Greece Farmakas, N; Xenopoulou, T; Levisianou, D; Karakaidos, D; Karis, C; Xatzis, D; Dimitris, H; ‘‘Agios Panteleimon’’ General Hospital, Pediatrics and Allergy and Clin. Immunology service, Nikea-Piraeus, Greece

Introduction: We wished to investigate the clinical manifestations, and etiology, of anaphylaxis admissions to our Hospital, in children up to 14 years old, living in a particular industrial urban area, close the major port of the country (Pireus). Methods: Retrospective review of Hospital charts, from 1999 to 2007. r 2008 The Authors Journal Compilation

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Results: Among 9 000 admissions, in children aged 2.5 to 14 years old, 80 (0.88%) were due to anaphylaxis in 48 boys (60%) and 32 girls (40%). The clinical manifestations are depicted in the Table. Regarding aetiology, the most common cause was food (n 5 30, 37.5%). Twenty-two (26.9%) of the events were attributed to medications, and hymenoptera stings caused anaphylaxis in 6 cases (6%). No cause was identified in 18 cases (22.5%). In 3 cases, contact with Latex, olive tree leaves, and seaweed led to anaphylaxis, and one case was exercise-induced anaphylaxis. The break down of food causes showed: nuts, followed by various fruits, and shellfish. Penicillin (n 5 12) and NSAIDs (n 5 10) were the most common drug-associated cases. In the vast majority of the cases (n 5 75, 97.5%) there was no previous history of any adverse reaction to a particular causative agent, while in 35% (n 5 28) and 37.5% (n 5 30), acute urticaria and asthma, respectively, reported in the Past Medical History. History of atopy was present in 35% (n 5 28) of their mothers and in 22.5% (n 5 18) of their fathers. Total IgE level 4100 had 48 (60%) of the cases. The management of the cases was carried out as outpatients with epinephrine, followed by antihistamines, and, thirdly, by cortocosteroids. Conclusions: Anaphylactic reactions are common in this particular population. The leading cause of these events is food, followed by medications. A considerable number of cases was not associated with skin manifestations. Positive, personal and/ or family history of atopy, was found in several cases. As a first line of defence was the use of epinephrine Clinical manifestations

n

%

Urticaria Angioedema Dyspnea GI manifestations Cough/voice hoarseness Hypotention Without skin manifestation Dizziness Paleness Nasal congestion Fainting Lacrimation Feeling of impending doom ‘‘Difficulty in swallowing’’

60 48 43 25 22 11 10 6 6 4 4 2 2 1

75 60 53.75 31.75 27.5 13.75 12.5 7.5 7.5 5 5 2.5 2.5 1.25

295 Anaphylaxis due to sheep and goat’s milk cheese Laıń, S; Pe´rez-Bustamante, S; Davila, G; Fuentes, V; Zapatero, L; Alonso, E; Martıńez-Molero, M Hospital Materno-Infantil Gregorio Maranõń, Pediatric Section, Madrid, Spain

The existence of a high degree of crossreactivity between caseins from cow’s, sheep’s and goat ’s milk has been reported. However, sheep’s and goat’s milk allergy is uncommon and does not usually involve allergic cross-reactivity to cow’s milk. We describe two different cases of anaphylaxis due to sheep and goat’s milk cheese. Methods: CASE 1: A 3-year-old non atopic girl experienced an anaphylactic reaction after eating sheep and goat’s milk cheese. She presented generalised itching, eyelid swelling, dry cough and wheezy dyspnea. After the episode, she had tolerated cow’s milk and cow’s milk dairy products. CASE 2: A 6-year-old girl with antecedents of cow’s milk allergy was treatment successfully with an specific oral tolerance induction treatment. One year later, fifteen minutes after the ingestion of sheep’s milk cheese she presented rhinitis, dry cough, wheezy dyspnea and generalised itching.Skin prick tests (SPT) were carried out using cow’s milk and its proteins. Prick by prick tests with cow’s milk, cow’s, sheep’s and goat’s milk cheese, specific IgE measurements, SDS-PAGE immunoblotting, and inhibition tests were performed to study the pattern of IgE-binding proteins and the potential cross-reactivity between caseins. Results: 1. Skin prick tests and Specific IgE.(Table). SPT (mm)

SPT (mm)

Case 1

Case 2

Cow casein 3 2 ALA 42 BLG Negative Cow’s milk Negative Sheep’s milk Goat’s milk Cow cheese Sheep cheese Goat cheese

Prick by PricK (mm) Case 1

43 32 Negative 31 Negative ND ND Negative 20 15 14 8

Prick by Specific Prick IgE (mm) (KU/L) Case 2 Case 1

75 ND ND Negative 10 5 11 4

Specific IgE (KU/L) Case 2

3.64 20.5 0.88o0.35 0.71 0.6 3.02 3.23 4100 18.6 4 100 20.5

2. Case 1: Immunoblotting showed two Ig E-binding bands in goat casein at 20 and 14 kDa, several bands between 14 and 45 kDa in sheep’s milk and no bands in cow’s milk. 3. Case 2: SDS-PAGE Immunoblotting was carried out using cow’s milk and sheep’s milk, showing multiple bands for both. In the SDS-PAGE-Immunoblotting inhibition with sheep’s milk proteins using cow’s milk, cow’s casein, and sheep’s milk as inhibitory phase showed a band of 30 kDa compatible with sheep’s casein molecular weight to which was only inhibited by the sheep’s milk. Conclusion: We report two cases of food allergy due to sheep’s and goat’s milk with good tolerance to cow’s milk. Specific caseins were probably the main allergens causing sensitisation in our patients.

Background: Cow’s milk allergy is the most frequent cause of food allergy in children.


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296 Breast milk anaphylaxis – a case report Palma Carlos, S1; Matos, V2; Loureiro, V2; Leiria Pinto, P1 Dona Estefaˆnia Hospital, Immunoallergy Departement, Lisbon, Portugal, 2Dona Estefaˆnia Hospital, Clinical Pathology Departement, Lisbon, Portugal

1

Background: Cow’s milk allergy (CMA) is the most common food allergy in children. In rare cases, infants with CMA don’t tolerate breast feeding. In some studies beta-lactoglobulin (BLG) was detected in breast milk. A great intra and inter-individual breast milk BLG concentration seems to exist, without relation with mother’s daily cow milk intake. After intense investigation through available documentation, it was not possible to find any reported case about breast milk anaphylaxis or loss of tolerance to it. Case report: A 27 months old girl having CMA with urticaria since 4 months old is being followed in our department since 5 months old. Skin prick test (SPT) were positive to milk, BLG, alpha lactoalbumin (ALA) and casein (CAS). Total IgE was high (115 KU/L) and specific IgE were elevated (milk 17.5 KU/L, BLG 16.9 KU/ L, CAS 4.55 KU/L and ALA 15.2 KU/L). She was breastfeed since birth until 9 months old. Despite cow’s milk avoidance diet had been performed, SPT and specific IgE were positive. At 25 months old, her mother had a baby and starts breastfeeding him. One month later, the mother gave her daughter 200 cc of breast milk. The child immediately began coughing followed by urticaria, angioedema, vomiting, shortness of breath and stridor. Medical treatment with epinephrine, b2 agonist and antihistamines was required. Breast milk SPT was positive. Mother’s cow milk daily intake was 500 cc or equivalent. Discussion: The natural evolution of breast milk tolerance in children with CMA that had tolerated breast feeding in early infancy is unknown. The mechanisms involved in breast milk tolerance in children with CMA, are poorly understood. In this case, a loss of breast milk tolerance in a CMA was observed after an eviction period. This was a serious clinical picture and the clinic shall be aware to advise children’s parents for this possibility. 297 Anaphylaxis to fish gelatin Kuehn, A; Hilger, C; Hentges, F CRP-Sante´, Laboratory of Immunogenetics and Allergology, Luxembourg, Luxembourg

Background: Fish gelatin represents a useful alternative to porcine or bovine gelatins. The risk of allergic reactions to gelatin in general and fish gelatin in particular is considered to 124

be low. We report a 12-year-old boy with known fish allergy, who suffered from a severe anaphylactic reaction with angioedema, urticaria and severe asthma after eating from a barbecue composed of animal meat, but no fish, and marshmallows. Methods: The patient’s history was examined. Skin prick tests with commercial meat and fish extracts including cod, salmon and tuna were performed. Specific IgE were determined by ImmunoCap method. Fish muscle proteins and gelatins were separated using SDS-PAGE and tested by IgE-immunoblotting. From 8 commonly consumed fish species, 12 recombinant parvalbumins and 8 native parvalbumins as naturally occurring isoform mixtures were chromatographically purified to homogeneity. The patient’s IgE-binding to these parvalbumin preparations and to different gelatins was analyzed for IgE-titer in ELISA experiments. Results: The patient had strongly positive skin tests to house dust mites, grass pollen, cod, salmon and tuna. Total IgE was 700 kU/L. Specific IgE was 63 kU/L for dermatophagoı¨ des pteronyssinus, 64 kU/L for meadow grass pollen, 65 kU/L for tuna, 32 kU/L for salmon and 16 kU/L for cod. IgE-immunoblotting showed IgE-binding to several fish parvalbumins at ca. 10 kDa and to gelatin proteins of different molecular size. The quantitative ELISA with recombinant parvalbumins gave IgE-binding values below those found in the commercial CAP system for the specific fish extracts. The difference was mostly significant for tuna where about half of the IgE-reactivity was not directed to tuna parvalbumin. This was consistent with the finding that the patient had specific IgE to fish gelatin as shown in ELISA assay. Conclusion: For some fish allergic patients, the ingestion of fish gelatin-containing products, as for instance marshmallows, harbors a risk for severe anaphylaxis. A primary source of fish gelatin is skin from tuna, a fish species considered less allergenic because of low parvalbumin content. As shown in this clinical case, it is highly recommended to detail the IgE reactivity of fish allergic patients and distinguish between IgE binding to recombinant, purified parvalbumins and gelatin.

298 Use of epinephrine in anaphylaxis emergency episodes Moro, M1; Tejedor, M1; Mugica, M2; Rosado, A2; Vila, C2; Cardenas, R2 1 Fundacion Hospital Alcorcon, Allergy, Alcorcon, Spain, 2 Spain

Background: The use of epinephrine in episodes of anaphylaxis is low (15–75%). We have assessed the clinical reasons why Journal Compilation

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physicians of Emergency Unit of our hospital utilized epinephrine in anaphylaxis episodes. Methods: Patients with anaphylaxis episodes were identified, in the computerized clinical records of our hospital, among patients who were attended in the Emergency Unit of our hospital in the years 2004 and 2005, using alpha-numeric sequences which identify acute allergy syndromes. We have noticed that this strategy has 95% of sensitivity to identify these patients. We showed 433 anaphylaxis episodes. A binomial logistic regression model was constructed to explore variables related to the use or non use of epinephrine among patients with anaphylaxis episodes who were attended in the Emergency Unit of our hospital. Results: Epinephrine was used in the 11.5% (95% CI 8.7–14.9) of anaphylaxis episodes, although there was a lineal relationship between severity of anaphylaxis and percentage of epinephrine use (mild 4.1%–95% CI 1.9–7.6-, moderate 10.2%–95% CI 5.9–16and severe 44.6%–95% CI 31.3–58.5-, Po 0.001). In the model of logistic regression, severe anaphylaxis (O.R. 16.8), lower respiratory system involvement (O.R. 3.5) was associated to a higher use of epinephrine. On the other hand, ages between 15–40 years (O.R. 0.1), and having drug anaphylaxis (O.R. 0.21) was associated to lesser use of epinephrine. In the next table, we show the odds ratios of epinephrine use related to different clinical variables. 95% lower limit

95% Upper limit

0.03 0.15 0.06

0.35 1.35 1.16

0.30

2.60

16.82 0.68

3.40 0.18

83.31 2.52

3.53

1.41

8.84

2.28

0.98

5.29

0.79

0.24

2.55

1.57

0.66

3.71

0.41 0.43 0.21 0.18

0.11 0.12 0.06 0.03

1.53 1.52 0.74 1.17

Odds ration Age 0–15 years Age 16–40 years Age 41–60 years Age 460 years Mild Anaphylaxis Moderate Anaphylaxis Severe Anaphylaxis Cardiovascular involvement Lower respiratory system involvement Upper respiratory system involvement Previous cardiovascular illness Previous pulmonar illness Idiopathic Anaphylaxis Food anaphylaxis Drug anaphylaxis Anisakis anaphylaxis

1 0.10 0.44 0.27 1 0.88

Conclusion: Out of them, severity of anaphylaxis was the principal reason to use epinephrine among patients with anaphylaxis episodes attended in Emergency Unit of our hospital. However drug anaphylaxis and ages between 16–40 years had a lesser use of epinephrine.



299 Evaluation of epinephrine treatment in anaphylaxis

sion, it questions the need for accessibility to multiple injections of epinephrine in the event of an anaphylactic reaction.

Wawrzkowicz, E; Luyt, D University Hospitals of Leicester, Children’s Allergy Clinic, Leicester, United Kingdom

Background: Anaphylaxis in children is most commonly caused by allergy to foods. Hospital treatment algorithms recommend intramuscular epinephrine and admission for observation post-treatment. Aims: The aim of this study was to evaluate treatment with epinephrine by observing the change in symptom severity of an anaphylactic reaction from onset through hospital stay and by assessing the relationship between administration of epinephrine and changes in symptom severity. Methods: We performed a retrospective audit of children o16 years old who were admitted to our institution between June 1997 and May 2005 with an acute anaphylactic reaction to food. Hospital notes were reviewed and data collected on signs and symptoms and medication administered from onset to admission and during hospital stay. Anaphylaxis was classified as mild if symptoms were cutaneous only and serious if other systems (e.g. respiratory) were involved. Patients: We evaluated 67 patient episodes in 46 boys and 21 girls; median age was 6 years and 6 months. Nineteen patients had had more than one admission; 28 had diagnosed asthma. Sixty-five episodes occurred following oral ingestion and 2 with skin contact only. The food trigger was identified as nuts in 27 patients, egg in 12, milk in 7 and fish and shellfish in one each; it was not identified in 2 patients. Results: Fifty-seven patient episodes were serious at onset; 14 were treated with epinephrine prior to arrival in hospital where 4 needed a further dose in hospital, as symptoms were still serious. Forty-three serious episodes were thus not treated preadmission; 29 received epinephrine in hospital whilst 14 resolved spontaneously. Ten patients had mild symptoms at onset, none were treated pre-hospital; symptoms progressed in 1 patient on admission who then received epinephrine. Two patients deteriorated later during hospitalisation where a further dose of epinephrine was deemed necessary. No biphasic reactions were observed. There were no fatalities. Conclusion: We have shown that most children with anaphylaxis to foods respond to a single dose of epinephrine even when presenting with serious symptoms. Many reactions resolve spontaneously without treatment. Admission for observation however remains necessary, as symptom severity need not be maximal at first assessment. As repeat does were not necessary pre-admisr 2008 The Authors Journal Compilation

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300 Adverse drug reactions (ADRs) to asthma therapy regimens in children and adolescences Fattahi, F1; Kourorian, Z1; Pourpak, Z1; Bemanian, M2; Gharagozlou, M2; Moin, M1 1 Immunology, Asthma and Allergy Research Institute, Medical Sciences/University of Tehran, Children Medical Center, Tehran, Islamic Republic of Iran, 2Medical Sciences/University of Tehran, Children Medical Center, Department of Immunology and Allergy, Tehran, Islamic Republic of Iran

Background: ADRs are a major burden on health care and could lead to morbidity and mortality. It is estimated that 5% of all patients suffer from an ADR. The asthmatic patients are not an exception. Moreover any chronic illness (including asthma) is a major risk factor for ADRs, which is probably, due to increased use of medication and polypharmacy. Indeed asthma medications use raises concerns over side effects, particularly in children and adolescents. We carried out a cross-sectional study to assess the nature and frequency of suspected ADRs among asthmatic patients and to identify the drugs involved in it. Methods: This study was conducted in the Department of Immunology and Allergy in Children Medical Centre during 12 weeks period (July-September 2005). All 133 patients younger than 20 years old admitted for following up their disease were questioned and evaluated for the presence of ADRs. An ADR documentation form was completed for all of patients. Results: Of studied asthmatic patients, 65.4% were male and 34.6% were female. The mean age was 6.33 7 3.75 years old. 247 numbers of drugs were consumed by the patients (1.85 numbers per patient). The most commonly used drugs were b2-agonists (35.2%), followed by inhaled corticosteroids (24.7%), ketotifen (23.5%) and second generation antihistamines (10.1%). In 23 of 133 patients (17.3%), 76 ADRs were detected. The most ADR was drowsiness (47.4%) followed by weight gain (14.5%) and palpitation (10.5%). Other side effects were less common. The most commonly culprit drug was ketotifen (61.9%), followed by second generation antihistamines (17.1%) and b2-agonists (13.2%). Other drugs were in next grades. The ADRs were mild or moderate in 92.1% of cases. Non-fatal adrenal insufficiency was reported in one case due to the long term use of corticosteroids. There was a significant relationship between ADR rate and number of regimens used.


Conclusion: These results suggest that ADRs in asthmatic patients are a significant public health issue. Concerning the common side effects of them and drowsiness which may produce problems in school attendance of children and adolescences, attention to appropriate prescription of drug such as ketotifen is necessary. Additionally, the number of drugs administered in patients was an ADR risk profile; therefore, education in appropriate use of drugs, including interpretation of safety information, is essential for patients and health-care providers.

301 Dermographism associated with a false positive drug hypersensitivity history in children Konstantinou, G1; Lourbas, G1; Zannikos, K2; Chatziioannou, A1; Giavi, S1; Douladiris, N1; Manoussakis, E1; Papadopoulos, N1 1 2nd Pediatric Clinic, Kapodistrian University of Athens, Allergy and Clinical Immunology Department, Athens, Greece, 22nd Pediatric Clinic, Kapodistrian University of Athens, Allergy Research Center, Athens, Greece

Background: Most of the children presenting to an Allergology department for drug hypersensitivity evaluation are proved to have a false positive history. Aim: To evaluate the presence of dermographism in children with a false positive history of drug hypersensitivity. Patients and methods: Sixty eight children (32 males) referred to our Department for drug hypersensitivity evaluation and further diagnosed as having a false possible history [cases] and 114 children (62 males) randomly selected from a generally school population without a previous history of any drug reaction [controls] were enrolled into this case-control study. Cases and controls were matched for age (9,5 7 3,2yo vs. 9,75 7 1,3yo respectively, t-test P 5 0,460) and gender (Fisher’s exact test P 5 0,654). All cases had a clear history of an urticarial and/ or maculopapular rash after antibiotic intake. Initially, they were examined for specific IgE (CAP System FEIA) and skin prick (SPTs) and intradermal (IDs) tests to the offending antibiotic(s) and to several others belonging to the same family. Afterwards (within a mean time of 13.7 months after the reaction), open challenges were offered if both SPTs and IDs were negative. Dermographism was objectively evaluated by testing with a calibrated dermographometer on the upper back. Any linear weal and flare reaction within 10 min after were considered as dermographism. Results: SPTs, IDs and open challenges were all negative suggesting a false positive drug hypersensitivity history. 53 out of these children (77.9%) were found to have dermo125


graphism comparing with only 3 (2.6%) of the controls (Fisher’s exact test Po0,0001). Symptomatic dermographism (defined as reaction at least to 36 gr/mm2 pressure) was rare since it was found in only 2 (2.9%) cases and 1 (0.9%) control. Conclusions: An idiosyncratic skin hyperreactivity, expressed as dermographism is frequently present in children with nonspecific skin manifestations attributed to drugs. It is not clear whether this skin hyperreactivity was present before the drug intake, caused by another stimulus, e.g. a parallel infection, or was induced from the drugs themselves, suggesting a new type of drug hypersensitivity.

302 Drug provocation tests in a pediatric population Silva, I; Chambel, M; Palma Carlos, S; Margarida Romeira, A; Martins, P; Leiria Pinto, P Dona Estefaˆnia Hospital, Immunoallergy Department, Lisbon, Portugal

Background: Drug hypersensitivity reactions to betalactamic antibiotics are commonly reported. Confirmation of the diagnosis should be based on clinical history, followed by skin tests and drug provocation tests (DPT). Despite this, intradermic skin tests are an invasive procedure, painful for young children and frequently difficult to perform. Objective: To describe the outcome of drug allergy investigation in a pediatric group of patients with a clinical history suggesting allergy to betalactamics. Material and methods: A retrospective analysis of clinical files from patients younger than 16 years old, with a suggestive clinical history of betalactamic allergy, tested in our outpatient clinic from 2001 to 2006. Results: A total of 110 patients were included, 65 (60%) were boys. The median age of the reaction was 3 years (p25–75 : 1–5 years) and the median age of DPT was 5 years (p25–75 : 3–7 years). 50% of the reactions occured in the first two days of treatment; half of the patients had symptoms in the 60 min following the last drug intake and 75% of patients recovered in 3 days, once they suspended it. Concerning the clinical presentation of the reaction, 91% had mucocutaneous symptoms, 7% gastrinstestinal and 5% respiratory. Anaphylaxis was reported in one patient. In the drug allergy investigation, 38 patients were submitted to skin tests to betalactamics, being positive in 27%. In those with a negative skin test result, who performed a DPT with the culpright drug, 8% had a positive reaction. 72 patients performed DPT without any previous skin test to 126

betalactams. The young age and the mild reaction described by parents were the main reasons for this clinical decision. From those, 47 children were challenged with the culpright drug and 8% had a positive reaction. From the 38 patients who were submitted to an alternative drug, 2,8% were positive. All the reactions during DPT were mild and easily controlled. Patients who performed skin tests were slightly older and presented more allergic respiratory symptoms than those who weren’t submitted to this procedure. Conclusions: DPT to betalactamics is a safe procedure in young children. In those with a mild reaction namely cutaneous, the decision of carry it out without previous skin tests seems a valid alternative.

24 h, 42.7% of them without any treatment. We made CAP in 8.7% of the reactions with negative results in all of them and skin tests methods in 50 cases (22.9%) being positive 12% (6 of them). The drug provocation was carried out in 89.4% of the cases, being positive 14.3% of them. Seventy five per cent of positive drug provocation tests were positive at home (delayed reactions). Conclusions: 14.7% of the children had positive allergologic tests. Only in four of them we could prove an allergic mechanism. Maculopapular rash is the most common clinical manifestation. Amoxicilin is the drug most frequently studied as a cause of these reactions. CAP determination is not usefull in this patients study. Because of the high positive results at the reprovocation we recommend its use.

303 Reassessment after two years of suspected adverse reactions to betalactam in Spanish children

304 Drug allergy in spanish children. Two years review

´ lvarez, M; Rodrigo Garcıá, G; Frıás Garcıá, M; Va´zquez A Quevedo Teruel, S; Bracamonte Bermejo, T; Echeverrıá Zudaire, L Severo Ochoa Hospital, Pediatric Allergy Unit. Pediatric Department, Madrid, Spain

´ lvarez, M; Quevedo Teruel, S; Rodrigo Garcıá, Va´zquez A G; Frıás Garcıá, M; Bracamonte Bermejo, T; Echeverrıá Zudaire, L Severo Ochoa Hospital, Pediatric Allergy Unit. Pediatric Department, Madrid, Spain

Objetive: Antibiotics are frequently used in childhood, especially betalactams, which are responsible for many suspicions of drug adverse reactions in a pediatric allergy consultation. We describe the kind of reaction, the drugs involved and the results of the allergologic studies done to the patients referred to our consultation for this cause. Material and methods: Histories of the patients referred to the pediatric allergy consultation for betalactams allergy suspicion from January 2006 to December 2007 were reviewed restrospectively. We reviewed epidemiologic data, the drugs involved, the characteristics of the reaction and the results of the carried out allergologic study. Outcomes: Two hundred and eighteen reactions were assessed. The average age of the patient was 5 years and 7 months. 49.1% were men. The time spent from the reaction to the first consultation was of 3 month (mean). 36.7% of the reactions took place in the first 24 h from the beginning of the treatment and 6.4% once it had concluded. The most frequent suspicious drugs were amoxicillin (56%), amoxiclavulanic (30.3%), penicillin V (6.4%) and cephalosporins (6.8%). 29.4% had had the drug for the first time. 94.5% had cutaneous symptoms (maculopapular rash (53.8%), urticary (41.2%), angioedem (12.6%)), 6,4% had had digestive symptoms, 0.45% had had respiratory symptoms and two patients suffered from anaphylaxis. The clinical manifestations had disappeared within

Objetive: Drug hipersensitivity reactions are common in pediatric age. There are not many studies done about them though. We will describe the kind of reaction, the drugs involved and the results of the studies done to the patients referred to our consultation for this cause. Material and methods: Histories of the patients referred to the pediatric allergy consultation for drug allergy suspicion from January 2006 to December 2007 were reviewed restrospectively. We reviewed epidemiologic data, the drugs involved, the characteristics of the reaction and the results of the carried out allergologic study. Outcomes: Two hundred and ninty six reactions were assessed. The average age of the pacients was 5 years and 7 months. 49.7% were men. 72.3% had only one drug involved in the reaction (rank 1–4). The time spent from the reaction to the first consultation was of 3 months (mean). 64.7% of the reactions took place in the first 48 h from the beginning of the treatment and 5.3% once it had concluded. The drugs more frequently involved were betalactam antibiotics 73.6%. (amoxicillin 56%, amoxiclavulanic 30.3%, penicillin V 6.4% and cephalosporins 6.8%), NSAID 15.5% (ibuprofen 78.3%, acetaminophen 10.9%, and others 10.8%) and macrolids 4.7%. The 29.7% of the children had had the drug for the first time. 93.6% of them had cutaneous symptoms (maculopapular rash 45.5%, urticary 42.3% and angioedem 18%) and 3 pacients suffered

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anaphylaxis. 42.2% of the patients did not need any treatment for the reaction. We studied 281 reactions. We made skin tests to 58 cases (prick test, patch test and intradermal skin test), 8 of them were positive (13.7%). A drug provocation test (DPT) was carried out in 94.2% of the cases, being positive in 35 children (12.5%). We carried out a reprovocation in the 22.7% of negative DPT and 10.9% of them were positive. 24.6% of the children had family history of drug allergy. Conclusions: 14.2% of the children presented positive allergologic study, only 5 of them had allergic mechanism demonstrated. DTP is the most commonly used test while cutaneous tests are only used in those cases with suggestive clinical manifestations of drug allergy. Amoxicillin, amoxiclavulanic and ibuprofen are the most frequently involved drugs in these reactions.

305 Diagnosis of allergy to beta-lactams in children with skin symptoms Caubet, J1; Kaiser, L2; Gervaix, A1; Eigenmann, P1 University Hospital of Geneva, Department of Pediatrics, Geneva, Switzerland, 2University Hospital of Geneva, Central Laboratory of Virology, Geneva, Switzerland

urticarial rash. Most of the patients were treated for ENT infections (otitis (60.7%) and pharyngitis (16.1%)). The antibiotics used were amoxicillin (48.2%), amoxicillin/ clavulanic acid (37.5%) and various cephalosporins (14.3%). In only 7 patients (12.5%) the skin rash was reproduced during the subsequent oral challenge test. Six of these patients had a delayed reaction (46 h after the first dose) and one an immediate reaction (within 30 min). No patient had experienced a more severe reaction than the initial one. Four patients with a positive challenge test had at least one positive IDR. Patch tests were always negative. Viral RNA for respiratory virus was found in 3/7 (42.8%) and 24/49 (48.9%) of the patient with a positive or negative challenge respectively. Conclusion: Skin rashes during beta-lactam treatments are a major problem for primary care physicians of children. In this situation, beta-lactam allergy is clearly overdiagnosed as the skin rash is only relatively rarely reproducible (12.5%). Oral challenge tests should be discussed in all children who develop a benign cutaneous eruption in relation to a treatment with beta-lactams.

1

Background: Urticarial or maculo-papular skin rashes are frequently observed in children treated by beta-lactam antibiotics. Although, such manifestations are thought to be mainly caused by the infection itself, a majority of these children are labeled as ‘‘penicillin allergic’’ without appropriate testing and are incorrectly deprived from any further treatment with penicillin and related antibiotics. Objectives: The aim of our study was to determine the etiology of urticarial and maculo-papular skin rashes occurring in conjunction with beta-lactams treatment. Methods: Children with an urticarial or maculo-papular rash during and up to 72 h after a treatment with a beta-lactams antibiotic were prospectively recruited. The patients were included at the emergency department of the Geneva University Hospital or referred during the acute episode by practicing pediatricians. During the first visit, a blood puncture (total white blood cell count and C-reactive protein) and a throat swab (for virus screening by RTPCR) were made. Two months later, all patients underwent allergy diagnostic skin tests (IDR and patch tests) and an oral challenge test with the incriminated antibiotic. Results: Fifty-six children (26 girls and 30 boys) were recruited. Thirty nine patients presented a maculo-papular and 17 an r 2008 The Authors Journal Compilation

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306 Allergic sensitisation to latex in an infantile general population. The 6 Cities Study Kalaboka, S1; Lavaud, F2; de Blic, J3; Charpin, D4; Raherison, C5; Kopferschmitt, C6; Caillaud, D7; AnnesiMaesano. I8 1 Inserm UMR-S707; UPMC Paris 6; Medical School StAntoine, Epidemiology of Allergic and Respiratory diseases, Paris, France, 2CHU Reims, Department of Pulmonology, Reims, France, 3Necker Hospital, Pediatric Pulmonology and Allergology, Paris, France, 4CHU Marseille, Department of Pulmonology and Allergology, Paris, France, 5Haut-Leveque Hospital, CHU Bordeaux, Department of Respiratory Diseases, Bordeaux, France, 6 CHRU Strasbourg, Department of Pulmonology, Strasbourg, France, 7CHU Clermont-Ferrand, Department of Pulmonology and Allergology, Clermont-Ferrand, France, 8Inserm UMR-S707; UPMC Paris6; Medical School St-Antoine, Epidemiology of Allergic and Respiratory diseases, Paris, France

Background: In spite of the relevance of latex allergy for sensitised individuals and its consequences in public health, due to its clinical expressions including anaphylactic shock and cross-reactions with certain food, latex sensitisation has been primarily investigated in symptomatic subjects or multioperated patients, health or household personnel and the workers in the latex industry. The purpose of this study was to estimate the prevalence of allergic sensitisation to latex in a general population-based sample of children, with a validated method and a standardised allergen. Methods: Within the framework of the 6 Cities (6C) Study, which is the French


contribution to the International Study of Asthma and Allergies in Childhood (7781 children in 6 French cities), the children selected to participate in the city of Reims were also tested (Skin Prick Test) for latex. The test was carried out at the school with the agreement of the Ethics committee. Prior to the test, the parents of the children had answered a relevant questionnaire, allowing to identify the children with a possible risk for anaphylactic shock (spina bifida, urologic malformations, multioperations...). Results: Among the children of the study, 755 children of Reims, aged from 9 to 11 years, underwent SPT with latex. The prevalence of allergic sensitisation to latex (mean diameter of the wheal 43 mm) was 0.66% (n 5 5). No accident occurred during the realization of the test. Conclusion: Our study made it possible to estimate for the first time in France the allergic sensitisation to latex in a population-based sample of school-aged children. This study proceeded without incidents thanks to a questionnaire on the child’s health history. After exclusion of children with contraindications for the test to latex, the prevalence was still of almost 1%. More studies are necessary to evaluate the utility of the systematic research of latex sensitisation, in order to prevent the latex related accidents and thus to better protect the sensitised children.

307 Low C4 levels with normal C1 INH levels in children with suspected hereditary angioedema Kocak, A; Dinleyici, E; Poyraz, H Eskisehir Osmangazi University, Faculty of Medicine, Department of Pediatric Allergy, Eskisehir, Turkey

Background: Hereditary angioedema (HAE) is a disorder characterized by episodic, highly localized, non-pitting edema that usually involves the extremities, but may affect the face, extremities, and gastrointestinal tract and generally manifests with recurrent episodes of soft tissue swelling without urticaria. Multiple parts of the body may be affected, including and especially HAE is a result of decreased levels or function of complement C1 esterase inhibitor. Serum C4 level is a good screening test for HAE and reduced levels of serum C4 have been considered a ubiquitous finding in HAE. Methods: Consecutive 13 children with suspected HAE were evaluated for serum C4 levels, C1 INH levels and clinical findings. Results: Thirteen consecutive children aged between 5 to 17.5 years who have a history of recurrent episode of soft tissue swelling were recruited. All of these patients have 127


respiratory system findings and cutaneous swellings. Two out of them had gastrointestinal findings such as recurrent colicky abdominal pain and one of this two cases admitted to our emergency unit mimicking

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acute abdomen. All patients have low serum C4 levels and (median 10, min-max 6– 14.8 mg/dl) and normal C1 INH levels (median 0.3, min-max 0.22–0.39). We could not perform the activity of C1 INH.

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Conclusion: All patients who are suspected of having HAE should have a C4 level measured. These patients C1 INH activity should be evaluated as well as C1 INH levels.


Poster Discussion Session 10 – Occupational Allergy in Health Care Workers and in Damp Environments

Poster Discussion Session 10 Occupational Allergy in Health Care Workers and in Damp Environments 308 Latex allergy in hospital professionals: a real problem Rodrigues-Alves, R1; Sousa-Uva, A2; Lima, M3; Pereira-Santos, C4; Branco-Ferreira, M1; Pereira-Barbosa, M1 1 Hospital Santa Maria, Immunoallergy Department, Lisbon, Portugal, 2Universidade Nova de Lisboa, Escola Nacional de Sau´de Pu´blica, Lisbon, Portugal, 3Centro Hospitalar de Lisboa Ocidental, Occupational Heath Department, Lisbon, Portugal, 4Instituto de Medicina Molecular/Faculdade de Medicina de Lisboa, Unidade de Imunologia Clıńica, Lisbon, Portugal

Background: Allergic reactions in latex sensitised individuals exposed to latex are very common and can be extremely severe. Consequently it is particularly important evaluate the risk of sensitisation in health care professionals. Our purpose was to quantify the prevalence of latex allergy in health care workers of a Portuguese central hospital. Methods: Four hundred and twenty six workers (88% exposed to latex in the workplace) answered to a standardized questionnaire to assess symptoms in last the 12 months associated to the use of latex gloves. Additionally, workers with respiratory, naso-conjunctival or cutaneous symptoms associated to gloves use, or aggravated in the workplace, performed skin prick tests (SPT) with standardized extracts of latex, avocado, banana, chestnut, kiwi and peach. Specific IgE quantifications for latex, Hev b1, Hev b3, Hev b5, Hev b6.01, Hev b6.02, Hev b8, Hev b9 and Hev b11 were performed in workers with positive SPT. Results: Rhinitis, wheezing and urticaria and/or eczema complaints, in last the 12 months, were 58%, 23% and 34%, respectively. About one third of the inquired workers referred the existence of at least one type of complaints related with the use of latex gloves (29% cutaneous complaints; 7% nasal complaints; 1% respiratory complaints). These symptoms were correlated with: female sex (OR 5 3,1); rhinitis in last the 12 months (OR 5 3,4); use 47 gloves/ day (OR 5 1,8) and use of gloves during 43 h/day (OR 5 2,2). From the 120 workers with complaints that accepted to perform SPT, five had positive SPT for latex and only one for the tested fruits. The presence of latex allergy (prevalence of 1,9%) was correlated to the presence of naso-conjuntival complaints (OR 5 28,1) and respiratory complaints (OR 5 3,7). Specific IgE quantifications for latex were positive (40,35 kU/L) in r 2008 The Authors Journal Compilation

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the five workers, and the majority of them were sensitised to Hev b6.01 and Hev b6.02. Conclusion: We documented a high prevalence of rhinitis, wheezing and urticaria/ eczema complaints associated to the use of latex gloves, in health professionals, which was not necessarily revealing of latex allergy diagnosis. The reduced prevalence of latex allergy comparatively to other studies could be related to the exclusive use of nonpowdered latex gloves in the hospital studied. This fact highlights, once more, the importance of this preventing measure.

309 Relationship between patterns of sensitivity to recombinant latex allergens and latex-related clinical symptoms in health care workers Tavares-Ratado, P1; Tomaz, M2; Ribeiro, H3; Lozoya, C4; Taborda-Barata, L1 1 University of Beira Interior, CICS – Faculty of Health Sciences, Covilha, Portugal, 2Viseu District Hospital, Allergy/Internal Medicine Department, Viseu, Portugal, 3 Viseu District Hospital, Occupational Medicine Department, Viseu, Portugal, 4Amato Lusitano Hospital, Allergy/Respiratory Medicine, Castelo Branco, Portugal

Background: Latex sensitisation is still of concern in health care workers (HCW). HCW are preferentially sensitised to Hev b2, Hev b5 and Hev b6.02. However, the relationship between sensitisation patterns and severity and spectrum of clinical latexrelated symptoms is currently unknown. Therefore, the objective of the present study was to determine the relationship between specific IgE responses to recombinant latex allergens and clinical responses to latex. Methods: Cross-sectional study carried out in a randomly selected population of HCW. Subjects filled out a specific questionnaire. Total serum IgE was determined using direct chemiluminescence (ADVIA Centaur, Bayer). Phadiatop, total latex- and recombinant latex allergen-specific IgE (rHev b 1,2,3,5,6.01, 6.02, 8, 9 and 11) were determined using ImmunoCAP 250 (Phadia). Clinical scores were used for classification of severity of latex-related symptoms. Mann-Whitney U, Chi-square test (with Bates’ correction) and Fischer’s Exact Test were used for statistical analysis. Results: Blood was randomly taken from 225 HCW. Sixty four had a positive questionnaire (28.4%). Of these, 10 (16%) had positive total latex (k82)-specific IgE. Four


patients were sensitised to Hev b5, Hev b 6.01 and Hev b 6.02, and 2 of these were also sensitised to Hev b 11. These 4 patients showed the highest severity of latex-related symptoms. Three patients were only sensitised to Hev b8 and 3 patients were solely sensitised to Hev b1, Hev b 3 or Hev b11. The patient with the broadest pattern of sensitisation (Hev b 1, 5, 6.01, 6.02 and 11) had the highest total latex-specific IgE level, and also the most significant clinical symptoms (eczema, nasal and ocular symptoms, dyspnoea and laryngeal oedema). A total sum of individual levels of specific IgE to recombinant latex allergens greater than 15 kUA/L was significantly associated with more severe symptoms. Sensitisation patterns were not related to years of surgical glove usage or general atopy. Conclusion: Our results suggest that the broader the response pattern to latex allergens is, the higher the possibility and severity of allergic reactions upon exposure. They also suggest that concurrent sensitisation to Hev b5, Hev b6.01 and Hev b6.02 is associated with more severe symptoms.

310 Latex allergy in hospital workers – followup of symptomatic subjects Folletti, I; Paolocci, G; Armadori, M; Bussetti, A; Siracusa, A University of Perugia, Clinical and Experimental Medicine, Terni, Italy

Background: Latex allergy (LA) manifests with a variety of clinical disturbances such as contact urticaria, angioedema, rhinoconjunctivitis, asthma, and anaphylactic reactions. Prevalence of sensitisation ranges from 2.8% to 17% in health-care workers and the main determinants of sensitisation and clinical symptoms are intensity of exposure, atopy, and pre-existing hand dermatitis. This follow-up study determined whether changing from powdered to powder-free latex gloves (LG) reduced workrelated symptoms of LA in previously surveyed hospital workers. Methods: In the first investigation we studied 235 (75.3% females) workers in Terni General Hospital, Italy. All subjects completed a questionnaire and underwent skin prick tests (SPT) for 8 common aeroallergens, two latex extracts, positive (histamine 10 mg/mL) and negative (normal saline) control solutions. Fifty three hospital work-

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ers with work-related latex symptoms where followed up 4.6 years after the first investigation and adoption of preventive measures, such as use of powder-free LG, to evaluate change in the prevalence of work-related symptoms. Results: Females had longer and more intense everyday exposure to LG, and had more positive SPT to latex than males. Irritative symptoms were 2–3 times more frequent in females. Intensity of occupational exposure to LG, as estimated by the number of pairs of gloves/day and hours/ day spent wearing them, was associated with irritative (Po0.05) and allergic symptoms (Po0.05). In subjects with positive SPT to common allergens the prevalence of allergic symptoms was 2.5 higher than in those with negative SPT (Po0.05). In subjects with positive SPT to latex the prevalence of allergic symptoms was 14.6 times higher than in those with negative SPT (Po0.001). Years of latex exposure and smoking habits were not significant predictors of workrelated latex symptoms. At follow-up after 4.6 years, 54.7% of 53 workers with workrelated latex symptoms used both powdered and powder-free LG, 37.7% used only latex free gloves and 7.6% stopped glove use. In 68% of subjects work-related symptoms were in remission or had improved. Improvement was greater in workers using powder-free gloves than in the others (Po 0.005). Conclusions: Our study shows that preventive measures, such as the use a powder-free latex gloves, are sufficient to reduce workrelated symptoms.

311 Does chlorhexidene pose an allergy risk for health care workers? Wicking, J1; Ekbote, A2; Nagendran, V2 Queen Elizabeth NHS Trust, Immunology, Biochemistry and Haematology, Woolwich, London, United Kingdom, 2 Epsom and St Helier University Hospitals NHS Trust, Immunology, Carshalton, United Kingdom

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Hospital acquired infections are common in the United Kingdom (UK), and in recent times hospitals have mounted vigorous campaigns encouraging frequent hand washing. We are aware that Healthcare workers are exposed to hand washing agents including chlorhexidine. Since its first use in 1954 there have been numerous documented cases of reactions to chlorhexidine. These have ranged in severity from contact dermatitis to anaphylaxis. Specific IgE mediated reactions (Type I hypersensitivity) are predominantly histamine mediated and present clinically as urticaria, angioedema, bronchospasm and anaphylaxis. It is now possible to measure Specific IgE to chlorhexidine (Phadia immunoCAP). To determine the 130

prevalence of allergy to chlorhexidine, a survey by questionnaire was carried of those most likely to be exposed to chlorhexidine. Staff working in the Emergency Department, Theatres and Gynaecology as well as those reporting to the Occupational Health Department at Queen Elizabeth Hospital, Woolwich, UK completed questionnaires. Fifty-three questionnaires were returned with nine potential reactors who reported they might have chlorhexidine allergy. All reported itchy skin following chlorhexidine use. Seven out of the nine had a co existing allergies, four of which were to latex. Two had hay fever and one had an allergy to shrimp. One of the nine staff had contact dermatitis and one had no allergies. Preliminary results show no specific IgE antibodies to chlorhexidine in those tested.

312 Exposure and atopy as independent risk factor of sensitisation to pancreatic enzymes among health care workers in a hospital Kim, S1; Lee, B1; Lee, J1; Park, H2 Eulji Hospital, Eulji university medical school, Internal Medicine, Seoul, Republic of Korea, 2Ajou University Medical School, Allergy and Rheumatology Department, Seoul, Republic of Korea

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Background: Enzyme powders inhaled at work can induce respiratory allergy in health care workers. It is important to know which risk factors contribute to the sensitisation of these enzymes in health care workers. This study evaluates the risk factors of sensitisation to enzyme powders in health care workers. Methods: A total of 220 health care workers (199 nurses and 21 pharmacists) who exposed to pancreatic enzyme powders (a-amylase and pancreatin) and 64 unexposed controls (office workers) in a hospital were analyzed using a questionnaire, and measurements of total serum IgE level and specific IgE to house dust mite and pancreatic enzymes (a-amylase and pancreatin). Logistic regression analysis was performed to obtain the adjusted odds ratios (OR) and 95% confidence intervals (CI) for the independent effect of risk factors on sensitisation to pancreatic enzymes. Results: The overall prevalence of sensitisation to pancreatic enzymes was 4.7%. Sensitisation to enzymes is significantly higher in pharmacists (28.6%) than in office workers (0%) and in nurses (5.0%). Occupational exposure and atopy were associated with an increased risk of these sensitisations, after adjusting for age and gender (adjusted odds ratio [OR], 11.5; 95% confidence interval [CI], 3.6B36.6, and OR, 3.2; 95% CI, 1.1B9.2, respectively). In contrast, working duration, and body mass index

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were not associated with sensitisation. In multivariate analysis, occupational exposure (OR, 11.0; 95% CI, 2.8B43.0), and atopy (OR, 5.6; 95% CI, 1.6B19.1) were associated with sensitisation to pancreatic enzymes. Sensitisations to pancreatic enzymes were not associated with work-related respiratory symptoms among the study population as a whole. Conclusion: Occupational exposure and atopy were the most important determinant of sensitisation to pancreatic enzymes in health care workers.

313 Occupational asthma caused by cefotiam in a nurse Lee, S; Yang, D; Son, C College of Medicine, Dong-A University, Internal Medicine, Busan, Republic of Korea

Occupational asthma has been defined airway inflammation, hyperresponsiveness, and reversible airway obstruction related to exposure in the workplace. Cephalosporins are well known agents that may cause occupational asthma and there have been a few reports suggesting that the pathogenesis may be IgE-mediated. To the best our knowledge, this is the first report of cefotiam-induced occupational asthma. A 36 year-old female, a nurse, visited our allergy department for evaluation of occupational asthma. She had worked in surgical inpatients’ ward, and handled a few kinds of antibiotics for four years. At that time, she had developed paroxysmal cough, dyspnea and chest tightness for two months. Her symptoms used to be worsened during and shortly after her work and subsided several hours after work. However, her symptoms were subsided spontaneously, because she was brought to outpatient’ department for recent 3 years. Therefore, she had only mild dyspnea on exercise. She had no history of previous allergies. Skin prick test (SPT) to 80 common allergens showed negative responses and methacholine bronchoprovocation test revealed not positive response. Skin prick test with cefotiam showed a strong positive response and specific bronchoprovocation test with cefotiam demonstrated an early asthmatic response. One day after bronchoprovocation test with cefotiam, methacholine bronchoprovocation test showed positive response. In conclusion, the pathogenetic mechanism of cefotiaminduced occupational asthma may be an IgE-mediated allergic reaction by the strong positive reaction in skin test and early asthmatic response in cefotiam bronchoprovocation test. Further studies will be needed to evaluate exact pathogenetic mechanism of cefotiam-induced occupational asthma.



314 Formaldehyde-induced asthma in healthcare workers (about 2 cases) Cherif, J1; Benzarti, A2; Daghari, F2; Mehiri, N1; Mamlouk, H2; Beji, M1; Ben Jemaa, A2 1 Hospital, pulmonology and allergology, Tunis, Tunisia, 2 Hospital, Occuational diseases department, Tunis, Tunisia

Formaldehyde is a very widely used chemical in hospital particularly in disinfecting agents and cleaning products. It represents a strong allergen and a low weight molecule that can be easily combined with other proteins to form additional antigens resulting in the production of specific antibodies eliciting thereby acute and chronic health related problems. Only few cases of formaldehydeinduced asthma in healthcare workers were reported in the literature. We herein report two cases of 43 and 56 years old nurses working since an average of 30.5 years. The first patient is working in an emergent department and manipulating formaldehyde when disinfecting and sterilizing materials. She has developed rhinitis and conjunctivitis then dyspnoea with sibilance. Spirometry was normal. Unspecific provocation test to methacholine was positive at 400l, total IgE were high, skin prick test and RAST to formaldehyde were positives. The second case concern a polyvalent nurse responsible in sterilizing diverse materials with formaldehyde. She has developed progressive dyspnoea with sibilance. Spirometry was normal. Non specific provocation test to methacholine was negative, total IgE antibodies were high and RAST to formaldehyde were negatives. However, skin prick test and specific provocation with formaldehyde were positives. Healthcare workers are at risk for work-related asthma. Healthcare provides need to recognize this risk, even thought induced formaldehyde asthma is rare due probably to undiagnosed cases. Prevention is essential and exposure should be avoided.

Methods: Personal samples (n 5 413) in various dental jobs (clinical, administration, laboratory and auxiliary) as well as airborne area (n 5 116) and water (n 5 403) samples from dental handpieces were collected in 5 academic dental institutions. The chromogenic-1000 limulus amoebocyte lysate (LAL) assay was used to determine endotoxin levels. Exposure metrics were developed on the basis of individually measured exposures and average levels within each job category. Multiple linear and logistic regression models were constructed. Results: There was a two-fold variation in personal airborne endotoxin from the least (administration) and most exposed (laboratory) jobs (geometric mean levels: 2.38 vs. 5.63 EU/m3). In the linear models, institution explained the most variability in endotoxin in air (r2 5 0.25) and water (r2 5 0.29), followed by sampling month in air (r2 5 0.19) and water (r2 5 0.27). A model explaining the greatest variability (adjusted r2 5 0.41) included building age, number of dental units per institution, ambient temperature, air velocity, endotoxin levels in water, job type, dental unit model type and dental unit age. Significant predictors of high (upper tertile:45.84 EU/m3) versus low airborne endotoxin exposure (lowest tertile:o2.23 EU/m3) were dental unit age (OR 5 25.6), clinical dental job (OR 5 17.7), building age (OR 5 1.6), number of dental units per institution (OR 5 1.2). Conclusion: Aside from job type, dental institutional characteristics are important predictors of elevated aerosolised endotoxin levels in dental health care settings.

316 Exposure of health-care staff to biological dust in indoor air: the case of microbes and mites associated with moisture damage Pennanen, S1; Sahlman, M1; Reiman, M1; Putus, T2 Finnish Institute of Occupational Health, Kuopio, Finland, 2National Public Health Institute, Finland

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315 Workplace determinants of endotoxin exposure in dental health care settings Singh, T1; Mabe, O1; Bello, B2; Jeebhay, M3 National Institute for Occupational Health, NHLS, Immunology & Microbiology, Johannesburg, South Africa, 2National Institute for Occupational Health, NHLS, Epidemiology & Surveillance, Johannesburg, South Africa, 3University of Cape Town, Occupational and Environmental Health Research Unit, Cape Town, South Africa

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Background: Aerosols generated during dental procedures have been reported to contain endotoxin from bacteria contamination of dental unit water lines. This study investigated the determinants of airborne endotoxin exposure in dental health care settings. r 2008 The Authors Journal Compilation

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Every year occupational diseases caused by microbes and mites are diagnosed among Finnish health-care centre staff. This study assessed the mite and microbe exposure and sensitisation of workers in health-care centres. Four health-care centres with mould damage or some other indoor air problem participated the study. Samples of settled dust were analysed for mites, microbes and allergens and air samples for microbes. All the workers in the health-care centres completed a symptom questionnaire and gave a blood sample for mite-specific and microbe-specific IgE and IgG antibody analysis.Mites were found in 47% of dust samples at densities ranging from o14 to 67


mites per gram of dust. Most of the mites were storage mites. The counts of viable moulds and bacteria in indoor air were low, but the samples nevertheless contained some indicator species typical of moisture-damaged buildings (e.g. Aspergillus versicolor, Aspergillus fumigatus). In dust samples, the number of viable fungi ranged from 102 to 105 cfu/g and that of viable bacteria from 105 to 108 cfu/g. The concentrations of mite and fungi allergens were below the detection limit. Irritation symptoms and respiratory infections were common among the study subjects. Five per cent of the subjects were sensitised to mites and 4% to fungi. The sensitisation correlated significantly with doctor diagnosed asthma (Po0.05). Sensitisation to fungi correlated significantly also with cough and phlegm production (Po 0.001). In conclusion, the results indicated that exposure to mites and microbes is possible in health-care centres, and that the levels of exposure are sufficient to cause symptoms and clinical disease.

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Abstract withdrawn.

318 Hypersensitivity pneumonitis caused by molds in a hairdresser Segura, N; Sobrevia, M; Cubero, J; Fraj, J; Goienetxe, E; Cola´s, C Clinical University Hospital, Allergy Department, Zaragoza, Spain

Background: Hypersensitivity pneumonitis is an allergic lung disease related to repeated inhalation of a variety of etiologic agents. A 29 year-old woman, hairdresser who works in her own damp business, was attend in emergency room and then admitted in hospital for three days. She presented a history of cough and fever lasting one month. Molds have been described as a cause of Hypersensitivity pneumonitis. Aspergillus contaminated humidifiers and heating ventilation systems. Methods: We request Blood test and precipitating antibodies. We studied the patient after working in her hairdresser’s for two hours in the morning. In the afternoon in the emergency room of our hospital we performed a Physical examination, Chest Xray and request another Blood test. The next morning we asked for Pulmonary function tests and a diffusion capacity study. Results: Physical examination revealed fever. Blood test showed severe leukocytosis and neutrofilia. Chest X test without radiological signs. Pulmonary function tests and the diffusion capacity were normal. Precipi-

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tating antibodies to A. fumigatus were detected in the patient’s serum. Conclusion: The patient reported that the hairdresser’s had dampness. After getting another place to work as a hairdresser she is asymptomatic. The results of the investigations carried out in our patient indicated that she probably had developed hypersensitivity pneumonits caused by indoor molds (Aspergillus species).

319 Mould-induced occupational asthma: from asthma-like symptoms to clinical asthma Karvala, K1; Toskala, E2; Luukkonen, R3; Nordman, H1 Finnish Institute of Occupational Health, Team of Occupational Medicine, Helsinki, Finland, 2Finnish Institute of Occupational Health, Team of Control of Hypersensitivity Diseases, Helsinki, Finland, 3Finnish Institute of Occupational Health, Team of Statistical Services, Helsinki, Finland

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Background: Building-related moisture and mould damage are significant indoor air problems in a growing number of countries. Numerous studies confirm the association between exposure to indoor air dampness and moulds and different health outcomes. The evidence that building dampness and moulds aggravate pre-existing asthma is plausible, and there is limited evidence for an association between the exposure and the induction of new asthma. We followed up patients earlier diagnosed with asthma-like symptoms for possible later development of clinical asthma. Methods: In 1995–2004, altogether 2196 patients, who had work-related symptoms appearing in damp and mouldy indoor environments, were examined at the Finnish Institute of Occupational Health (FIOH). In this follow-up study, a questionnaire comprising sections of present symptoms, health and working conditions, was offered to these patients in the beginning of 2007 (2–15 years after the examinations). The questionnaire had 60% (1306/2196) participation. Originally, 570 respondents had been diagnosed with asthma (either occupational or non-occupational asthma) at FIOH, and 536 respondents had had workrelated asthma-like symptoms (cough, dyspnoea or wheezing) without objective evidence of asthma. Later development of clinical asthma was assessed by the question: Have you got physician-diagnosed asthma after the examinations at FIOH? Results: There were 536 (536/1306) patients with asthma-like symptoms in the baseline. From these, 67% were still working and 20% had moisture damage at the present workplace. Altogether 18% (98/536 patients), reported having got a physician diagnosis of asthma later. Conclusion: A significant proportion (18%) of our patients suffering from work-related 132

asthma-like symptoms had developed clinical asthma diagnosed by a physician. If examinations are performed at a symptomatic stage, before the development of lung function deterioration, the clinical asthma may easily be labelled non-occupational without considering the possibility of occupational asthma. To avoid the risk of occupational asthma being unrecognized due to prior negative examinations, it is important to arrange regular health surveillance of patients with work-related asthmalike symptoms and repeat the clinical assessment if consequent deterioration of lung function occurs. Missing out the correct diagnosis will leave the workplace ignorant of the health risks.

320 Cleaners asthma: assessment of airway inflamation using induced sputum and flow cytometry during specific inhalation challenges with chlorine Madero, M1; Olivares, M1; Fernandez-Nieto, M1; SańchezGarcia, S1; Sastre, B2; Del Pozo, V2; Quirce, S1; Sastre, J1 1 Fundacioń Jimeńez Diaz, Allergy, Madrid, Spain, 2 Fundacioń Jimeńez Diaz, Immunology, Madrid, Spain

Background: Cleaning substances are associated with worsening or development of occupational asthma. Sodium hypochlorite (bleach) is the chemical substance more frequently associated to this disease. The pathophysiology is poorly known. Methods: Six patients who work as cleaners either in houses or office buildings and 3 control patients with asthma without exposure to cleaning products were studied. All were females with a mean age of 43,5 years for cleaners and 39 years for asthmatic controls. All cleaners reported asthma symptoms (cough, dyspnoea or wheezing) with occupational exposure to bleach. Spirometry, methacholine challenge and induction of sputum were performed on a control day and 24 h after specific inhalation challenge (SICs). Blood and sputum samples were obtained after giving written informed consent and analysed using Flow cytometry. Results: Atopy was present in 3 cleaners and 2 asthmatic control patients. Baseline methacholine test was positive in 3 cleaners and 2 asthmatic controls. Sputum eosinophilia (43%) was present in all patients. SICs in cleaners elicited two late and one dual asthmatic responses. PC20 methacholine significantly decreased 24 h after SIC in 3 cleaners and one control patient. A mild increase in eosinophils (12,6 vs. 15,8%), activated basophils (0,57 and 1,33%), and activated eosinophils CD69 1 (5,98 and 6,61%) percentages was found in the cleaners group 24 h after SIC. No differences in asthmatic controls were found.

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Conclusion: This preliminary data demonstrate a positive bronchial challenge to chlorine in some cleaning workers. No significant changes in sputum cells were observed after challenges except for activated basophils.

321 Exercise-induced respiratory symptoms and exercise-induced bronchoconstriction in female cleaners Minov, J1; Karadzinska-Bislimovska, J1; Vasilevska, K2; Risteska-Kuc, S1; Stoleski, S1; Mijakoski, D3 1 Institute of Occupational Health, WHO Collaborating Center, Dpt for Cardiorespiratory Functional Diagnostics, Skopje, Macedonia, Fyrom, 2Institute of Epidemiology and Biostatistics, Dpt for Biostatistics, Skopje, Macedonia, Fyrom, 3Institute of Occupational Health, WHO Collaborating Center, Allergy Center, Skopje, Macedonia, Fyrom

Objective: To evaluate prevalence and characteristics of exercise-induced respiratory symptoms (EIRS) and exercise-induced bronchoconstriction (EIB) in female cleaners. Methods: We performed a cross-sectional study including 43 female cleaners aged 26 to 57, with duration of exposure 5 to 24 years. In addition, 45 female office workers aged 27 to 58 were studied as a control. Evaluation of exposed and unexposed workers included completion of a questionnaire, skin prick tests to common aeroallergens, spirometry, and constant submaximal exercise challenge test (ECT) on cycle ergometer. Results: The prevalence of EIRS did not differ significantly between cleaners and office workers (44.2% vs. 37.8%, P40.05), and inability to get deep breath was the most frequent individual EIRS in both groups (23.2% and 17.8%, respectively). We found similar EIB prevalence in both cleaners and office workers (9.3% vs. 6.7%; P40.05). Bronchial reaction to exercise, expressed as a mean fall index FEV1, was significantly greater in ECT positive cleaners (23.4% vs. 16.1%, Po0.05). EIB was strongly linked to atopy and positive family history for asthma in both groups, whereas its relation to daily smoking just missed significance in cleaners (P 5 0.07). Bronchial reaction to exercise was significantly greater in smoking cleaners than in smoking office workers (9.4% vs. 4.5%; Po0.05). EIRS were weakly associated with EIB, with a large proportion of false positive and a low proportion of false negative results in both examined groups. Conclusions: Our data demonstrated similar prevalence of EIRS and EIB in both cleaners and office workers and greater EIB severity in cleaners. EIRS were not reliable in identifying EIB in both examined groups.



322 Occupational allergy in strawberry greenhouse workers Patiwael, J1; Vullings, L2; de Jong, N1; van Toorenenbergen, A3; Gerth van Wijk, R1; de Groot, H1 1 Erasmus MC, Department of Allergology, Rotterdam, the Netherlands, 2Agricultural Occupational Health and Safety Advisory Services STIGAS, De Meern, the Netherlands, 3Erasmus MC, Department of Clinical Chemistry, Rotterdam, the Netherlands

Background: Some industries are particularly associated with a high prevalence of occupational disease. Glasshouse workers are one of the occupational groups at risk, because exposure to a variety of sensitisers and irritants, like pollen, other plant allergens, moulds and mites is unavoidable. Pollen from flowers and bell pepper plants cultivated in greenhouses for example, are known causes of occupational allergy. In the last decade, the strawberry cultivation in the Netherlands shifted from open field


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horticulture towards greenhouses. Consequently, employees in this branch of crop growing are also highly exposed to (potential) allergenic agents. No occupational allergy in this branch of horticulture has however been described before. In the first part of the study, the presence of work related allergic symptoms in strawberry greenhouse employees was explored. Secondly, we investigated whether these work related complaints were due to an IgE mediated allergy, elicited by a source in strawberry greenhouses. Methods: First, a questionnaire survey concerning work related allergic symptoms among strawberry workers in the Netherlands was carried out. In the second part of the study, three workers with work related symptoms were investigated more in detail. Skin tests, serum specific IgE tests with home made extracts of strawberry pollen and other possible allergenic agents of the


strawberry glasshouse environment were executed. Furthermore, immunoblots and nasal provocations with strawberry pollen extract were carried out. Results: Twenty-nine of seventy-five questionnaire respondents (38,7%) reported work related symptoms. Sensitisation to strawberry pollen was confirmed by skin test, serum specific IgE and immunoblots in all three employees with work related complaints. Nasal provocation validated clinically relevant allergy to these pollen in two of three subjects. Conclusion: Allergic symptoms attributable to the workplace are present among a substantial proportion of strawberry greenhouse employees. A new IgE mediated occupational allergy to strawberry pollen has been established.

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Poster Discussion Session 11 – Peanut, Wheat and Other Food Allergies in Children

Poster Discussion Session 11 Peanut, Wheat and Other Food Allergies in Children 323 Specific IgE to recombinant peanut allergens: diagnosis and prognostic value Codreanu, F; Jacquenet, S; Collignon, O; Morisset, M; Astier, C; Renaudin, J; Moneret-Vautrin, D; Kanny, G EA 3999:Allergic diseases:diagnosis and therapeutics Central Hospital University Hospital, Internal Medicine,Clinical Immunology,Allergology, Nancy, France

Background: Peanut allergy (PA) is a severe persistent food allergy. In a preliminary study, we have showed the high diagnosis value of specific IgE (sIgE) to rAra h 2 and the fact that polysensitisation to majors peanut allergens could be predictive of severity. The aim of this study was first to establish on a largest series the sensibility and specificity of sIgE to rArah1, rArah2, rArah3 for diagnosis of PA and secondly to establish a prognostic score of severity of accidental exposure. Method: Ninty four patients allergic to peanut with positive double blind placebo controlled food challenge (DBPCFC) were recruited. ImmunoCAP (Phadia, Swedish) to peanut (f13), rArah1, rArah2, rArah3, rArah8 were performed at the same time. Clinical history and skin prick-tests (SPT) to nuts, legumes were also noted. The severity of PA was scored using the scale previously described. 40 allergic patients to birch and grass pollens without PA and 50 non atopic patients were included as control. Results: In patients with PA, 100% of ImmunoCAP were positive to peanut (f13), 99% to rArah2 (93/94), 79% to rArah1, 66% to rArah3 and 48% to rArah8. In allergic patients to pollens, 55% were positive to peanut (22/40), 82.5% to rArah8 and only 1 patient to rArah2 (0.11 kU/L). None sIgE were detected in 50 non atopic patients. Severity of PA was not statistically different in asthmatic and non asthmatic patients. Severity was not different between monosensitised patients to peanut and polysensitised to other nuts and/or legumes. There was no correlation between the severity of accidental ingestion and severity of DBPCFC. The severity score of the first accident and the DBPCFC were not correlated to polysensitisation to recombinant allergens. Results were similar if we combined threshold dose and severity of DBPCFC. However, considering all SPT to nuts and legumes, total IgE and spIgE to recombinant allergens, we can isolate severity score of the first allergic accident. 134

Conclusion: spIgE to rArah2 improve diagnosis of PA with an excellent sensibility and specificity. Using for routine diagnosis will lead to improve specificity to 44% for ImmunoCAP to peanut to 97% for rArah2, with a sensibility of 99%.

324 Peanut allergy and allergic airway inflammation Hughes, J1; Brown, T2; Edgar, D3; Shields, M1 Royal Belfast Hospital for Sick Children, Department of Child Health, Belfast, United Kingdom, 2Ulster Hospital, Pediatrics, Belfast, United Kingdom, 3Royal Hospital, Immunology, Belfast, United Kingdom

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Background: Asthma is a major risk factor of anaphylactic deaths in children with peanut allergy. Peanut allergy is a lifelong condition but some children outgrow their coexistent asthma. It is currently not known whether children who have outgrown their asthma symptoms have on-going eosinophilic airways inflammation and should be considered for continuation of their inhaled corticosteroids. Exhaled nitric oxide (420 ppb) is recognised as a non-invasive marker of eosinophillic airways inflammation. Aims: The aim of our project was to examine the levels of exhaled nitric oxide in peanut allergic children. In particular we wished to determine if those with a history of asthma outgrown or those with no asthma have elevated exhaled nitric oxide levels. Methods: Children with peanut allergy were recruited at the Ulster Hospital and Royal Belfast Hospital for Sick Children, Northern Ireland. Exclusion criteria included a recent exacerbation of asthma. Exhaled nitric oxide levels (eNO) were measured using the NIOX Mino in all children. A value of ‘20’ ppb indicated an elevated eNO. Results: Ninty four children were enrolled over a 9 month period, age range 4 to 15 years (median 10 years), 62% male. 30 (32%) had no history of asthma, 7 (7%) had outgrown asthma, 20 (21%) had at least 1 wheezing episode within the last year but were not taking any regular asthma medication and 37 (40%) had treated asthma. All those with outgrown asthma had elevated levels of eNO, and 80% of children with wheeze within the last year but no regular asthma medication had elevated eNO levels. Outgrown asthma and wheezing episodes within the last year had higher levels of eNO Journal Compilation

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than no asthma or current treated asthma (P o0.05). Conclusions: Exhaled nitric oxide levels were elevated in children with a history of asthma outgrown and those with current ‘untreated’ asthma. This would suggest ongoing allergic airways inflammation. Our study gives a rationale for checking eNO in children with peanut allergy. Consideration should be then be given to starting inhaled corticosteroid therapy in peanut allergic children with elevated exhaled nitric oxide levels.

325 Serological and clinical characteristics of children with peanut sensitisation in an Asian community Chiang, W1; Pons, L2; Kidon, M1; Liew, W1; Goh, A1; Burks, W2 1 Kerdang Kerbau Women’s and Children’s Hospital, Pediatric Allergy, Immunology and Rheumatology, Singapore, 2Duke Children’s Hospital, Pediatric Allergy and Immunology, United States

Background: The incidence of food allergy in general and peanut allergy in particular is on the rise. In previously published data, 27% of children presenting to our allergy clinic with symptomatic food hypersensitivity, had a positive skin prick test for peanuts. Objective: We aimed to review the clinical presentation of peanut sensitivity in Asian food allergic children and the correlation with the specific IgE response to the major peanut allergens. Methods: All children presenting to the Singapore Children’s hospital allergy clinic, from March 2003 to June 2006, with peanut sensitisation documented on skin prick testing were eligible for inclusion. A voluntary detailed questionnaire was sent out to parents and responders were invited to participate in the study. Thirty-one patients of 62 patients were recruited and serum was obtained for laboratory evaluation of IgE specific response to native Ara 1, native Ara 2 and recombinant Ara 3 with an ELISA assay. Results: Thirty-one patients participated in this study. Of these 18 patients had previously documented reactions to peanuts, while 13 had a positive SPT with no previous clinical reaction. Eighty-three per cent of our patients with peanut sensitisation on skin prick testing were found to have specific IgE directed against one of the tested major peanut allergens. The



frequency of the major allergenic peanut protein in this population ranks Ara h 2 in 87.1%, Ara h 1 in 87.1% and recombinant Ara h 3 in 54.8% of the peanut sensitised patients. One way ANOVA of the mean peanut wheal size was performed against the subsets of patients who have had previous reactions to peanuts or who actively avoid (reactors/avoiders) peanuts as compared to children who are now tolerant of the peanut protein. The mean wheal size is 9.25 and 4.5 mm respectively (Po0.032). By logistic regression, the reactors/avoiders were more likely to recognize the presence of Ara h 1 and Ara h 2 (Po0.042, OR 12.5) than the tolerant patients. Conclusions: Asian children with peanut sensitisation respond to the same major allergenic proteins as do their western counterparts. The low published incidence of peanut allergy in Asia may represent a ‘lag time’ in presentation. As the Western world has seen an epidemic of peanut allergy in the last decade, we may likewise see a rise in food allergies in general and peanut allergy in particular in the East. Component resolved diagnosis for tailored immunotherapy in the future maybe considered.

326 T cell responses to major peanut allergens in children with and without peanut allergy Flinterman, A1; den Hartog Jager, S1; Hoekstra, M2; Bruijnzeel-Koomen, C1; Pasmans, S1; van Hoffen, E1 1 UMC Utrecht, Dermatology/Allergology, Utrecht, the Netherlands, 2UMC Utrecht, Pediatrics, Utrecht, the Netherlands

Background: Peanut-specific T cell responses involved in patients with peanut allergy are poorly understood. Objective: To investigate T cell responses towards major peanut allergens in peanutallergic subjects compared to non-allergic controls. Methods: Nineteen peanut-allergic children (PA), 7 non-allergic peanut-sensitised children (PS) and 11 non-atopic adults (NA) were included. The primary response to crude peanut extract (CPE) was measured by direct stimulation of PBMCs with CPE. In addition, CPE-specific short-term T cell lines were generated and subsequently stimulated with CPE and purified Ara h1, Ara h2, Ara h3 and Ara h6. Proliferation and production of IL-13, IFN-g, IL-10, and TNF-a were analyzed. Results: Primary proliferation of PBMCs to CPE was comparable in all groups. The primary cytokine response to CPE was comparable between PA and PS, with increased production of IL-13, IFN-g and TNF-a compared to NA. Production of IL-10 was not observed. In short-term T cell r 2008 The Authors Journal Compilation

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lines, the proliferative response to CPE and major peanut allergens was stronger in PA than in PS and NA. The response was highest after Ara h3 stimulation, and lowest after Ara h2 stimulation. Only PA had a cytokine response to major peanut allergens, characterized by IL-13 production. Conclusion: T cell responses to CPE in PA and PS children were characterized by both Th1 and Th2 cytokines. Only PA children showed enhanced Th2-type responses to the major peanut allergens.

327 Percutaneous sensitisation to food allergens in a child with severe atopic dermatitis Grilo, M; Afanas, N; Soares, H; Lopes, A; Guerra, P; Bonito Vitor, A Saõ Joaõ Hospital, Pediatric Immunoallergology Unit, Porto, Portugal

Background: Atopic dermatitis is a common allergic disease affecting young children with sensitisation to several food and inhalator allergens. Recently, has been referred the importance of percutaneous sensitisation to food allergens in the aetiology of this disease mediated by Langherhans cells like antigen presenting cells. Methods: We present a young child with severe atopic dermatitis that has multiple sensitisations to food allergens probably due to percutaneous contact. Results: A nine months old boy, asymptomatic until age of two months old, when he started eritematous descamative itchy skin lesion in face, hair, thorax, legs and arms. He was exclusively breastfeeding until four months old. At this age he started glutenfree lacteal soft food and artificial formula milk with worsening skin lesions and weight lost. At seven months he changed to extensively hydrolysed formula milk. Several emollient cosmetics containing peanuts and oat extracts were used since two months old due to severe and worsening atopic dermatitis. Laboratory investigation revealed high elevation of total IgE (1089 kU/L) and of specific IgE to peanuts (89.1 kU/L) wheat (4100 kU/L) alfa lactalbumin (20.3 kU/L) and oats (16.2 kU/L). He started eviction of the referred cosmetics and eviction dietary with slow but good clinical improvement. Conclusion: This patient had an early contact with oats and peanuts contained ointments that may induce a sensitisation even before he ingested this particular food. The authors highlighted the risk of the early application of peanuts and oats allergens containing cosmetics in severe atopic dermatitis in infants.


328 A supervised learning approach to identify potentially useful components in wheat allergy diagnosis Soeria-Atmadja, D1; Eriksson, C2; Turjanmaa, K3; ¨ nell, A2; Holmquist, I2; Dahlstro¨m, J2; Hammerling, U1; O Poorafshar, M2; Gustafsson, M4 1 National Food Administration, Division of Toxicology, Uppsala, Sweden, 2Phadia AB, Research and Development, Uppsala, Sweden, 3Tampere University Hospital, Allergy Unit, Dept. of Dermatology, Tampere, Finland, 4Uppsala University, Department of Medical Sciences, Uppsala, Sweden

Background: In the clinic it is often difficult to tell, without challenge, whether IgE sensitisation to wheat reflects a food allergy or not. The aim of this study was to evaluate the applicability of a range of components and protein fractions with a potential of being useful for the clinical diagnosis of wheat allergy. Subjects: Serum samples were collected from 65 children, with ages ranging from 3 months to 7 years, all challenged with wheat. Provocation separated test subjects into two clinical groups: A) children with acute symptoms (n 5 26) and B) children either without symptoms or those of delayed type (n 5 39). Methods: Gliadins and water soluble proteins were purified according to standard procedures. The fractionated proteins were immobilized in triplicates onto a capillary flow membrane. In total, specific IgE abs to 30 proteins/fractions were recorded for each patient using an anti-IgE ab conjugated to a fluorescent marker. To identify a potentially discriminative set of proteins/fractions, supervised multivariate data analysis was performed on the array data according to the following protocol:1. Randomised division of subjects in a training set for model building and a test set2. Supervised identification of the protein/fraction combination that best discriminates between groups in the training set3. Design of a supervised classifier with training set using best protein combination4. Evaluation of predictive performance of classifier using the test set5. Evaluation of robustness by repeating step 1–4 50 times with varying selection of patients in training/test sets. Results: Frequencies of proteins occurring in combinations that best discriminated between groups A and B over 50 iterations were recorded. Eleven of the components/ fractions were never selected in this process and 10 had a low frequency range (2–10%). Those of highest occurrence frequencies were timothy allergen Phl p 4, a marker for cross-reactive carbohydrate determinant (CCD) and wheat a-Amylase inhibitor 2. Average sensitivity and specificity over 50 iterations was 64% and 76%, respectively. Conclusion: Clearly, analysis of multivariate data generated from protein micro arrays 135


needs specialized computational methods. The presented methodology seems useful for discarding components with limited clinical importance. More test subjects are, however, needed to identify single or combinations of components for a robust prediction of wheat allergy.

329 Usefulness of specific IgE antibodies to x5 Gliadin in the diagnosis and follow up of Japanese children with wheat allergy Shibata, R1; Nishima, S1; Petersson, C2; Tanaka, A3; Borres, M2; Morita, E4 1 Fukuoka National Hospital, Department of Pediatrics, Fukuoka, Japan, 2Phadia AB, Medical Department, Uppsala, Sweden, 3Phadia K.K., Scientific Affairs, Tokyo, Japan, 4Shimane University School of Medicine, Department of Dermatology, Shimane, Japan

Background: Wheat is a staple food in the western world and wheat allergy is a common disease in childhood. It is known that there is a weak correlation between serum levels of specific IgE antibodies to wheat and the outcome of oral wheat food challenges. The gliadins have been implicated in the IgE-mediated allergy to ingested wheat and O 5-gliadin represents a major role in wheat-dependent anaphylaxis. This study investigates if specific IgE antibodies to o-5-gliadin is useful in the diagnosis and follow up in wheat allergic children. Patients and methods: Serum samples from 88 children sensitised to wheat were collected. Sixty-seven children were classified as wheat allergic and 21 as non-wheat allergic determined by case history and/or wheat challenge. Out of these, 29 children with wheat allergy were investigated also at a second time for determination of wheat tolerance or not. Serum specific IgE ab to wheat and o-5-gliadin were related to a physician’s diagnosis of wheat allergy and challenge symptoms. Results: Elevated levels of sIgE- o-5-gliadin-ab were found in 47% of the non-wheat allergic children and in 88% of the wheat allergic children and the levels o-5-gliadin specific IgE antibodies differed significantly between the two groups; (o0.35–100.0 kU/L) median 1.59 kU/L for wheat allergic children and (o0.35–1.8 kU/L) median 0.36 kU/L for non-wheat allergic children (Po0.001). No significant difference were observed regarding wheat specific-IgE antibodies (o0.35–100.0 kU/L) median 23.1 kU/ L for wheat allergic children and (o0.35– 81.5 kU/L) median 10. kU/L for non-wheat allergic children. In the subgroup of children with a follow up visit, levels of sIgE-o-5gliadin-ab was at first and second visit for the group with persistent wheat allergy median 2.11 and 2.51 kU/L respectively. Corresponding figures for the group with

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outgrown allergy were 1,82 and 0,34 kU/L respectively (Po0.05). Conclusion: The presence of sIgE-o-5-gliadin-ab is related to reaction level to wheat challenge outcome in wheat sensitised children.

slower achievement of tolerance and increased risk of asthma.

330 The prognosis of wheat allergy in children

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Kotaniemi-Syrjanen, A; Palosuo, K; Jartti, T; Kuitunen, M; Pelkonen, A; Ma¨kela¨, M Helsinki University Central Hospital, Department of Allergology, Helsinki, Finland

Background: Wheat is a common food causing allergy in infants and small children, yet studies on the natural history of wheat allergy are lacking. We aimed to elucidate the natural history of wheat allergy and to define risk factors for persistent wheat allergy, and to evaluate the development of respiratory allergy in wheat allergic children. Methods: The development and subsequent loss of wheat allergy, clinical findings, skin prick test (SPT) reactivity, and the development of allergic rhinoconjunctivitis and asthma were charted retrospectively in 28 children with wheat allergy proven by the open oral challenge at the median age of 21 months. Results: All 21 children with SPT-positive wheat allergy experienced skin symptoms during the diagnostic wheat challenge, while appearance of gastrointestinal symptoms only (n 5 4) was associated with SPT-negative wheat allergy (P 5 0.002). Wheat was tolerated by 59% (n 5 16/27) of the children by age 4, 69% (n 5 18/26) by age 6, 76% (n 5 19/25) by age 8, 84% (n 5 21/25) by age 10, and 96% (n 5 23/24) by age 16. Sensitisation to gliadin with a SPT wheal diameter of at least 4 mm at the time of the diagnostic challenge was associated with a slower course of recovery: wheat was tolerated at the median age of 4.6 years in those with a SPT wheal diameter of at least 4 mm (n 5 7), and at the median age of 3.7 years in those with a wheal diameter of less than 4 mm to gliadin (n 5 17) (P 5 0.046). A SPT wheal diameter of at least 3 mm to gliadin ever (n 5 14/28) was associated with the later development of asthma (n 5 12/28) (predictive value (PV) 64%; odds ratio (OR) 6.6; 95% confidence interval (CI) 1.2–35.4; P 5 0.022). SPT reactivity to wheat (n 5 21/28) was associated with later SPT reactivity to birch pollen (n 5 16/26) (PV 80%; P 5 0.001), and the development of allergic rhinoconjunctivitis (n 5 22/28) (PV 90%; OR 12.7; 95% CI 1.6–102.3; P 5 0.021). Conclusion: Practically all children with wheat allergy tolerate wheat by teen age. Sensitisation to gliadin is associated with

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331 The predictive value of specific immunoglobulin E levels in corn allergy Estrada-Reyes, E1; Toledo-Bahena, M1; Nava-Ocampo, A2 Hospital Angeles Metropolitano, Allergy Dept, Mexico City, Mexico, 2Pharma Reasons, Statistical Department, Toronto, Canada

Background: Cereals are the major components of the human diet throughout the world. The literature provides few cases of food induced allergic reactions to maize in general population. Due to high consumption patterns of corn in Mexico we have not epidemiology studies, neither cut off points for corn allergy in children. Aim: To evaluate the diagnostic capacity of specific IgE in predicting the putcome of oral food challenge and to determine decision point for the specific IgE levels to corn. Patients: Our prospective study included 41 patients consecutively with food allergy suspicious, considering an allergic reaction with the following symptoms (urticaria, atopic dermatitis, vomiting, diarrhea, abdominal pain, rhinitis, wheezing). All the children underwent specific IgE and food oral challenges. Patients were between 9 months and 11.5 years (median 2.34), 25 were boys (60.9%) and 16 were girls (39.1%). Specific IgE. The blood test was before the oral challenge. Serum samples from all patients were analyzed for specific IgE. We investigated sera concentration titres to corn. Determined by Pharmacia CAP-system FEIA method. Food Oral Challenges: Percentage Sensitivity Specificity PPV% NPV%

96 77 80 66

Patients were investigated according to the EAACI guidelines, with a 6 h interval between challenges. We started with a dosage 0.5 g the dose was increased by a factor 2 every 20 min until the entire meal was eaten or until symptoms appeared. The maximum dosage 50 g. Data analysis: Software: StatsDirect v. 2.6.1 (Cheshire, United Kingdom) Two-by-two tables were used to calculated the sensitivity, specificity, positive predictive value, negative predictive value. We plotted (ROC) curves for specific IgE. Area under the curve was calculated to quantify the accuracy of the single test and to compare the diagnostic value. Results: The diagnostic challenge test was carried out in 29 children (70%). 8 (19.5%)



presented urticaria, 16 (39%) atopic dermatitis increasing, 29 (70%) wheezing, 10 (24%) diarrhea. The analysis of the ROC curve makes it possible to determine the optimal decision point. This point was 7.5 Ku/L finding an area under the curve 0.789, a positive predictive value 80% and negative predictive value 66% with sensitivity 96% and specificity 77%. Conclusion: Demostration of food specific antibodies support the existence of allergy to corn, however a definitive diagnosis can only established after the challenge test, but this cut off points will be useful in order to document diagnosis and tolerance.

332 Food allergy to beans in a Brazilian child Jacob, C1; Caixeta, L1; Yonamine, G1; Pastorino, A1; Fomin, A1; Moschione-Castro, A2 1 Brazil, 2Faculty of Medicine University of Saõ Paulo, Department of Pediatrics, Brazil

Background: Among Spanish children, sensitivity to legumes is the fifth most prevalent food allergy. Allergy to beans in Brazilian children has not yet been reported, although it is frequently consumed by this population. Case report: An 8-year-old Brazilian boy was referred to our institution with a history of four episodes of angioedema and hyperemia of the lips, without systemic reaction, after contact with boiled bean, since 6 months of age. The mother referred reactions also with lentil and pea (coughing, itching of the throat and vomiting) and soy (vomiting). He had never ingested chickpea or other types of bean. The skin prick test with industrialized extracts showed the following wheal diameter: bean 3 mm, lentil 1 mm, soy 1 mm and peanut 0 mm.The prick to prick test with boiled legumes showed: bean 16 mm with pseudopods, pea 6 mm, chickpea 6 mm, lentil 6 mm and white bean 4 mm. It was performed open challenges with boiled chickpea, pea, lentil, white bean and bean. The patient presented angioedema and hyperemia of the lips, after 5 min of bean ingestion and angioedema on the right eye and discrete angioedema on the lips after 15 min of white bean ingestion. Conclusions: The authors point out this uncommon IgE mediated reaction to beans and the cross-reactivity to other legumes, detected by prick to prick using boiled foods from the same botanical family and to white bean in the open challenge. Despite the exclusion of the allergen from the diet of the child, it is important to notice the persistence of the allergy. This is the first report of allergy to beans in our country and the reactions with the boiled beans may suggest this processing can change the beans allergenicity. r 2008 The Authors Journal Compilation

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333 Identification of soybean cross-reactive allergens by analysis of IgE reactivity in sera from soy sensitised children from the BAMSE cohort Sjolander, S1; Bernhardsson, F1; Perborn, H1; Kull, I2; Ostblom, E2; Hallner, E2; van Hage, M3; Wickman, M2; Ahlstedt, S4; Poorafshar, M1 1 Phadia AB, R&D, Uppsala, Sweden, 2Karolinska Institute, Dep of Occup and Env Healt, Stockholm, Sweden, 3Karolinska Institute, Clin Immunol and Allergy Unit, Dep of Medicine, Stockholm, Sweden, 4Phadia AB and Karolinska Institute, Center for Allergy Res, Uppsala and Stockholm, Sweden

Background: The predictive value of soyspecific IgE antibody (ab) levels for soy allergy has been difficult to assess. One reason for this may be the close botanical and proteome relationship between soy, other legumes and pollens. Identification of the cross-reactive allergens may clarify the reason for the relatively low clinical relevance of the presence of soy IgE ab and even suggest improvement of test algorithms. Objective: To identify cross-reactive allergenic components by analysis of IgE ab responses in low level soy sensitised children without symptoms. Materials & methods: All children (n 5 40) not sensitised to soy (o0.35 kU/L) and without reported soy-related symptoms at the age of 4, that had developed soy sensitisation at the age of 8 (geometric mean (gm) level 0.93 kU/L), were selected from a birth cohort from northern Stockholm, Sweden (BAMSE, n 5 2 600). Symptoms from peanut were reported for 12 children at 4 and/or 8 years of age. Sera from 19 children without reported symptoms from soy/peanut and not sensitised to soy (o 0.35 kU/L) at either 4 or 8 years of age, was used as controls. The level of IgE ab to soy, peanut, birch and timothy crude extracts as well as to major allergenic components thereof was analysed in sera collected at 4 and 8 years of age, using ImmunoCAPs for extracts and a protein microarray for components. Results: At the age of 8, a majority of the children in the soy sensitised group also had high IgE ab responses,450 kU/L, to timothy (n 5 7), peanut (n 5 13) and birch (n 5 17). In the control group only one child was sensitised (timothy). The microarray analysis revealed that in 4 sera, sensitisation to Ara h 1 and 3 was correlated with the presence of IgE ab to the homologous components in soy, glycinin and b-conglycinin, whereas sensitisation to Bet v 1 in 13 sera, was correlated with sensitisation to the homologous soy protein Gly m 4. The upcoming sensitisation to soy at 8 years of age could be detected at minute levels already at 4 years of age in the soy sensitised group (gm level 0.027 kU/L), and was


significantly higher than in the control group (gm level 21 10 6 kU/L). Conclusion: IgE cross-reactivity to peanut and birch are of importance for low level soy sensitisation. Component analysis showed that the sensitisation was represented by cross-reactions from for example Ara h 1 and 3 and Bet v 1.

334 Sunflower seed allergy: a rare food allergy in children Marta, G; Bonito Vitor, A Saõ Joaõ Hospital, Pediatric Immunoallergology Unit, Porto, Portugal

Background: Sunflower belongs to the family of Compositae. The whole seeds are used in breads and for garnishing bakery products or as livestock, bird, and poultry feed. Edible sunflower seed oils are ingredients of cooking, salad oils and margarine. In south Europe countries like Portugal and Spain dry salty sunflower seeds are also consumed as appetizer. Although allergy to sunflower seed and oil is a relatively rare occurrence, several cases of sunflower seed allergy have been observed. Sunflower seed can cause severe anaphylactic reactions in some susceptible individuals. Methods: We describe one case of sunflower allergy, a rare condition in children. Results: A five year old boy, with trisomy 21, no other allergies described, developed oral irritation including tongue and lip swelling after consuming bread with sunflower seeds. He had a previous history of sunflower seed bread sporadic ingestion. Skin prick was positive to commercial sunflower seed extracts. IgM, IgG, IgA and total IgE levels were normal. Conclusion: We described a particular clinical case of singular sunflower food allergy in a young child. This allergy is uncommon and may be due to sensitisation not only to the seed but also retained protein in the oil. This is particularly relevant as sunflower oil is a common ingredient in foods.

335 Allergen profiling of five different kiwi fruit cultivars Oberhuber, C1; Bublin, M2; Bulley, S3; Fritsche, P4; Hofstetter, G2; Ballmer-Weber, B4; Testolin, R5; Hoffmann-Sommergruber, K2 1 Biomay, Vienna, Austria, 2Medical University of Vienna, Department of Pathophysiology, Vienna, Austria, 3 HortResearch (Mt Albert), Flavour Biotechnology Group, Auckland, New Zealand, 4University Hospital Zurich, Allergy Unit, Department of Dermatology, Zurich, Switzerland, 5University of Udine, Dipartimento di Scienze Agrarie e Ambientali, Udine, Italy

Background: Since its introduction to the European market in the 1980s, green kiwi

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fruits (Actinidia deliciosa) cv. Hayward became more and more popular, followed by the yellow fleshed cultivar A. chinensis cv. Hort 16A, Zespri Goldt, in 1999. In the last years many studies demonstrated the different allergenic potential of green and gold kiwi fruits. Thus, allergenic risk assessment of well known kiwi cultivars and those, not yet on the market was performed providing essential information for the allergic consumer. Methods: Kiwi fruit protein extracts from A. deliciosa cv. Hayward and cv. Summer 3373, A. chinensis cv. Jintao, and A. eriantha cv. 114 and cv. 96 were separated by SDSPAGE followed by Coomassie staining. To identify relevant IgE binding components in the respective kiwi cultivar, IgE immunoblotting with individual sera from 16 kiwi fruit allergic patients was performed. Results: SDS-PAGE analysis of the five kiwi fruit extracts revealed distinct protein patterns with abundant, clearly resolved bands in the range from 10 to 30 kD and very weak bands above 30 kD. In the range of 20–30 kD clear quantitative and qualitative differences of protein patterns were visible in all five kiwi fruit extracts. Actinidin, the major kiwi allergen was the most prominent band in cv. Hayward, but almost invisible in cv. Summer (both A. deliciosa). IgE immunoblotting with 16 kiwi fruit allergic patients’ sera showed several IgE binding components between 11 and 72 kDa. Although the overall IgE binding capacity of the five kiwi fruit extracts tested was weak, several IgE binding bands were detected. In cv Summer and cv Hayward (A. deliciosa) up to five IgE binding components were determined. In cv. 96 and cv. 114 (A. eriantha) four allergens in the range of 17–55 kDa were detected. Whereas only three IgE binding proteins from cv. Jintao (A. chinensis) kiwi fruit extract were verified. Conclusion: In the recent past increasing evidence of IgE mediated allergic reactions to green and gold kiwi fruits was reported. We confirmed the presence of IgE binding components in commercial available kiwi fruit cultivars, as well as in cultivars not yet on the market. However, obvious differences in the allergenic profiles of the individual kiwi cultivars were detected. Pre-market testing could identify hypoallergenic cultivars with a benefit for kiwi fruit allergic consumers.

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336 Impact of peanut allergy on family life Dunbar, H; Luyt, D University Hospitals of Leicester, Children’s Allergy Clinic, Leicester, United Kingdom

Background: Peanut allergy is an important pubic health problem as its increase in prevalence means that near 2% of the childhood population are at risk of allergic or anaphylactic reactions. These risks or reactions to trace amounts of peanuts impose severe lifestyle restrictions on affected children as they have to carefully avoid all nut contact and have epinephrine to hand should inadvertent exposure trigger a life threatening reaction. There is limited research into the psychological impact of these risks and restrictions. Objectives: This study aimed to explore the impact of a diagnosis of peanut allergy on family life; thereby assessing the experiences of the affected child, his or her siblings and their parents. Methods: A small-scale exploratory study was conducted within a qualitative framework, influenced by the constructivist paradigm of research inquiry. A strategy of purposeful sampling was adopted to select 3 families for more in-depth study using a collective case study design. Inclusion criteria were that the affected child was a minimum of 7 years old, and had at least one parent and an older sibling willing to take part in the study. Data was collected separately from the parents and affected child using semi-structured interviews. A focus group was run for the siblings from the 3 families. Analysis of the data was by a process of unitising and categorising to identify patterns and themes for discussion. Results: Three themes were identified.1. The emotional journey that the families travel from diagnosis, through coping with daily life, to taking ownership of the condition. Sub-issues were fear and parents losing control as children became independent.2. The need to maintain a safe environment with avoidance strategies and adaptations made to lifestyle. A sub-issue was anxiety about cross-contamination because of poor labelling.3. The cultivation of an environment of awareness around the condition. There was a real feeling of isolation and lack of support. Conclusion: The study found that families have to make adaptations to their lifestyle but simultaneously seek to achieve as normal a life as possible for their children.

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For children diagnosed at an early age living with the condition was normal for them. Parental responses differed according to coping strategies. Families expressed that the development of teaching packages, review of labelling practices and manufacturing processes by the food industry would help them adapt more easily.

337 The implicit association test in food allergy Dunn-Galvin, D1; DunnGalvin, A2; O’B. Hourihane, J2 University College Cork, Applied Psychology Department, Cork, Ireland, 2University College Cork, Pediatrics and Child Health, Cork, Ireland

1

Background: The impact of a restricted environment on children’s emotional and psychosocial development is an important area of study in food allergy. The Implicit Association Test (IAT) measures associative links between environmental triggers and psychological states. Objective: To validate a novel measure to assess implicit attitudes in food allergy. Method: This study examined implicit and explicit attitudes towards environment in parents of food allergic children (N 5 60) and matched controls (N 5 30). The IAT was used to measure the differential association of a target category (environmental stimuli) with an attribute dimension (anxious vs. relaxed). A study specific questionnaire (SSQ) was used to measure explicit attitudes. Results: A larger effect size for the IAT (d1.02; M0.52, SD0.51, Po0.05) shows that higher levels of perceived anxiety were measured on the IAT than the SSQ (d0.31; M0.40, SD1.27, Po0.05). Higher perceived anxiety was found for mothers versus fathers and parents of teens versus parents of children. An interaction effect was found, with mothers of teens exhibiting highest anxiety levels (F(2,60) 5 8.401, Po0.05, partial eta squared 5 0.157). Matched controls showed lower levels of anxiety across domains. Conclusions: The novel use of an implicit measure will clarify and provide more depth to previous research findings in this area. Further, the implicit association test is highly mobile, economical and quick to use, providing an immediate result without need for scoring keys, analytical charts or statistical calculations.


Poster Discussion Session 12 – Mechanisms of Allergic Disease

Poster Discussion Session 12 Mechanisms of Allergic Disease 338 Effect of Parietaria judaica pollen on human lung microvascular endothelial cells Taverna, S; Flugy, A; Colomba, P; Barranca, M; De Leo, G; Alessandro, R Universita` di Palermo, Biopatologia e Metodologie Biomediche, Palermo, Italy

Parietaria judaica belongs to the Urticaceae family, is commonly found in urban and rural areas and its pollen is one of the main causes of allergy in the Mediterranean area. The small dimensions of Parietaria judaica pollens (13–17 mm) allow their easy access in the high respiratory tract where they contact the epithelial cells. Pollen extracts contain allergens as well as enzymes with proteolytic activities which can alter the epithelial integrity contributing to the sensitisation and pathogenesis of allergy. Following the disruption of the epithelial barrier, a variety of proteins and/or bioactive compounds contained in the pollen grains may transverse the epithelial sheet and come in contact with stroma cells including endothelial cells. The present study is designed to assess if Parietaria judaica pollens contains bioactive compounds able to elicit a functional response in endothelial cells. We have demonstrated by western blot, immunofluoreescence and Real Time PCR analyses that addition of aqueous pollen extract to human lung microvascular endothelial cells (HMVEC-L) induces a modification of cell morphology accompanied by cytoskeletal rearrangements, an alteration of monolayer integrity with loss and delocalization of adhesion molecules belonging to EC adherens junctions, an increase of E-selectin and ICAM-1 as well as an increase of production of Il-8, an important inflammatory mediator. To investigate on functional role of the observed increase of E-Selectin and ICAM-1 expression in HMVEC-L treated with pollen extract, we tested the ability of PMNs to adhere to the endothelial monolayer. We shows a dose-dependent increase of adhesion of granulocytes to cells after a 6 h treatment with 250 mg/mL of pollen extract. Transendothelial permeability measurement in HMVEC-L has indicated that these cells may form more intact tight junction than other endothelial cells types. Consistently, we observed that HMVEC-L confluent control monolayer did not permit the passage of FITC-dextran. However, after 6 h of treatment with pollen extract, a significant increase in cell permeability was r 2008 The Authors Journal Compilation

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registered. These findings suggest that pollen may affect directly endothelial cells modulating critical EC function related to the allergy pathogenesis. We believe that our results could provide important indications to develop new therapeutic strategies for allergic patients.

339 Timothy grass pollen major allergen Phl p 1 activates respiratory epithelial cells by a non-protease mediated mechanism Ro¨schmann, K1; Ulmer2, A; Suck3, R; Ko¨nig, P4; Petersen, A5 1 Research Center Borstel, Molecular and Clinical Allergology & Cellular Immu, Borstel, Germany, 2 Research Center Borstel, Cellular Immunology, Borstel, Germany, 3Allergopharma, Joachim Ganzer KG, Reinbek, Germany, 4University of Lu¨beck, Institute for Anatomy, Lu¨beck, Germany, 5Research Center Borstel, Molecular and Clinical Allergology, Borstel, Germany

Background: Allergic diseases affect a large part of the population in industrialized countries, with about 30% occurence of type I allergies. Group 1 allergens (e.g. Phl p 1, the major allergen of Timothy grass pollen Phleum pratense) cause IgE-reactivity in about 95% of allergic subjects and exist in all grass species. The respiratory epithelium represents a first line of contact of the immune system with the environment including airborne allergens. It functions as physical barrier and serves as important immunological regulation system, which is also involved in allergic reactions like asthma. We study the interaction of Phl p 1 with human respiratory epithelium in detail to elucidate the contribution of epithelial cells to the initiation and development of allergic reactions. Methods: Cells of the alveolar line A549 cells were incubated either only with purified Phl p 1 or Der p 1, or pre-incubated with antibodies or inhibitors, supernatants were assayed for IL-6 and IL-8 release via ELISA, mRNA expression was determined via real time-PCR. Proteolytic activity was determined via MTT-test, zymogrammes and an ex vivo model of the murine trachea. Results: We determined that Phl p 1 activates respiratory epithelial cells measured by release of IL-8 and IL-6 from A549 cells. The activity of the allergen cannot be inhibited after pre-incubation with antibodies blocking the TLR2- and TLR4-signalling pathways, indicating that bacterial contaminations are not responsible for the activation of the cells. A complete deletion


of the biological activity of Phl p 1 after proteinase K digestion indicates that the allergen itself interacts with the epithelium. After pre-incubation with different inhibitors we were able to show the involvement of the signal transduction elements p38, ERK and NFkB in the activation of epithelial cells by the allergen. Furthermore, we were able to show that Phl p 1, in contrast to other major allergens like Der p 1 from the house dust mite, does not exert any proteolytic activity. Conclusion: Our results show that n Phl p 1 induces the release of IL-8 and IL-6 and the expression of TGF-b in alveolar and bronchial epithelial cells. Bacterial contaminants could be excluded to be responsible for this effect. In direct comparison with rDer p 1 we were able to show that nPhl p 1 does not exert any proteolytic activity. The underlying mechanism of activation of respiratory epithelium by Phl p 1 is still under invesitgation.

340 Impact of aqueous birch pollen extract on natural killer T cells in vitro and in vivo Bewersdorff, M1; Braun, A1; Jakob, T2; Behrendt, H1; Mempel, M1 1 Division of Environmental Dermatology and Allergy – Helmholtz Zentrum Mu¨nchen, ZAUM – Center for Allergy and Environment, TUM, Munich, Germany, 2University of Freiburg, Allergy Research Group, University Medical Center, Freiburg, Germany

Background: Natural Killer T cells (NKT cells) represent a subset of T cells, which are rapidly activated in innate as well as adaptive immunity. The invariant T cell receptor of NKT cells recognizes the MHCclass-I-like CD1d molecule on antigen presenting cells (APC) which presents lipids and glycolipids such as a-Galactosylceramide (a-GalCer) which induces the production of substantial amounts of inflammatory cytokines, e.g. IFN-g and IL-4 in NKT cells. It has been already shown, that aGalCer treatment can abrogate the symptoms of asthma in sensitised and challenged mice when applied 1 h before the first OVAaerosol challenge. Because aqueous birch pollen extract (Bet.-APE) has been shown to modulate the function of APC by bioactive lipid mediators we wondered whether these lipid mediators are able to influence the outcome of a-GalCer treatment in vivo. Methods: To this end, liver and spleen mononuclear cell (LMC/SMC) cultures

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and myeloid dendritic cell (mDC) -NKT-cell co-cultures were analyzed for production of various cytokines using a-GalCer as model antigen in the presence of varying concentration of Bet.-APE. In addition to cell culture experiments we established a murine model of pulmonary hypersensitivity in which the effects of Bet.-APE on a-GalCer mediated allergy protection were investigated. Results: In in vitro LMC and SMC cultures, IFN-g secretion was dose-dependently reduced by Bet.-APE. At the same time, Bet.APE did not significantly change the production of IL-4 and IL-13 resulting in a net propagation of TH2 cytokines. Similar results were observed when hybridoma NKT cells were stimulated with Bet.-APE and co-cultured with a-GalCer loaded mDC. In contrast, mDC pre-incubation with Bet.-APE did not influence significantly TH1/TH2 dichotomy but rather suppressed a-GalCer induced cytokine production by NKT cells. Our results suggest that NKT cell function is directly influenced by Bet.-APE and not mediated through modulated mDC function. a-GalCer treatment in vivo ameliorated airway inflammation, but was not altered by Bet.APE treatment. Interestingly, cytokine secretion of a-CD3 re-stimulated SMC cultures from a-GalCer-treated mice was reduced and finally abrogated in Bet.-APE treated mice. Conclusion: These findings point to a systemic role of Bet.-APE on a-GalCer-treated spleen NKT cell function.

341 A novel recombinant mannose-specific banana lectin is a potent modulator of the proliferation response in CD3+, CD4+, and CD8+ populations of human PBMCs Gavroivc-Jankulovic, M1; Brckalo, T2; Poulsen, K3; Bobic, S1; Lindner, B4; Petersen, A5 1 Faculty of Chemistry, Biochemistry, Belgrade, Serbia and Montenegro, 2Universitat Pompeu Fabra (DCEXS), Molecular Immunopathology Unit, Barcelona, Spain, 3 University of Aarhus, Institute of Medical Microbiology and Immunology, Aarhus, Denmark, 4Borstel Research Center, Division of Immunochemistry, Borstel, Germany, 5 Borstel Research Center, Division of Molecular and Clinical Allergology, Borstel, Germany

Introduction: Recognition of cell-surface carbohydrates by lectins has wide implications in various immunological processes. A mannose-specific lectin from banana fruit revealed immunomodulatory potential by a strong IgG4 antibody response and T cell proliferation in humans. As a protein resistant to proteolysis in the GIT it has been proposed as a potentially useful carrier for oral antihapten immunization. Due to a remarkable structural microheterogeneity of natural lectin, the aim of this work was to

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produce a recombinant counterepart and to explore its immunomodulatory potential on several cell populations of human PBMCs: monocytes, B, T, and NK cells. Methods: The gene of banana lectin was cloned, sequenced and a recombinant protein was produced in E. coli. The protein structure of rBanLec was characterised in detail and compared with nBanLec by peptide mapping by using a high-resolution ESI FT-MS. Biolocical activity of rBanLec was confirmed by inhibition of lectin binding to immobilized HRP with monosaccharides. For the cell proliferation assay, LPS and LPS-related molecules were removed from rBanLec preparation using the Polymixin B. Freshly isolated peripheral blood mononuclear cells from heparinized blood of healthy volunteers, after labeling with CFSE, were cultured in the presence of various concentrations of nBanLec, rBanLec, and Con A lectin. Seven days after the stimulation the cells were stained with phycoerythrin (PE)-labeled anti-CD3, antiCD4, anti-CD8, anti-CD19, and anti-CD56 monoclonal Ab and analyzed by fluorescence-activated cell sorted. Results: The obtained BanLec cDNA revealed a novel isoform of banana lectin. Natural and recombinant BanLec marked all analyzed cell populations however, proliferation was detected of only CD3 1, CD4 1, and CD8 1 cells among PBMCs. The optimal proliferation response was achieved with the concentration of 2 ı` g/mL of both lectins although rBanLec induced higher T cell proliferation response: 56.65 7 15.95% CD3 1 cells, 52.23 7 14.02% CD4 1 cells, and 53.74 7 10.86% CD8 1 cells, when compared to nBanLec: 31.19 7 6.02% of CD3 1 cells, 34.44 7 4.37% of CD4 1 cells, and 33.26 7 4.90% of CD8 1 cells. Conclusion: Certain plant lectins have been investigated as agents for specific targeting of molecules to a mucosal epithelium. As a biologicaly active lectin rBanLec could be explored as antigen delivery system circumventing the use of classical adjuvants for modulation of the mucosal immune response.

342 Angiotensin converting enzyme-gene polymorphisms in normal subjects, atopic individuals and patients with anaphylaxis to venom, foods & drugs Varney, V1; Warner, A1; Ghosh, A2; Nicholas, A2; Sumar, N3 1 St Helier Hospital, Immunology, Surrey, United Kingdom, 2St Helier, Immunology, Surrey, United Kingdom, 3St Helier, Immunology, Surrey, United Kingdom

Background: Circulating angiotensin-2 levels (A2) protect the circulation against

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sudden falls in blood pressure. A2 is generated by the enzymatic action of angiotensin converting enzyme (ACE) on angiotensin1. The ACE genes have 2 allellic forms, with the presence ‘I’ or absence ‘D’ of a 287 base pair intron of the gene. The deletion ‘DD’ genotype is associated with high A2 generation, and the insertion ‘I I’ with low levels. Aim: To examine whether these 2 inherited polymorphisms have a different profile in patients with past anaphylaxis compared to control groups. Method: A total of 286 subjects were analysed. 118 had previous anaphylaxis, 119 were healthy controls, 49 were atopics without any previous anaphylaxis. DNA extracted from EDTA blood was analysed for ACE gene polymorphisms using polymerase chain reactions, followed by gel electrophoresis to identify the genotypes. Results: See table below including P values from chi squared analysis. ACE

DD

ID

II

genotype

Healthy

Atopics

Controls

Anaphyl- Cutaaxis

neous and RS

Healthy

53

44

22

Controls

(45%) (37%) (18%)

P 5 0.142 P 5 0.009 P 5 0.053

N 5 119(%) Atopic

15

controls

(31%) (53%) (16%)

26

8

P 5 0.142

P 5 0.425 P 5 0.025

N 5 49(%) Anaphylaxis 30

58

30

P 5 0.009 P 5 0.425

N 5 118(%) (25%) (50%) (25%) Cutaneous

15

and

(56%) (44%)

12

0

P 5 0.025 P 5 0.053

Respiratory anaphylaxis Airway

13

angioede-

(14%) (52%) (34%)

46

30

P 5 0.001 P 5 0.024

P 5 0.001

ma&CVS collapse

2 subjects were deleted from sub-analysis due to treatment with ACE-inhibitors No statistical differences were found between ‘Atopics’ & HC, but significant differences in the genotype frequency were seen between subjects with anaphylaxis and HC. Dividing the anaphylaxis group into 2 subgroups, there was a significant difference between those with airway angio-oedema 7 cardiovascular collapse (AACVS) and HC, but not between HC & cutaneous & mild respiratory symptoms (CRS) only. The AACVS & CRS subgroups were significantly different; AACVS collapse showing increased II genotype. No statistical difference was found between ‘anaphylaxis and ‘atopics’, but ‘atopics’ were significantly different from the 2 AACVS & CRS subgroups. Conclusion: The results show a significant change in the genotype frequency between the HC and subjects who have suffered ‘anaphylaxis’, particularly those with AACVS. These changes show a reduction in DD genotype & increases in ID & II.



343 Satratoxin positive Stachybotrys chartarum activates inflammatory cytokine response in synergy with lipopolysaccharide Leino, M1; Kankkunen, P1; Rintahaka, J1; Wolff, H2; Alenius, H1; Matikainen, S1 1 Finnish Institute of Occupational Health, Health and Work Ability/Unit of Excellence for Imm, Helsinki, Finland, 2Finnish Institute of Occupational Health, Health and Work Ability, Helsinki, Finland

Background: Our previous studies show that a damp building mould, Stachybotrys chartarum, induces inflammation in mice airways. Macrophages and neutrophils were the main responding cell types found in the lung infiltrate. Fungal recognition by Tolllike receptors (TLRs) and C-type lectins (CLRs) in macrophages results in activation of antifungal defence and pro-inflammatory response. In this report, we have studied the effect of a satratoxin producing strain of Stachybotrys chartarum on pro-inflammatory cytokine production in human macrophages. Methods: Human macrophages were differentiated with GM-CSF from monocytes obtained from leukocyte-rich buffy coats of healthy blood donors. Macrophages were stimulated with roridin A, a satratoxin producing and a non-producing S. chartarum strain with and without LPS. RNA was extracted from the cells and cytokine mRNA expression was measured using real-time PCR. Cytokine secretion was measured with ELISA and caspase 1 and 3 activation was studied with Western blotting. Results: Our results show that a satratoxin producing S. chartarum strain activates the expression of IL-1b and IL-18 genes with cooperation with lipopolysaccharide (LPS, a TLR4 ligand). Moreover, the satratoxin producing strain activated caspases 1 which is needed for proteolytic processing of IL-1b and IL-18 and further secretion of the biologically active cytokines. Secretion of IL-1b and IL-18 was enhanced when macrophages were stimulated with S. chartarum and LPS simultaneously. In addition activation of caspase 3, an executioner caspase of apoptosis, was also seen. Caspase 1 and 3 activation was not seen with a non-satratoxin producing S. chartarum strain, suggesting that fungal toxins are essential for the activation of inflammatory responses. Roridin A, a mycotoxin related to satratoxin, activated caspase 1 and 3 in human macrohages. In addition, when roridin-A was costimulated with LPS an enhanced secretion of IL-1b and IL-18 was seen. Conclusion: Our results show that S. chartarum-produced mycotoxins are involved in activation of both inflammatory response as well as apoptosis in human macrophages. Pro-inflammatory response induced by myr 2008 The Authors Journal Compilation

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cotoxins was dependent on co-exposure of macrophages with LPS. Therefore, our results suggest that synergistic effect of mycotoxins and bacterial cell wall components may contribute to inflammatory lung disorders seen in moisture building exposed patients.

344 S. aureus and S. aureus-derived lipoteichoic acid attentuate immediatehypersensitivity reactions independent of TLR2 Mrabet-Dahbi, S; Metz, M; Maurer, M Charite-Universitaetsmedizin Berlin, Department of Dermatology and Allergy, Berlin, Germany

Current allergy treatment relies in large parts on blocking the effects of symptom-causing mediators (e.g. histamine and leukotrienes). In contrast, the use of non-pathogenic microbial agents, e.g. probiotics, is a novel approach to control allergy development. Because IgE/mast cell (MC) interactions significantly contribute to allergic inflammation, we asked whether S. aureus and staphylococcal-derived products, common trigger factors of several allergic disorders, can selectively inhibit MC activation. First, we passively sensitised C57BL/6 mice intraperitoneally with DNP-specific IgE in the presence or absence of heat-inactivated S. aureus. Then, we monitored the anaphylactic response to DNP challenge by rectal temperature measurements. Both experimental groups displayed a fast and virtually identical drop of core body temperature. However, S. aureus-treated mice began to recover significantly faster from anaphylaxis. To identify the signals involved in this response, we next tested the S. aureus cellwall products lipoteichoic acid (LTA) and the lipopeptide homologue MALP-2 showing both a similar downregulation of anaphylactic responses as S. aureus treatedmice, with LTA-treatment resulting in stronger inhibition. Interestingly, LTA and MALP-2 were only found to be beneficial when applied before as opposed to after the allergen challenge. To further elucidate the protective effects of LTA we quantified the levels of MC mediators by ELISA. The amount of histamine was severely reduced in the peritoneal lavage fluid of IgE 1 LTAtreated mice 30 min after challenge (18.75 ng/ mL vs. 30.5 ng/mL in IgE 1 vehicle/DNP group; P 5 0.01). Also, de-novo synthesized leukotrienes were measured in the supernatants of IgE 1 LTA/DNP-stimulated peritoneal MCs (PMCs). Notably, LTA dramatically decreased leukotriene levels as compared to IgE 1 vehicle/DNP-stimulated PMCs (66 pg/mL vs. 6251 pg/mL; P 5 0.05) indicating that this pathway was affected as well. To clarify the underlying mechanisms,


IgE 1 LTA-treated PMCs were subjected to FACS analysis of FcepsilonRI expression, which was downregulated by almost 60% (P 5 0.003). Intriguingly, this process occurred independently of TLR2, as FcepsilonRI expression was also downregulated by LTA in TLR2-deficient PMCs (70% reduction; P 5 0.002). Collectively, these data suggest that S. aureus-derived LTA may be a feasible therapeutic target to control MCmediated type-I allergic reactions.

345 Dendritic cells treated with a combination of TLR4 and TLR7/8 ligands support the sequential differentiation of IL-10, IFN-g and IL-17 secreting CD4 1 T cells Lombardi, V; Van Overtvelt, L; Horiot, S; Moingeon, P Stallergenes, R&D, Antony, France

Background: Toll-like receptors (TLRs) recognize conserved microbial motifs and regulate the induction of appropriate immune responses against potential pathogenic agents. Triggering various TLRs on DCs initiates distinct patterns of gene expression and cytokine secretion in naı¨ ve CD4 1 T cells, leading to Th1, Th2, Treg or Th17 differentiation. Methods: We tested a combination of TLR4 (LPS from E. coli) and TLR7/8 (R848) ligands on human monocyte-derived DCs subsequently co-cultured with allogeneic naı¨ ve CD4 1 T lymphocytes. Patterns of cytokine production were analysed by ELISA and cytofluorometry, and gene expression was determined by quantitative PCR analysis. Results: Human monocyte-derived dendritic cells stimulated with a combination of TLR4 and TLR7/8 agonists produce high levels of IL-10, IL-12 and IL-23. We observed an unexpected variability in DC responses to such stimuli, depending on individuals. DCs responding to a TLR4-7/ 8 stimulation support a sequential, as opposed to a parallel differentiation of naı¨ ve CD4 1 T cells into IL-10, IFN-g, and eventually IL-17-producing cells. In this model, induction of IL-17-production cells is blocked by an anti-IL-23 antibody. Conclusion: Altogether, our results illustrate the synergy between TLRs in inducing a sequentially orchestred balance of regulatory and proinflammatory cytokines by naı¨ ve CD4 1 T cells. We also document the dramatic heterogeneity of DC reactivity to TLR-mediated stimulation between individuals.

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346 Presymptomatic differences in toll-like receptor function in infants who develop allergy Prescott, S1; Noakes, P1; Chow, B1; Breckler, L1; Thornton, C2; Hollams, E3; Van den Biggelaar, A3; Ali, M1; Tulic, M1 1 University of Western Australia, School of Pediatrics and Child Health, Perth, Australia, 2University of Swansea, School of Medicine, Wales, United Kingdom, 3 Telethon Institute for Child Health Research, Division of Cell Biology, Perth, Australia

Introduction: This study explored the hypothesis that impaired function of Toll-like receptor (TLR) microbial recognition is a key factor in allergy development. Method: Cord blood mononuclear cells samples (n 5 111) were cultured either alone or with optimal concentrations of TLR ligands: lipoteichoic acid (TLR2), polyinosinicpolycytidylic acid (TLR3) lipopolysaccharide with interferon gamma (IFN-g) (TLR4), flagellin (TLR5), Imiquimod R837 (TLR7) and CpG (TLR9). Supernatants were analyzed for cytokine responses. Questionnaires, clinical assessment and skin prick testing were used to assess allergy risk (maternal allergy) and allergy outcomes (food allergy and atopic dermatitis) at 12 months of age. Results: Maternal allergy (n 5 59) was associated with significantly higher neonatal responses (particularly IL-12 and IFN-g) to TLR2, TLR3 and TLR4 activation. Consistent with previous reports, neonates who subsequently developed allergic disease (n 5 32) had lower Th1 IFN-g response to mitogens (phytohaemaglutinen). However these infants had significantly higher TLR mediated responses than those without subsequent disease (n 5 75), notably inflammatory TNF-a; and IL-6 responses to TLR2, TLR2, TLR4 and TLR5 activation. The presence of pets in the maternal household was associated with significantly higher neonatal IFN-g responses to all TLR ligands tested (TLR2, 3, 4, 5, 7, 9). Conclusion: Allergic disease was associated with increased (rather than decreased) perinatal TLR responses. Further studies are needed to determine how these responses track in the postnatal period and if this relative hyperresponsiveness is a product of intrauterine influences including maternal atopy, and/or functional genetic polymorphisms. 347 Elevated exhaled nitric oxide detected in the absence of asthma symptoms: does it reflect airway inflammation? Baldassarre, S1; Van Muylem, A1; Sele, J2; Henket, M2; Louis, R2; Michils, A1 1 Hoˆpital Erasme, Pneumology, brussels, Belgium, 2CHU Sart-Tilman, Pneumology, Lie`ge, Belgium

Background: Exhaled nitric oxide (FENO) is considered as a reliable marker of 142

eosinophilic airway inflammation in patients experiencing respiratory symptoms. In this study, we investigated whether elevated FENO observed in some patients suffering from allergic rhinitis but no asthma symtoms is associated with signs of eosinophilic airway inflammation. Methods: PC20 AMP as well as sputum eosinophil count and chemotactic activity for eosinophils in sputum supernatants, were evaluated in two groups of patients exhibiting high FENO measured according to ATS/ ERS recommendations: 20 patients with uncontrolled asthma and 23 patients with allergic rhinitis but no asthma symptoms. Results: Both groups exhibited similar FENO levels (81 7 34 ppb and 86 7 22 ppb-p 5 0.09). Median sputum eosinophil counts were found to be 10% and 1% in patients with uncontrolled asthma and patients with allergic rhinitis but no asthma symptoms (P40.001). Sputum supernatants from both groups exhibited a significant chemotactic activity for eosinophils (chemotactic indexes: 2.8 and 3.9-Normal o1.6). PC20 AMP averaged 25 mg/mL and 200 mg/ mL in patients with or without uncontrolled asthma (Po0.001). In both groups, FENO was reduced by 50% after a two-week course of anti-inflammatory treatment (budesonide 800 mg daily). Conclusion: Elevated FENO observed in the absence of asthma symptoms (mostly in patients with allergic rhinitis) seems to be part of an airway inflammatory process that is limited in such a way that it does not result in a significant eosinophilic airway infiltration, a substantial airway reactivity nor respiratory symptoms that usually characterize bronchial asthma.

348 Leukotriene biosynthesis inhibition and direct 5-lipoxygenase noncompetitive inhibition by the Cys-LT1 receptor antagonists montelukast and zafirlukast Macchia, L1; Rossi, M1; Rucco, A2; Fabiano, M1; Giliberti, L1; Ramires, R1; Caiaffa, M1 1 University of Bari, Department of Allergology and Clinical Immunology, Bari, Italy, 2University of Bari, Allergology and Clinical Immunology, Foggia, Italy

Leukotrienes are powerful lipid mediators involved in inflammation, cell-cell communication and other important physiological and pathological conditions, including asthma and allergic diseases. 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of leukotrienes, belongs to a family of enzymes with a highly conserved structure and a wide distribution throughout most of the eucaryote spectrum, from plants, to higher vertebrates, to humans.Here we report that montelukast and zafirlukast, structural antagonists of the cysteinyl-leuko-

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triene receptor Cys-LT1, also exert a substantial inhibitory effect on leukotriene biosynthesis and 5-LO activity in various in vitro models, at concentrations in the lower micromolar range. The profiles of inhibition by the two leukotriene antagonists were similar but not identical. Thus, we found that montelukast inhibited leukotriene biosynthesis in the human mast cell line HMC1, in human PMNL (stimulated with the Ca ionophore A23187) but also in rat mast cells RBL-1 (incubated with A23187 and exogenous arachidonic acid), with an IC50 between 2.5 and 10 mM. Moreover, montelukast directly inhibited human recombinant 5-LO, although incompletely and at a slightly higher concentration. Kinetic experiments revealed that the inhibition was of the noncompetitive type, suggesting that montelukast binds a yet undefined allosteric site on 5-LO.Moreover, the drug had no effects on other enzymes of the leukotriene cascade, viz. LTC4-synthase and LTA4-hydrolase. Nor did montelukast inhibit two other mammalian lipoxygenases studied: the human platelet 12-LO and the porcine granulocyte 12-LO. On the other hand, zafirlukast also inhibited leukotriene biosynthesis in the HMC-1 cell and PMNL models with an IC50 x 10 mM, whereas slightly higher concentrations were needed for inhibition in the RBL1 model. Zafirlukast also inhibited the recombinant 5-LO activity but was inactive on human platelet-type 12-LO and porcine leukocyte-type 12-LO.

349 Manumycin and asukamycin inhibition of IL-1beta and IL-18 release from human macrophages by caspase-1 blocking Striz, I1; Krasna, E1; Petrickova, K2; Brabcova, E1; Kolesar, L1; Slavcev, A1; Jaresova, M1; Petricek, M2 1 Institute for Clinical and Experimental Medicine, Department of Immunology, Prague, Czech Republic, 2 Academy of Sciences of Czech Republic, Institute of Microbiology, Prague, Czech Republic

Cytokines of the IL-1 family play a critical role in modulating the innate and adaptive immune responses and represent important factors by which macrophages interact with epithelial cells. These cytokines including IL-1 beta and IL-18 are synthesized in a precursor form and require proteolytic processing by caspase-1 to become fully active. The aim of our study was to assess the effect of two streptomyces-derived antibiotics on caspase-1 mediated release of IL1beta and IL-18 from THP-1 monocyte/ macrophage cell line. Furthermore, the capacity of IL-1 related cytokines to induce chemokines was tested in epithelial cell lines of respiratory and renal origin. The THP-1 cells were cultured in RPMI1640 with 5% fetal calf serum and then stimulated with



TNF alpha (20 ng/mL) under serum free conditions in the presence or absence of manumycin and asukamycin (both at 0.3 mg/mL). Synthetic caspase-1 inhibitor AcYVAD-CHO was used as a control. The concentrations of cytokines in culture supernatants were measured by ELISA (IL-18, MBL) or Luminex (IL-1 beta, R&D). In additional experiments, the efect of recombinant IL-1 beta and IL-18 (both at 50 ng/ mL) on chemokine induction was tested in respiratory (A549) and renal epithelial cell lines using cDNA array system and the mRNA data were confirmed by measurement of chemokine levels using Luminex. IL-1 beta was not detectable in culture supernatants of unstimulated THP-1 cells but appeared in response to TNF alpha (4.96 7 0.59 pg/mL). Both manumycin (0.34 7 0.48 pg/mL) and asukamycin (1.06 7 0.81) inhibited IL-1 beta release induced by TNF alpha. IL-18 was found to be constitutively produced (14.68 7 7.83 pg/mL) and the release was doubled by TNF alpha (30.98 7 2.21 pg/mL) and inhibited to basal values by both manumycin (18.04 7 10.21 pg/mL) and asukamycin (12.96 7 2.32 pg/mL). In repetitive experiments, epithelial cells constitutively expressed mRNA for CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 and in response to IL-1 beta also CCL2, CCL4, CCL5, and CCL20 mRNA appeared. In contrast, the induction of epithelial cells by IL-18 showed only moderate changes compared to a potent effect of IL-1 beta stimulation. We conclude from our study that 1) manumycin and asukamycin represent potent blockers of caspase-1 mediated release of IL-1 beta and IL-18 and 2) IL-1 beta and IL-18, although related in a molecular structure and signalling pathways, differ in the capacity to induce epithelial CXC and CC chemokines.

350 Rupatadin inhibits mediator secretion from human mast cells Vasiadi, M1; Kalogeromitros, D2; Kempuraj, D1; Theoharides, T1 1 Tufts University School of Medicine, Pharmacology and Experimental Therapeutics, Boston, United States, 2 Medical School, University of Athens, Allergy Unit, ‘‘Attikon’’ University Hospital, Athans, Greece

Background: Mast cells are involved in allergic inflammation by secreting histamine, platelet activating factor (PAF), proteases, vascular endothelial growth factor (VEGF), and several cytokines including interleukin (IL)-6, tumor necrosis factoralpha (TNF-a) and IL-8. Certain histamine1 receptor antagonists have been reported to inhibit histamine and tryptase secretion, but the effect on inflammatory cytokine release


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from human cultured mast cells is not well known. Methods: We investigated the effect of rupatadine, compared to the flavonoid quercetin, on secretion of IL-6 stimulated by IL-1 from human leukemic mast (HMC1) cells, as well as secretion of histamine, IL8 and VEGF stimulated by substance P (SP) from LAD2 cultured human mast cells. Results: Rupatadine (10–50 mM) added 10 min prior to the trigger, reduced IL-6 release from HMC-1 cells with the strongest inhibition of about 80–100% at 50 mM. A preincubation time course showed that there was no difference whether rupatadine was added 10 min or 24 h prior to stimulation. Rupatadine (50 ı` M) inhibited histamine, IL8 and VEGF release from LAD2 cells by almost 100%, with about 20% inhibition at 10 ı` M. Rupatadine on equimolar concentrations was more potent than quercetin in inhibiting histamine, IL-8 and VEGF release, but less so for IL-6 release. Cell viability was acceptable. Conclusion: Rupatadine, a potent histamine-1 receptor antagonist that also blocks PAF actions, is shown to inhibit IL-1induced secretion of IL-6 and SP-induced histamine, IL-8 and VEGF release from human mast cells, a beneficial action in treating nasal and skin allergic inflammation.This work was supported by GRUPO URIACH (Barcelona, Spain).

351 Histamine receptors on human dendritic cells influence the Th2 response in grass pollen allergy Lundberg, K1; Larsson, K1; Sissela, B1; Greiff, L2; Borrebaeck, C1 1 Immunotehchnology, Department of Immunotechnology, Lund, Sweden, 2Lund University Hospital, Department of Otorhinolaryngology, Lund, Sweden

Introduction: Histamine is an important inflammatory mediator that is released in the airways during an allergic response and current antihistamine drugs work by blocking the histamine receptors. Four different histamine-receptors (HR), HR1-4, have been identified on monocyte-derived dendritic cells (DCs), however, the role of these receptors on allergen presentation to CD4 1 memory T has not been established. Methods: The cell surface expression levels of HR1-4 were investigated on MoDCs, as well as on myeloid BDCA1 1 DCs (mDCs) and plasmacytoid CD123 1 DCs (pDCs) from human peripheral blood. To study the functional properties of HR1, MoDCs from grass pollen allergic donors were pulsed with Phl p extract with or without a HR1 antagonist for 48 h. Thereafter, autologous CD4 1 memory T cells were added


and cocultured with DCs for 7 days, after which T-cell polarization and proliferation were monitored. Similarly, the effect of HR1-4 antagonists on allergen-presentation by primary DCs from allergic individuals was investigated by monitoring the T cell cytokine response and proliferation after DC/T cell coculture. Results: Addition of HR1 antagonist to Phl p – stimulated MoDCs inhibited T-cell proliferation and production of the Th2 cytokines IL-4, IL-5 and IL-13, whereas the level of IFN-g was unaltered. Furthermore, the HR1-4 antagonists were shown to have different effects on either mDCs or pDCs. Conclusion: Histamine influenced the ability of DCs to regulate Th2 responses, which suggests that current antihistamine drugs also function by down-regulating the Th2 responses, through a direct interaction with DCs.

352 A dual role of nerve growth factor in airway epithelium injury and repair Sonar, S1; Schwinge, D1; Kilic, A1; Yildirim, A2; Seidler, K1; Renz, H1; Nockher, A1 1 Biomedical research center, clinical chemistry, Marburg, Germany, 2University Hospital of the Philipps-University, Clinic of Internal Medicine (Respiratory Medicine), Marburg, Germany

Background: Epithelium injury and repair constitute important events ensuing inflammatory airway diseases. Nerve growth factor (NGF), a prominent member of the neurotrophin family, has increasingly been shown to play a role in immune functions pertaining to the development and pathogenesis of inflammation in airway diseases. We hypothesized a dual role for NGF, as an important player in mediating both inflammation and repair during airway epithelial injury. Method: In vitro method to determine the role of NGF in wound healing in bronchial epithelial cells was employed. The expression of its receptor, TrkA was shown with the help of Immunofluorescence microscopy. For the in vivo analysis, the clara cell population of epithelial cells lining the airway was denuded using naphthalene. Trangenic mice overexpressing NGF in the lung was further analysed to strengthen the data. Results: In cultured airway epithelial cells, proliferation and wound closure was dependent on NGF production and expression of its receptor TrkA. In mice, treatment with naphthalene resulted in the denudation of Clara cells within the bronchial epithelium, accompanied by a local inflammatory response. During acute epithelial damage, inflammatory cell recruitment and TNFalpha fnlevels were significantly decreased upon inhibiting NGF signalling. Using

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transgenic mice constitutively overexpressing NGF (NGFtg) in the lung, we could also show that NGF is directly involved in epithelial proliferation and regeneration. After injury, NGFtg mice exhibited higher proliferative capacity in the lung and earlier reappearance of Clara cells compared to wildtype mice. Moreover, lungs of reepithelializing NGFtg mice showed a significantly increased matrix turnover as demonstrated by elevated expression of ECM proteins and regulation of matrix metalloproteinases. Conclusion: This study demonstrates for the first time a dual role for NGF, being necessary to initiate an inflammatory response after injury as well as for epithelial recovery via repair and remodelling of the wounded tissue. Thus, the airways early response to injury also relies upon NGF to remodel and recuperate the airway’s integrity.

353 TGF-beta isoforms and receptors in chronic sinus disease Van Bruaene, N; Perez-Novo, C; Gevaert, P; Holtappels, G; De Ruyck, N; Van Cauwenberge, P; Bachert, C University Hospital Ghent, ENT, Ghent, Belgium

Background: Recent research suggests that TGF-beta, a multifunctional and pleiotropic growth factor, plays a key role the pathogenesis of airway disease. TGF-beta has important anti-inflammatory properties, and acts as a master switch in the induction of fibrosis. Three TGF-beta isoforms have been described, however TGF-beta 1 is the most studied isoform. Knowledge on the distribution of the other isoforms is limited. In addition, little is known regarding TGFbeta receptor expression in chronic sinus disease. In this study, we analyzed the expression of TGF-beta isoforms 1, 2 and 3, together with its receptors I, II and III in chronic rhinosinusitis with or without polyps. Material and methods: Sinus tissue from patients undergoing endoscopic sinus surgery for chronic rhinosinusitis was collected. Inferior turbinates from patients undergoing

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septoplasty or rhinoseptoplasty were collected as controls. The samples obtained from CRSwNP (n 5 13), CRSsNP (n 5 13) and controls (n 5 10) were analyzed by means of immunohistochemistry, ELISA and RT-PCR for three TGF-beta isoforms 1, 2 and 3 and TGF-beta receptor 1, 2 and 3. Results: TGF-beta 1 was significantly higher in CRSsNP versus controls, together with Tbeta RI. In contrast, TGF-beta 1 was significantly lower in CRSwNP versus controls, in line with the low expression of Tbeta RII, while TGF-beta 2 was found higher in CRSwNP versus controls. In addition, Tbeta RIII was significantly higher in CRSsNP and significantly lower in CRSwNP versus controls. Conclusion: As Tbeta RI and RII are both required for TGF-beta signaling, a decreased expression of Tbeta RII together with low TGF-beta 1 protein concentration in CRSwNP indicates suppressed TGF-beta signaling. In strong contrast, in CRSsNP, the increased Tbeta RI expression concurred with high TGF-beta 1 protein expression. Tbeta RIII, which act as an enhancer for Tbeta RI and RII binding, was found decreased in CRSwNP and higher in CRSsNP compared to controls, further supporting the decreased TGF-beta signaling in CRSwNP. These data clearly support the hypothesis that CRSsNP and CRSwNP are distinct disease entities, as indicated by the differential regulation of TGF-beta protein and receptor expression. This is reflected by the typical extracellular matrix remodeling patterns observed, characterized by pseudocyst formation in CRSwNP, and fibrosis in CRSsNP.

354 Specific adjuvants prime dendritic cells to induce regulatory T cells Sibiryak, D; van Capel, T; Kapsenberg, M; de Jong, E AMC University of Amsterdam, Department of Cell Biology and Histology, Amsterdam, the Netherlands

effecter cells or Th2 cells) to innocuous inhaled proteins (1.e. antigens or allergens). Currently the only curing therapy in the treatment of this disease is specific allergen immunotherapy (SIT), based on frequent parental application of allergen. SIT is thought to be effective through the induction of allergen-specific regulatory T cells (Tregs) that suppress the activity of the harmful allergen-specific Th2 cells. A major drawback of SIT is the risk of anaphylactic reactions evoked by binding of allergen to immunoglobulin E and delivery of biologically active compounds from mast cells. This unwanted reaction is prevented by application of very low starting doses. The efficacy of SIT may be further improved by applying adjuvants in SIT vaccines which may amplify the induction of Tregs. Therefore, we investigated the effects of candidate adjuvants on the ability of dendirtic cells to induce Tregs as well as the phenotype of the induced Tregs. Monocyte-derived DCs were primed with 1-alpha, 25-dihydroxi vitamin D3, Cholera toxin B (CTB), Heatkilled Listeria monocytogenous (HKLM) in the absence or presence of LPS and used to stimulate naı¨ ve CD4 1 Th cells. When T cells were resting, they were analyzed for their capacity to suppress proliferation of bystander Th cells as well as their expression of CD25, CD127, CTLA4 and intracellular Foxp3 protein. Specific candidate adjuvants did indeed induce the development of Tregs. Moreover, the induction of CD25 1 Foxp3 1 cells among CD4 1 T cells in cultures treated with adjuvants was enhanced in comparison to control cultures. To distinguish between CD25 1 activated and regulatory T cells, we also analyzed the expression levels of CD127, which is marginally expressed by Tregs. We observed a slight increase in quantity of CD127dim/ Foxp3 1 Tregs in adjuvants-treated cultures. This data shows that specific adjuvants may induce the development of Tregs from naı¨ ve precursors and this approach shows promise for there use in SIT vaccines.

Allergic asthma results from uncontrolled reactivity of immune cells (1.e. type 2

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Poster Discussion Session 13 – Immunotherapy: Different Viewpoints on Clinical Aspects

Poster Discussion Session 13 Immunotherapy: Different Viewpoints on Clinical Aspects 355 Attitude of allergists in managing polysensitised patients Ciprandi, G1, Alesina, R2, Ariano, R3, Aurnia, P4, Borrelli, P5, Cadario, G6, Capristo, A7, Carosso, A8, Casino, G9, Castiglioni, G8, Cesinaro Di Rocco, P10, Colangelo, C10, Di Gioacchino, M11, Di Paola, M12, Errico, G13, Fiorina, A14, Gambuzza, F4, Gangemi, S15, Gasparini, A16, Giugno, R17, Iemoli, E18, Isola, S15, Maniero, G19, Marengo, F6, Mazzali, P20, Minelli, M21, Mosca, M22, Pellegrino, R23, Piconi, S18, Pravettoni, V24, Quaglio, L19, Ricciardi, L15, Ridolo, E25, Sillano, V26, Valle, C20, Varin, E27, Verini, M28, Zambito, M29, Puccinelli, P30, Incorvaia, C31, Scurati, S30, Frati, F30 1 San Martino Hospital, Internal Medicine Department, Genoa, Italy, 2Policlinico San Matteo, Pneumology, Pavia, Italy, 3General hospital, Allergy Department, Bordighera, Italy, 4ASL 7, Allergy Service, Ragusa, Italy, 5 Regional Hospital, Allergy Service, Aosta, Italy, 6San Giovanni Battista (Molinette) Hospital, Allergy Department, Turin, Italy, 7Federico II University, Pediatric Allergy, Naples, Italy, 8ASL 4, Allergy Service, Turin, Italy, 9 Ospedale Piedimonte Matese, Allergy Service, Caserta, Italy, 10Ospedale Spirito Santo, Allergy Service, Pescara, Italy, 11G. D’Annunzio University, Department of Medicine and Ageing Sciences, Chieti, Italy, 12ASL 3, Allergy Service, Catania, Italy, 13Ospedale F. Fallacara, Allergy Service, Triggiano, Italy, 14ASL 2 Savonese, Pneumology and Allergy, Savona, Italy, 15Policlinico Hospital, Allergy Department, Messina, Italy, 16General hospital, Allergy Service, Cernusco sul Naviglio, Italy, 17 Gravina Hospital, Allergy Service, Caltagirone, Italy, 18 Luigi Sacco Hospital, Allergy Service, Milan, Italy, 19 General Hospital, Pediatrics, Pinerolo, Italy, 20San Paolo Hospital, Allergy Service, Milan, Italy, 21Ospedale Campi Salentini, Allergy Service, Lecce, Italy, 22 Policlinico San Matteo, Dermatology and Allergy, Pavia, Italy, 23Martini Hospital, Allergy Service, Turin, Italy, 24Ospedale Maggiore Policlinico Mangiagalli Regina Elena IRCCS, Allergy Department, Milan, Italy, 25 University Hospital, Clinical Sciences Department, Parma, Italy, 26Legnano General Hospital, Allergy Service, Corsico, Italy, 27Ospedale Maggiore Policlinico Mangiagalli Regina Elena IRCCS, Pediatrics, Milan, Italy, 28 Ospedale Clinicizzato, Pediatrics, Chieti, Italy, 29ASL 6, Allergy Service, Palermo, Italy, 30Stallergenes, Scientific Department, Milan, Italy, 31ICP Hospital, Allergy Department, Milan, Italy

Background: The condition of polysensitisation is, especially in adult patients, the most common to encounter in the routine activity of allergists. However, it is poorly known how polysensitised subjects are managed regarding drug treatment and particularly allergen specific immunotherapy (SIT). A survey on the attitude of allergists in treating polysensitised patients was conducted in Italy. Methods: A total number of 439 entered the study. Of them, 272 completed the first year of observation. They were 123 males and 149 females, of a mean age of 28.7 years (SD 12 years). The clinical manifestation at study start was rhinitis in 49.3% of pts, asthma in 3.7%, rhinitis and asthma in 47%. Rhinitis was slight intermittent in 3.7% of cases, moderate intermittent in 18.7%, slight persistent in 17.9%, and moderate persistent r 2008 The Authors Journal Compilation

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in 59.7%. Asthma was intermittent in 20% of cases, slight persistent in 40%, moderate persistent in 30%, severe persistent in 10%. Results: Two-hundreds-forty-four patients were polysensitised, 25% of them with two positivities, 26% with three positivities, 15% with four positivities, 10% with five positivities. Most frequently positive allergens were grass pollen (81%), Parietaria pollen (48%), dust mites (46%), Betulacee pollen (38%), olive pollen (32%), mugwort (28%), cat (22%), and dog (17%).SIT was prescribed to 163 pts (64%) with one allergen extract, to 55 (21%) with two allergen extracts, to 6 (2.4%) with four allergen extracts, and to 3 (1.2%) with five allergen extracts. SIT was performed in 39.8% of cases with dust mites, in 39.8% with grass pollen, in 17.2 with Parietaria pollen, in 9.4% with olive pollen. In 71% of treatments there was no interruption, in 15% one interruptions, in 8% two interruptions, in 3.2% three interruptions, in 1.8% more than three interruptions. At initial evaluation patients with slight rhinitis were 9%, while were 30% after 1 year (Po0.0001). Patients without or with slight asthma were 74%, while were 83% after 1 year (Po0.004). Significant reductions of allergic symptoms and of drug consumption were found after SIT. A significant improvement was observed in quality of life. Conclusion: Most polysensitised patients are treated by SIT with one or two allergen extracts. The findings of this study suggest the adequacy of this kind of treatment, since a significant improvement in the clinical status of such patient was found.

356 Comparative benefit-risk balance of subcutaneous immunotherapy for seasonal allergic rhinitis DuBuske, L1; von Weikersthal-Drachenberg, KF2 Brigham and Women’s Hospital, Allergy, Boston, United States, 2Allergy Therapeutics plc, International Medical, Worthing, United Kingdom

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Background: The decision to administer subcutaneous immunotherapy (SCIT) for seasonal allergic rhinitis (SAR) necessitates a careful consideration of the possible benefits and risks associated with this mode of therapy. Here the efficacy and safety aspects of SCIT are summarized compared with other interventions in the treatment of SAR.


Methods: Efficacy and safety data for the treatment of SAR with SCIT were compared with similar data for antihistamines, steroids and a leukotriene antagonist to assess the extent of symptomatic relief and the adverse event profile associated with each treatment strategy. Well-designed, randomised, controlled studies were selected to form the basis of evidence considered in this investigation. Results: In placebo-controlled randomised trials, antihistamines and the leukotriene antagonist gave statistically significant improvements in clinical outcome over treatment periods of 2–4 weeks. The magnitude of these improvements was often small with a reduction in symptoms from baseline of 15–40% in most studies, and a difference of 4 to 12% compared to that achieved with placebo. Intranasal corticosteroids were associated with a significantly greater reduction in symptoms compared with antihistamines. SCIT produced improvements in symptom scores of approximately 25% greater than placebo, and was the only therapy to provide a lasting effect after cessation of therapy. Concurrent usage of symptomatic medications was also significantly reduced. Adverse events varied with treatment type. Antihistamines were associated with sedation and cognitive effects while intranasal corticosteroids were associated with local events such as epistaxis and nasal irritation. SCIT was associated with injection site reactions including erythema, swelling and itch and late systemic reactions such as conjunctivitis, rhinitis and wheezing with an accepted, although rare, possibility of anaphylaxis. Conclusion: When administered according to appropriate guidelines, in appropriatelyselected patients, SCIT is an effective and relatively safe treatment for patients having SAR which has been inadequately controlled with other therapy. SCIT has demonstrated efficacy in patients of all ages, and, unlike symptomatic medications, continues to be effective after the treatment course has finished. Adverse events with SCIT are predominantly of mild to moderate intensity with local reactions generally easily manageable. The risk of anaphylaxis inherent with each SCIT injection suggests that development of briefer courses of SCIT employing extract formats less likely to induce anaphylaxis should be pursued.

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357 Easy administration of SLIT during the first Year (The EASY Study) – assessment of patient satisfaction with SLIT by using the validated Treatment Satisfaction Questionnaire for Medication (TSQM) in patients with rhinoconjunctivitis to grass and rye allergens Wolf, H1; Schnitker, J2; Worm, M3 ALK-SCHERAX Arzneimittel GmbH, Clinical Development, Wedel, Germany, 2Institut fu¨r Angewandte Statistik GmbH, Biometrician, 33647 Bielefeld, Germany, 3 Allergie Centrum Charite´, Klinik fu¨r Dermatologie und Allergologie, 10117 Berlin, Germany

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Background: Sublingual immunotherapy is appropriate for home treatment. Objective of this study was to investigate patient satisfaction and compliance during the first year of treatment. Methods: In an open, observational study data for 206 patients with allergic rhinoconjunctivitis with or without asthma to grasses and rye were recorded in 35 allergists’ offices from June 2005 to January 2007. Patients treated with SLIT using single dose containers (SLITones 200 STU/mL, daily, ALKAbello´, DK) were evaluated for the first year of SLIT with control visits every 3 months. Patient satisfaction was recorded using the validated ‘Treatment Satisfaction Questionnaire for Medication’ (TSQM, 14 questions in the domains effectiveness, side effects, easiness of use and global satisfaction) that was completed after 2 and 11 months of SLIT, respectively. Before SLIT demographic data, allergy history and diagnostic data were recorded by the physician. Symptoms and medication use were assessed for the grass pollen season before SLIT and for the first season with SLIT. At the control visits patients were asked about regular intake of SLIT, easiness of handling and tolerability. The data were evaluated for the two cohorts o18 (N 5 106) and 18 years (N 5 100). Results: The TSQM mean values ( 7 SD) for patient satisfaction after about 11 months of SLIT were comparable to results for treatments of other chronic diseases for effectiveness (67.37 7 24.83) and easiness of use (83.53 7 18.39), and higher for side effects (98.45 7 7.42) and global satisfaction (78.61 7 17.89); mean values were higher for all domains in patients o18 years; about 93% of the patients adhered to the scheduled visits, about 86% did apply SLIT at regular intervals. Compared to the previous grass pollen season symptoms improved in about 70% of the patients with higher rates in the cohort o18 years; 80.7% used no or less symptomatic medication during grass pollen season after initiation of SLIT. Tolerability was very good. Conclusion: In this study patient satisfaction with SLIT using single dose containers

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was high overall and regarding side effects and was higher rated in children and adolescents than in adults. For effectiveness and easiness of use the assessment was similar to treatments of other chronic diseases.

inhalant allergens. Therefore a specific education of the patients and general practitioners regarding the aims of immunotherapy like symptom reduction, avoiding new sensitisations, and preventing asthma is essential.

358 Patients’ compliance during specific allergen immunotherapy – frequency of dropouts and underlying reasons

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Jurilj, M1; Schuster, C1; Fru¨hauf, J1; Horn, T2; Aberer, W1; Sturm, G1 1 Medical University of Graz, Department of Environmental Dermatology and Allergy, Graz, Austria, 2 ALK, Alk-Abello`, Linz, Austria

Background: Specific immunotherapy represents the only causal treatment for immediate-type allergy. To achieve longterm protection, immunotherapy should be administered for at least 3 years, in Hymenoptera venom allergy for 5 years. The aim of our study was to gain detailed information regarding the dropouts during immunotherapy and the reasons for stopping this therapy prematurely. Methods: Six hundred and seventy eight patients have been included in this study from January 2001 until June 2007. Patients were classified into four groups: in birch pollen-, grass pollen-, Hymenoptera venomand house dust mite allergic patients. Dropouts were detected by the order list of the supplier company Alk – Abello` Austria; reasons for early discontinuation of therapy were collected by telephone enquiry. Results: One hundred and thirty four out of 678 (19.8%) patients discontinued specific immunotherapy before the recommended time of therapy. In detail, 22 of 39 (56.4%) house dust mite allergic patients, 32 of 97 (33.0%) grass pollen allergic patients and 17 of 79 (21.5%) birch pollen allergic patients terminated their therapy early. The lowest number of dropouts (63 of 463; 13.6%) was in the group of Hymenoptera venom allergy. The most common causes of dropouts were lack of time (41.7%), tiredness (18.7%), local itching (14.6%) and missing therapeutical effect (12.5%). Other forms of treatment (homeopathy and acupuncture) were reasons for 16.7% of patients discontinuing specific immunotherapy. 10.4% of patients were advised against specific immunotherapy by their general practitioner. Conclusion: The majority of the Hymenoptera venom allergic patients completed the treatment despite of a therapy period of 5 years. By comparison, in house dust mite or pollen allergic patients more dropouts could be observed. This might be based on the higher risk for life-threatening symptoms after Hymenoptera stings compared to inconvenient, but not severe reactions to

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Abstract withdrawn.

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Abstract withdrawn.

361 Proof of concept for a carry-over effect of co-seasonal SLIT in patients with grass pollen allergic rhinoconjunctivitis Ott, H1; Sieber, J2; Baron, J1; Merk, H1 University Clinic RWTH, Department of Dermatology & Allergology, Aachen, Germany, 2Stallergenes GmbH, Medical Department, Kamp-Lintfort, Germany

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Background: Efficacy and safety of a 3 years treatment period with sublingual allergen application in allergic rhinoconjunctivitis (AR) has been demonstrated recently. Additionally, we evaluated the carry-over effect in this proof-of-concept study for the seasonal treatment regimen. Methods: Two hundred and thirteen patients (99m, 114f, mean age 32.2 years) with AR against grass pollens (mean disease duration 12.8 years) were randomised 2 : 1 to verum or placebo. A baseline season with only anti-symptomatic treatment was followed by seasonal SLIT treatment for 3 years and 1 follow-up season without SLIT. At the beginning of each season, SLIT treatment with a 5-grasses mixture titrated under medical observation within 90 min (0– 30–90–150–300 IR) followed by a daily intake of 300 IR for the whole season. Symptoms, medication intake and adverse events (AE) were documented daily in a diary for each pollen season of the 5-year observation period. Results: Data from 184 patients were analyzed. The treatment duration was approximately 3 months in each season. There was a significant difference in the level of the mean combined score compared to baseline (symptoms and medication) for verum (V) versus placebo (P) already in the first season (V 0.97, P 1.28, P 5 0.0427) which continued in season 2 (V – 0.95, P 0.06, P 5 0.0401), season 3 (V – 1.35, P 0.56, P 5 0.0019) and the follow-up season (V – 1.76, P – 1.19, P 5 0.0508). A more pronounced difference was seen in the symptom score in season 1 (V – 0.03, P 1.28 P 5 0.0366), season 2



(V – 0.89, P 0.91, P 5 0.0235), season 3 (V – 1.02, P 1.32, P 5 0.0004) and the follow-up season (V – 1.94, P – 0.3, P 5 0.0153). Intake of rescue medication was very low (n.s. for V vs. P in all seasons). Titration and daily SLIT intake were well tolerated. Most frequent reactions were mild to moderate local itching and burning in the oral cavity. Serious systemic or anaphylactic reactions were not reported. There was a consecutive reduction in the frequency and severity of side effects from season to season. Conclusion: Seasonal sublingual immunotherapy with ultra-rush titration is effective with significant differences versus placebo from the first treatment season on with increasing magnitude of efficacy over consecutive treatment seasons. The obtained data are indicative for a carry-over effect of co-seasonal SLIT. Overall, the treatment was well tolerated without serious systemic side effects.

0.001), and no significant systemic side effects were observed. In 9 patients with severe atopic dermatitis who had baseline SCORAD values same or above 40, the SCORAD values significantly decreased from 55.9 7 3/4 11.0 at baseline to 38.2 7 3/4 19.2 at 6 months (P 5 0.01) and 28.1 7 3/4 14.0 at 12 months (P 5 0.008, compared to the baseline). Conclusion: In this uncontrolled pilot study, combined treatment with allergen-specific immunotherapy and a histamine-immunoglobulin complex resulted in significant clinical improvements in patients with atopic dermatitis. Additional controlled studies are needed to confirm the clinical usefulness of this modified allergen-specific immunotherapy for atopic dermatitis.

362 Treatment of atopic dermatitis with a combination of allergen-specific immunotherapy and a histamineimmunoglobulin complex

Barberi, S1; Caminiti, L1; Vita, D1; Passalacqua, G2; Pajno, G1 1 Allergy Unit – University of Messina, Department of Pediatrics, Messina, Italy, 2Allergy and Respiratory Diseases – University of Genoa, DIMI, Genoa, Italy

Nahm, D1; Jeon, S2 Ajou University School of Medicine, Department of Allergy and Rheumatology, Suwon, Republic of Korea, 2 Yonsei-Ajou Pediatric Hospital, Allergy Clinic, GwangJu-City, Republic of Korea

Background: Sublingual Immunotherapy (SLIT) is effective in selected patients with IgE mediated rhinitis and/or asthma, but little is known about the long term benefit after discontinuation. Methods: We performed a randomised controlled trial of the discontinuation of SLIT with Parietaria judaica in children with seasonal asthma and rhinitis, who previously received a 3-year SLIT. In the fourth year of this trial, the seasonal symptomsmedication scores were reassessed. Symptoms were assessed for chest, nose and eyes and pulmonary function test was also performed. Results: The scores for seasonal symptoms and the use of rescue antiallergic medication remained low after the discontinuation of immunotherapy in those children treated with active SLIT in comparison with children treated with placebo. We observed a statistical difference either in symptoms score P 5 0.04 and in rescue medication score P 5 0.02 between the two groups. A significant difference was also reached in the forced expiratory flow 25–75 between two groups on third years after SLIT discontinuation, but not before this time (P 5 0.35). Conclusion: Sublingual Immunotherapy for Parietaria pollen for 3 years induced prolonged clinical remission accompanied by a marked reduction of the use of rescue medication.

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Background: Allergic responses to common environmental agents are believed to be involved in the development of atopic dermatitis, but clinical usefulness of allergen-specific immunotherapy in the treatment of atopic dermatitis is controversial. We evaluated the clinical usefulness of combined treatment with allergen-specific immunotherapy and a histamine-immunoglobulin complex in patients with atopic dermatitis. Methods: Twenty patients with atopic dermatitis and hypersensitivity to house dust mites whose clinical conditions had not been effectively controlled by current standard medical therapies were treated with a combination of allergen-specific immunotherapy using house dust mite extract and a histamine-immunoglobulin complex for 12 months. The primary efficacy outcome was the change in the standardized clinical severity scoring system for atopic dermatitis (SCORAD) values at 6 and 12 months in comparison with the values at baseline. Results: In 18 patients who completed all 12 months of treatment, the SCORAD values significantly decreased from 43.6 7 3/4 15.9 (mean 7 3/4 SD) at baseline to 27.8 7 3/4 18.3 at 6 months and 18.3 7 3/4 14.9 at 12 months (Wilcoxon signed-rank test, Po r 2008 The Authors Journal Compilation

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363 Long term clinical efficacy of sublingual immunotherapy in children with parietaria allergy


364 Insect venom immunotherapy with 50 mg venom: safety and efficacy in Greek children Konstantinou, G1; Chatziioannou, A1; Kalobatsou, A1; Koutli, M1; Giavi, S2; Lourbas, G1; Douladiris, N1; Manoussakis, E1; Papadopoulos, N1 1 2nd Pediatric Clinic, Kapodistrian University of Athens, Allergy and Clinical Immunology Department, Athens, Greece, 22nd Pediatric Clinic, Kapodistrian University of Athens, Allergy and Clinical Immunology Department, Thessaloniki, Greece

Background: Venom immunotherapy (VIT) has been shown to be an effective and safe treatment for preventing sting-induced anaphylaxis in persons with a history of systemic reactions to insect stings. A remaining problem is the relative effectiveness and safety of different immunotherapy (IT) protocols used in respect to maintenance dose, injection interval and duration. Objective: To describe a modified rash VIT protocol with a maintenance dose of 50 mg lasting for 5 years and to evaluate its safety and efficacy in preschool and school children. Patients and methods: Fifty three children 9.5 7 3.2 years old with a history of at least one anaphylactic reaction to hymenoptera stings underwent VIT between 1995–2005. The identification of the offending insect(s) was based on patient’s report and documented with in vivo (SPTs and IDs) and in vitro (RAST/CAP) test results. A modified rash outpatient protocol lasting for 5 weeks until an ending maintenance dose of 50 mg was followed (commercial extracts from ALK Abello`) according to clinical history and test results. After the maintenance dose was achieved the followed injection intervals were 4 weeks for the first year, 5 for the 2nd and 3rd year and 6 for the last 2 years. Results: The distribution of the performed VIT was: 24 children to Apis mellifera (AM), 19 to Vespula species (Vs), 1 to Polistes species (Ps), 5 to mixed-Vespid [2 sensitised to both Vs and Dolichovespula maculate (DM), 2 sensitised to both Vs and Dolichovespula arenaria (DA) and 1 to Vs, DM and DA], 2 to both AM and Vs and 2 to both AM and Ps. In two of these children (sensitised to AM) the maintenance dose was increased to 100 mg per dose due to systemic reactions (one during the increasing period and one 2 1/2 years after reaching the maintenance dose). The remaining 51 children tolerated the administered dose without side effects. 21 (41.18%) of them reported sting from the offending, for the anaphylaxis, insect (15 while being on maintenance dose and before the end of IT and 6 one to seven years after finishing IT) all without symptoms apart from mild local reactions. Conclusion: VIT with 50 mg maintenance dose seems to be safe and effective enough 147


to produce tolerance to children with hymenoptera venom hypersensitivity. The efficacy is enhanced by the fact that our sample includes only children with anaphylactic reactions and that the time period between VIT initiation and a new sting is sort for the hypersensitivity to be outgrowth.

365 Short term efficacy of sublingual spray immunotherapy Palma-Carlos, A Clinical Allergy Immunology Center, CAIC, Allergology, Lisbon, Portugal

Background: In the last few years sublingual immunotherapy (SLIT) has gained increasing popularity at least in Europe and all allergen producers laboratories have launched sublingual drops. Sublingual sprays have a larger mucosal contact than drops or tablets increasing also the rate of absorption. Therefore this method of dispensing drugs and allergens can also be applied to SLIT. The efficacy of this technique must be assayed. Methods: Fourty patients with respiratory allergy, allergic rhinitis, asthma or allergic rhinitis asthma syndrome have been included in the trial. Informed consent has been obtained from all patients. SLIT spray has been given in a standardized schedule from 4 vials in progressive concentrations A-1/125, B 1/25, C 1/5, D 1/1. For mites the vaccine was a standardized at 5 mcg/mL for Derp 1 1 Derp2 and 5 mcg/mL, for Derf1 1 Derf2 and Derf1. Cummulative dose at the end of induction was for Derp 1 around 490 mcg (DIATER Laboratories, Spain). The composition of SLIT-spray was chosen in each case by clinical history, skin tests and specific IgE. In children less than 10 years half-concentration of SLIT-spray was employed. The procedure has been

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carefully explained at all the patients. Daily application for VIALS A,B,C from 1 to 4 puffs each day. Vial manutention dosage 1 puff daily. This dosage being attained only after 13 days and continued 2 months more. A booklet with instructions was given to the patients and a visual scale from 0 to 5 points registered in a second appointment to evaluate facility, convenience, practicability, clarity of instructions and general evaluation of treatment. Side effects were registered for all the patients for each dosage. Symptomatic therapy was allowed during trial with anti-histamines nasal steroids, and formoterol-budesonide when necessary. Results: Short-term efficacy of immunotherapy was good in all patients either for house dust mites allergy or pollens. Evaluation by the patients give on a visual scale an average of 3.5 on 5 points (70%). Medical evaluation was of good (12) or very good (24) results in the patients even in the patients treated during pollen season. Only 1 patient withdrawed from the trial due to the tongue swelling and nauseous state. SLIT spray is effective generally well tolerated and very convenient for the patients. Larger series of SLIT-spray must be evaluated in order to confirm these preliminary results.

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366 The effect of preseasonal and perennial immunotherapy on asthma symptoms and airway inflammation in patients allergic to grass pollen Bochenska-Marciniak, M1; Tworek, D2; Kuprys-Lipinska, I2; Kuna, P2 1 Medical University of Lodz, Division of Pneumonology and Allergy, Lodz, Poland, 2Medical University of Lodz, Division of Internal Diseases, Asthma and Allergy, Lodz, Poland

Background: Immunotherapy is the only treatment that may change natural course of allergic dieseases. Clinical observations suggest that perennial immunotherapy (PIT) is more effective compared to preseasonal immunotherapy (PSIT). The aim of our study was to compare the effect of PIT and PSIT on asthma symptoms and airway inflammation in subjects allergic to grass pollen. Methods: Patients allergic to grass polen suffering from rhinoconjuctivitis with or without concomitant seasonal asthma were included. After baseline season of observation they were radomized to receive PSIT or PIT in a double-blind manner for 3 years. The effect of immunotherapy on asthma symptoms was evaluated using diaries. Exhaled nitric oxide (FeNO) was measured outside the pollen season at the beginning of treatment and during the pollen season in the first and third year of immunotherapy. Results: The data collected from 48 patients were analyzed (25 in PSIT group and 23 in PIT group). Ten patients (40%) in PSIT group and 16 (69%) in PIT group sufferd from seasonal asthma. There was significant difference in asthma symptoms score between PIT and PSIT groups during the 2nd (0.23 7 0.2 vs. 0.44 7 0.24; P 5 0.015) and 3rd (0.2 7 0.22 vs. 0.32 7 0.17; P 5 0.02) pollen season. No significant difference in FeNO levels between the PIT and PSIT group was found at any timepoint. A trend toward lower levels of FeNO during the third year compared to the first year of immunotherapy was observed in both groups. Conclusion: PIT seems to be more effevtive than PSIT in improving asthma symptoms in patients allergic to grass pollen.


Poster Discussion Session 14 – Infection and Allergy – from Methodology to Risk Factors

Poster Discussion Session 14 Infection and Allergy – from Methodology to Risk Factors 367 Comparison of nasal sampling methods for the detection of viral pathogens

368 Primary nasal epithelial cells express different levels of Toll-like receptors

Spyridaki, I1; Christodoulou, I1; de Beer, L2; Carlsen, K3; L^drup-Carlsen, K3; van Drunen, C2; Hovland, V3; Kowalski, M4; Kurowski, M4; Molenkamp, R5; OlszewskaZiober, A4; Papadopoulos, N1 1 University of Athens, Second Department of Pediatrics, Athens, Greece, 2Academic Medical Center, ENT Research Laboratories, Amsterdam, the Netherlands, 3 National Hospital HF, Voksentoppen BKL, Oslo, Norway, 4 Medical University of Lodz, Department of Immunology, Lodz, Poland, 5Academic Medical Center, Department of Clinical Virology, Amsterdam, Norway

van Tongeren, J1; Guitink, A1; Luiten, S1; Fokkens, W1; van Drunen, K1; de Jong, E2 1 Academic Medical Centre Amsterdam, Otorhinolaryngology, Amsterdam, the Netherlands, 2 Academic Medical Centre Amsterdam, Cell Biology and Histology, Amsterdam, the Netherlands

Background: Respiratory viruses are the major cause of the common cold, as well as asthma exacerbations. The presence of rhinovirus (RV) or other respiratory viruses may be associated with 80–85% of asthma exacerbations in children, and over 50% in adults. Although detection methodologies have considerably improved during the last few years, techniques for obtaining nasal secretions may considerably influence the detection rates. The aim of our study was to compare the efficacy and patient discomfort between 4 different techniques for obtaining nasal secretions. Methods: Nasal secretions from 58 patients with symptoms of a common cold from 3 clinical centres (Amsterdam, Lodz, Oslo), were obtained by 4 different methods: aspirate, brush, swab, and wash. In each patient all 4 sampling procedures were performed and the patient discomfort was evaluated by a visual scale (scale 1–5) after each procedure. Single pathogen RT-PCRs for RV, influenza and adenovirus were performed in all samples. Results: A specific viral cause of respiratory tract infection was determined in 38 patients (65.5%). The detection rate for any virus was 55% in washes, 49% in aspirates, 40% in swabs and 42% in brushes. The degree of discomfort reported was 2.53 for swab, 2.67 for wash, 2.71 for aspirate and 3.58 for brushings. Conclusion: Nasal washes yield the highest rate of viral detection without a excessive patient discomfort. However, differences were relatively small, suggesting that the optimal sampling method must be balanced with compliance, costs and time required to collect the specimens.


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Background: Our nasal mucosa is constantly exposed to air pollutants, airborne allergens, and (non)-pathogenic micro-organisms. Epithelial cells form the outer lining of the airway mucosa and are the first to encounter these environmental factors. Epithelial cells not only play a passive role as physical barrier, but should be considered as an active component of the mucosal defense system. Limited data on the expression and functional activity of Tolllike receptors (TLRs) in primary nasal epithelial cells is currently available. Method: We isolated primary epithelial cells from non-allergic healthy individuals and TLR expression was analyzed by RT-PCR. Furthermore, nasal primary epithelial cells were stimulated with TLR-agonists to investigate the functional activity of the TLRs through determining the release of different pro- and anti-inflammatory cytokines and chemokines by ELISA. Results: Primary nasal epithelial cells express different levels of all ten TLRs, with relative high mRNA levels for TLR 1, TLR3, TLR4, TLR5, TLR6 and TLR 9. Surprisingly, only stimulation of TLR3 by poly(I:C) (dsRNA) and to lesser extent of TLR2 by peptidoglycan resulted in increased secretion of IL-6, IL-8, RANTES, IP-10, MIP-1a, MIP-1b, and G-CSF. High mRNA expression of TLR4 was not sufficient to respond to LPS. Conclusion: In contrast to their expression levels of TLRs primary nasal epithelial cells only respond to stimulation of TLR3 with poly(I:C) and to lesser extent of TLR2 to peptidoglycan. This study demonstrates that indeed nasal epithelial cells not only functions as a mechanical barrier but also are involved the defence against invading pathogens. Only some of the TLRs expressed in nasal epithelial are functionally active and this should serve as a reminder that conclusions based on mRNA expression only should be treated cautiously. Whether the expression and functional activity of the TLRs would be different during an active infection or in patients with allergic disease remains to be explored.


369 ECP, RANTES, and eotaxin in respiratory syncytial virus bronchiolitis as predictors of subsequent wheezing Kim, H1; Yoon, J2; Kim, J2; Lee, J2 The Catholic University of Korea, Medical College, Pediatric Allergy, Pucheon, Republic of Korea, 2Republic of Korea

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Background: Infants with respiratory syncytial virus (RSV) bronchiolitis are at increased risk of developing persistent wheezing and bronchial asthma later in life. Eosinophil-related-inflammatory mechanisms play a role in RSV bronchiolitis and asthma. Objective: The aim of our study was to find a relationship between eosinophil cationic protein (ECP), RANTES, eotaxin level in the nasophageal secreation of RSV bronchiolitis patients and subsequent wheezing. Method: ECP, RANTES, eotaxin level in nasopharyngeal secretion was determined in 28 children hospitalized with RSV bronchiolitis. 24 children were followed for two years after the RSV bronchiolitis. Result: Tweleve out of 24 children experienced subsequent wheezing verified by their physicians. Children who had subsequent wheezing show significantly higher level of ECP, RANTES, and eotaxin in the nasopharyngeal secretion than those who had not. There was a positive correlation between ECP and RANTES, ECP and eotaxin in RSV bronchiolitis patients. Conclusion: Nasopharyngeal ECP, RANTES, eotaxin may be useful in predicting the development of persistent wheezing in children affected by RSV bronchiolitis.

370 Increased risk of group A streptococcal upper respiratory trackt infections in pediatric atopic population Frey, D1; Li, X2; Jacobson, R1; Juhn, Y2 Mayo Clinic College of Medicine, Rochester, United States, 2Mayo Clinic, Rochester, United States

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Background: Our previous studies have demonstrated an increased risk of Invasive Pneumococcal Disease (IPD) and Group A Streptococcal (GAS) infections in pediatric asthmatic patients. The purpose of our study is to determine the influence of atopic disease other than asthma on the risk of developing Group A Streptococcal (GAS) infections of the upper respiratory tract during the first 18 years of life. 149


Methods: The study design was a retrospective cohort study which followed 340 subjects who participated in the Rochester Family Measles Study. We conducted comprehensive medical record reviews to determine atopic status, which was defined as a physician diagnosis of atopic dermatitis or eczema, allergic rhinitis, or hayfever. All laboratory test results of cultures, rapid antigen and PCR tests for GAS during the first 18 years of life were collected to compare the frequency of GAS episodes between atopic and non-atopic subjects. We also compared the frequency of negative GAS tests between the two groups. A Poisson regression model was fit to determine the association between atopic conditions and GAS infections. Results: Tweleve of 340 subjects, we enrolled 327 subjects who granted research authorization for medical record reviews. Of 327 subjects, 143 (44%) were positive for an atopic condition other than asthma. The incidences of GAS episodes for atopic and non-atopic subjects were 0.24 per personyear and 0.18 per person-year, respectively. The risk ratio adjusted for follow-up duration was 1.36 (95% CI: 1.10–1.67, P 5 0.004). The incidence of GAS episodes (P 5 0.79) and the incidence of tests for GAS infections (P 5 0.22) before and after diagnosis of asthma were not different. Other variables including gender, level of maternal education, and family history of asthma or atopy did not significantly influence the number of positive GAS episodes except Caucasian race and birth weight 42500 g. Conclusion: Children with atopic conditions other than asthma have an increased risk of upper respiratory GAS infection. This result does not appear to be due to a differential medical evaluation between individuals with and without atopic conditions (i.e. a detection bias). This result potentially suggests an immunogenetic mechanism underlying the risk of GAS infection (or colonization) in individuals with atopy, as opposed to a mechanism related to airway structure which may be more unique to asthmatic patients.

371 Epidemiological association of Tuberculosis infection with the allergy hygiene theory in Georgia Omiadze, K1; Khechinashvili, G2; Telia, A3; Machavariani, K3; Gogokhia, V3; Shavgulidze, M3; Mitskevich, N4 1 National Centre of TB and Lung Diseases, TB Department, Tbilisi, Georgia, 2Georgian Foundation of Tuberculosis and Lung Diseases, Tbilisi, Georgia, 3Tbilisi State Medical University, Allergy and Clinical Immunology, Tbilisi, Georgia, 4Iv. Javakhishvili Tbilisi State University, Immunology Department, Tbilisi, Georgia

Background: For a long time researchers have been interested in the effect of various 150

infectious pathologies on allergies. Aiming to find connections between the Hygiene Theory (HT) and a real life, investigators are mainly focused on developed country models. Developing countries are poorly studied, post-Soviet republics being a typical example. The HT has become an urgent issue in the post-Soviet countries, due to the high prevalence of tuberculosis (TB) and a dramatic rise in allergic diseases during the last two decades. Georgia is one of the clear examples of such an epidemiologic situation. Methods: The present case-control study involved weekly telephone calls to reveal and monitor allergic reactions. Extensive allergic examinations were conducted, if necessary. The Mantoux test was applied to detect M. tuberculosis infection (PPDRT23-Apisol, Parke-Davis, Morris Plains, NJ, USA). The LTD Biopharm ELIZA test system was used for specific IgE identification. The research data were statistically analyzed with the Epi-info software (Version 3.3.2., Atlanta, USA). Results: The frequency of atopic diseases in children of different age groups, infected with TB through family contacts, was distributed in the following way: 1998– 2001 years: children under 1 year – total of 53 cases for 2–7 years of follow-up; diagnosed atopic cases – 2. Children of 1–4 years – total of 123 cases for 2–7 years of followup; diagnosed atopic cases – 4. The confirmed frequency detected in the control group (uninfected children) was 10.1%. The frequency of diagnosed atopic diseases among patients infected through close TB contacts was much lower (3.4%) than in the uninfected children of the same age (10.1%). Conclusion: This study showed a statistically significant epidemiological inverse association between the family contact-related latent tuberculosis and the development of atopic diseases in children (0–4 years). Moreover, atopic diseases manifested later in TB-infected population compared to the uninfected community. The above-mentioned indicates that the TB infection in early childhood supresses atopic disease development and delays its clinical onset.

(MMR) vaccination is often refused by people having a notion that these infectious diseases are beneficial for a healthy development of a child’s immune system. This practice endangers herd immunity and is the cause of repeated outbreaks. As the clinical course of infections and also its possible impact on the development of atopy may be different in vaccinated and unvaccinated individuals, we explored in vaccinated and unvaccinated children associations of MMR infection with atopic disorders. Patients and methods: Using data from a previously conducted study on the relationship between the diphtheria-tetanus-pertussis-(inactivated) poliomyelitis vaccination in the first year of life and atopic disorders, the study population of 1872 8–12 year old was divided into children MMR-unvaccinated and children MMR-vaccinated in the first year of life. Within each group the association between MMR infections and atopic disorders (both as reported by the parents) was assessed. Results: We found a statistically significant positive association between measles infection and ‘‘any atopic disorder’’ (adjusted OR (95%CI): 1.77 (1.20–2.61)) in the MMRvaccinated group, mainly due to the relationship with eczema. For rubella there was a negative association with eczema and food allergy in the unvaccinated group: adjusted OR (95% CI): 0.57 (0.38–0.85) and 0.23 (0.07–0.76) respectively. All other associations were not statistically significant. Conclusions: We found a positive relationship between measles infection and any atopy in a group of MMR-vaccinated children and a negative association between rubella infection and eczema and food allergy in unvaccinated children. However, we cannot conclude that these relationships are causal. The negative association with rubella may be an artifact. This study shows no evidence for any protective effects from MMR diseases for the development of atopy and therefore supports conclusions found elsewhere that childhood vaccinations do not cause atopy.

373 372 Measles, mumps and rubella (MMR) infections and atopic disorders in MMRvaccinated and -unvaccinated children

Abstract withdrawn.

Bernsen, R1; van der Wouden, J2 United Arab Emirates University, Faculty of Medicine and Health Sciences, Community Medicine, Al Ain, United Arab Emirates, 2Erasmus MC – University Medical Center Rotterdam, General Practice, Rotterdam, the Netherlands

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Background: Vaccinations have been incriminated in the increase of atopic disorders. Especially the measles-mumps-rubella

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374 Imported subclinical house dust mite sensitisation in migrant populations from geohelminth-endemic developing countries Daschner, A; Elices, A; De Frutos, C; Valls, A Servicio de Alergia, Hospital Universitario La Princesa, Madrid, Spain

Background: It has been postulated, that parasite-burden in developing countries is inversely associated with atopy and asthma. The aim of this study was to compare the sensitisation profile of allergic immigrants proceeding from helminth endemic developing countries regions, with native patients of our region, in which no significant endemic helminth infection is known. Methods: We studied 49 consecutive immigrant patients from South-America and Philippines with rhinoconjunctivitis or bronchial asthma (RCBA) and 144 native RCBA patients in the same range of age (23–63 years). We analyzed the age of migration onset of RCBA symptoms. Skin Prick Tests (SPT) were performed against house dust mites (HDM, Dermatophagoides pteronyssinus, D. farinae, Tyrophagus putrescentiae), animal dander (cat, dog), 5 pollen mixtures of grass, tree and weed pollen and mould (alternaria alternata, Cladosporium herbarum). Results: Forty four out of fourty nine patients began to suffer RCBA after migrating to our region with a mean latency of 3.8 7 3.8 years. Mean age of symptoms initiation was 36.3 7 11.0 years and 30.6 7 8.8 years in the control group (P 5 0.005). Overall SPT reactivity was higher in immigrants than in native patients (95.5% vs. 75.0%; P 5 0.002). If only sensitised patients with RCBA were studied, HDM senistisation was significantly higher in immigrant patients (50% vs. 32%; P 5 0.04). 14/30 (46.7%) of immigrant patients with pollen associated only seasonal RCBA were also subclinically sensitised to HDM (only 21.7% of native patients with seasonal pollen associated RCBA; P 5 0.001). Conclusion: Due to geographic and climatic conditions, these results are to be interpreted in the special circumstance of a low prevalence of HDM sensitisation in Central Spain. The high prevalence of HDM sensitisation of our migrant patients could thus be due to previous sublinical sensitisation in their tropical and subtropical countries with more favorable conditions for the presence of different mite species, but other factors have to be taken into regard, like genetic factors or the higher prevalence of geohelminths, which could in turn be responsible for possible cross-reactive sensitisation against HDM.


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375 Allergy and common infectious diseases in Japanese women: baseline data from birth cohort study of national center for child health and development Ohya, Y1; Oishi, T1; Takayama, J2 National Center for Child Health and Development, Division of Allergy, Department of Medical Special, Tokyo, Japan, 2National Center for Child Health and Development, Department of Interdisciplinary Medicine, Tokyo, Japan

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Background: The persons with allergic diseases have been said to have some immunological characteristics. The aim of this study is to elucidate the association between allergic diseases and infections in Japanese pregnant women. Methods: This cross-sectional study included 1707 pregnant women who visited to the National Center for Child Health and Development in Tokyo Japan. They were asked to participate in this study by fulfilling the questionnaire including disease history, incidence of common cold and body weight and height. Current asthma, current atopic dermatitis and current allergic rhinitis were defined as being present if participants had history of receiving medication or treatment during the previous 1 year. Results: The past history of bladder infection was positively associated with current atopic dermatitis (OR: 1.353; 95% CI: 1.023–1.790) but was not with current asthma (OR: 1.596; 95% CI: 0.551–4.623), nor with current allergic rhinitis (OR: 1.281; 95% CI: 0.708–2.319). The history of tonsillectomy with recurrent tonsillitis was associated with their ever history of allergic rhinitis (OR: 1.804; 95% CI: 1.042–3.120), but was not with that of atopic dermatitis(OR: 1.014; 95% CI: 0.473–2.175), nor with that of bronchial asthma (OR: 1.896; 95%CI: 0.961–3.738). Annual frequency of being affected with common cold in the current asthmatics was higher than in the non current asthmatics (2.10 vs. 1.65), and that in ever asthmatics was higher in never asthmatics (1.90 vs. 1.63). No association was found between atopic dermatitis and common cold. Mean incidence of common cold after 12 years old in women with current allergic rhinitis was higher than in that without it (1.82 vs. 1.62). In women with ever allergic rhinitis, the incidence of common cold was also higher than in those without the history of allergic rhinitis (1.78 vs. 1.57). Conclusion: These findings suggest that pregnant women who had ever had asthma and/or allergic rhinitis might be prone to be affected with common cold. Bladder infection and recurrent tonsillitis showed association with current atopic dermatitis and ever allergic rhinitis, respectively.


376 Role of Staphylococcus aureus in severity of atopic dermatitis in children Kamali Sabzevari, S; Pourpak, Z; Shokouhi Shoormasti, R; Salehi, T; Moin, M Immunology, Asthma and Allergy Research Institute, Children Medical Center, Medical Sciences/Univers, Tehran, Islamic Republic of Iran

Background: Staphylococcus aureus has been identified as a possible trigger factor in atopic dermatitis (AD). S. aureus strains isolated from patients with AD are able to produce exotoxins with superantigenic properties, mostly staphylococcal enterotoxins B (SEB). To determine the relevance of IgE antibodies to SEB in children with AD and the severity of disease, this study was performed. Methods: Thirty children with AD entered this study; clinical severity was determined by the SCORAD index. Specimen for S. aureus culture was isolated from 3 different areas of the skin. Total serum IgE and IgE specific to SEB was measured by ImmunoCAP system. Results: Thirty children, 19 males and 11 females, the age ranged from 3 months to 8 years with AD entered this study. Among patients 13.7% were colonized with S. aureus producing staphylococcal enterotoxins B. Children colonized with toxigenic S. aureus strains had higher disease severity [SCORAD index of 65.1 7 18.5 in positive SEB vs. 23.9 7 16.9 in negative SEB group (P 5 0.005)]. Conclusion: Our results demonstrate a relationship between severity of disease in AD patients and IgE antibodies to SEB. Concerning to this association, it is strongly advised to perform prevention and treatment strategies for Staphylococcus aureus affecting in children with sever atopic dermatitis.

377 Sensitivity, specificity, positive and negative predictive values of diagnostic tests in Tricophyton allergy Palma-Carlos, A; Palma-Carlos, M Clinical Allergy Immunology Center, CAIC, Allergology, Lisbon, Portugal

Background: Tricophyton allergy has been reported 77 years ago. We have published in the last few years a large serie of confirmed cases. However the value of ‘‘in vivo’’ and ‘‘in vitro’’ diagnostic methods is not clear. We have determined the specificity, sensitivity, efficacy, positive and negative predictive values of current diagnostic tests in a group of 100 patients opposed to one equal control group. Material: One hundred patients with confirmed Tricophyton infection and allergy 151


have been studied. All these patients have a carefully clinical history and examination, skin tests in prick and intradermal for Tricophyton. Research of specific IgE has been done in 80 patients. A control group of 100 patients without clinical history of Dermatomycosis where skin tests and research specific IgE (80 patients) has been done was randomised from CAIC files. Statistical analysis has been calculated for sensitivity, specificity, efficacy, positive and negative predictive values for skin prick tests, intradermal tests and specific IgE. Intradermal tests have been reed at 20 min and 48 h and research of specific IgE done by UNICAP (Phadia). Results: Sixty seven patients had tinea pedis, 37 onychomicosis, 28 tinea cruris and 3 pruritus. Allergic manifestations were urticaria angioedema in 66, rhinitis in 15, asthma plus rhinitis in 11, eczema in 8 and conjunctivitis in 1. Prick tests were positive in 62 patients and intradermal in all 100. Specific IgE was positive in 54 out of 80 patients (67.5%). Skin prick tests and specific IgE were concordant in 63 cases and discordant in 17. In control group all tests were negative excepted delayed intradermal skin tests, positive in 58 cases. Best results for sensitivity (100%) were for intradermal tests, for specificity for prick tests and specific IgE (100%), for efficacy

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for intradermal tests (97%) for positive predictive value for prick tests and specific IgE (100%) for negative predictive value (100%) for intradermal tests. Discussion: A careful clinical history and clinical examination must be done in all patients with suspected Tricophyton allergy. Skin tests must be done in prick and intradermal and associated to the research of specific IgE for better results. Sensitivity is better for intradermal tests but results can be biased by false positives. Specificity is the same for skin prick tests and specific IgE. In practice skin tests in two steps, prick and intradermal and specific IgE must be combined for better diagnostic results. 378 Human immunodeficiency virus infection and acquired cold urticaria Popescu, F1; Dumitrescu, L2; Gheonea, C2 University of Medicine and Pharmacy, Department of Allergology, Bucharest, Romania, 2University of Medicine and Pharmacy Craiova, Department of Pediatrics, Craiova, Romania

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Acquired cold urticaria (ACU) has been previously described in three patients with human immunodeficiency virus (HIV) infection receiving zidovudine for significant CD4 cell depletion (but no AIDS-defining illnesses), and in one patient as a late

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cutaneous manifestation of AIDS, cryoglobulins, cryofibrinogen and cold agglutinins being not detected. The etiological role of HIV in the pathogenesis of cryoglobulinemia without ACU was suggested by the fact that antiretroviral therapy seems to decrease its prevalence. We present two cases of a HIV infection associated with a suggestive history of cold-induced urticaria. The first case is a heterosexual adult male with HIVassociated cryoglobulinemia and asymptomatic thrombocytopenia, in which ACU was the major presenting clinical manifestation that led to the discovery of HIV infection. The second case was a child with no family history of cold urticaria, and no personal history of conjunctivitis or arthralgias after general exposure to cold. In both cases we used the ice-cube test and the cold stimulation time test in the diagnostic workup, and there was no evidence of other coinfections that may be associated with ACU, such as hepatitis C and B viral infections, infectious mononucleosis, borreliosis, syphilis, or toxoplasmosis. The cold-induced cutaneous manifestations were treated with oral nonsedating H1-antihistamines.


Poster Discussion Session 15 – Highlights in Dermatological Research

Poster Discussion Session 15 Highlights in Dermatological Research 379 Long-term observation on clinical course and biochemical alterations in aspirinprecipitated skin reactions Podolec-Rubis, M1; Gielicz, A2; Setkowicz, M1; Mastalerz, L2; Sanak, M2; Szczeklik, A2 1 Jagiellonian University School of Medicine, Department of Dermatology, Krakow, Poland, 2Jagiellonian University School of Medicine, Department of Medicine, Krakow, Poland

Rationale: Special regulatory role of cysteinyl leukotrienes and prostaglandin D2 has been postulated in aspirin-induced urticaria. Objectives: Clinical observation was carried out during 4 years in patients with chronic idiopathic urticaria and aspirin hypersensitivity. Urinary levels of cysteinyl leukotrienes and prostaglandin D2 were measured at baseline and following aspirin challenge. Methods: The study population consisted of 22 patients with chronic idiopathic urticaria and aspirin hypersensitivity. Aspirin challenges were performed in 2002 (all positive) and repeated in 2006. Urinary concentrations of: 1) leukotriene E4 using immunoassay and 2) main prostaglandin D2 metabolite, 9a`11aˆPGF2, using gas chromatography/mass spectrometry were measured at the same time points. Measurements and main results: During 4 years observation period both the frequency and severity of spontaneous urticaria has decreased. Out of 22 patients who had positive aspirin challenge in 2002, 14 (63.6%) had positive reaction again in 2006. There was a tendency for increased urinary leukotriene E4 excretion after aspirin challenge in patients with positive result of aspirin test. Urinary leukotriene E4 concentration after aspirin challenge correlated with intensity of skin eruptions. At baseline main prostaglandin D2 metabolite value did not differ between the patients with positive and negative aspirin challenge. Following aspirin provocation, prostaglandin D2 metabolite increased in patients with positive aspirin challenge. The dose of aspirin had no effect on the magnitude of the response on the leukotriene E4 and prostaglandin D2 metabolite. Conclusions: Aspirin hypersensitivity manifesting as urticaria/angiooedema is characterized by fluctuations. Positive aspirin challenge is still observed in about 2/3 patients after 4 years. Aspirin-precipitated skin reactions might associate with changes in the urinary secretion of leukotriene E4 and prostaglandin D2 metabolite. Aspirin r 2008 The Authors Journal Compilation

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enhances urinary leukotriene E4 excretion in those patients.

380 Allergic contact dermatitis in swimming pool users Dalmau, G1; Ga´zquez, V1; Vilaplana, J2; Gaig, P1; Go´mez, C1; Navarro, S1; Canadell, L3 1 Hospital Universitari de Tarragona Joan XXIII, Allergy Unit, Tarragona, Spain, 2Hospital Clıńic de Barcelona, Dermatology Department, Barcelona, Spain, 3Hospital Universitari de Tarragona Joan XXIII, Pharmacology Department, Tarragona, Spain

Background: Bromine is a halogen that is particularly suitable for disinfecting water in swimming pools and spas, because it is efficient at eliminating not only bacteria, virus and fungi, but also organic impurities. Users of swimming pools disinfected with bromine often have problems of irritative contact dermatitis. The aim of this study was to assess whether bromide products used at swimming pools and spas can cause allergic contact dermatitis among its users. Methods: We report eight cases of users presenting eczematous cutaneous injuries after swimming in pools disinfected with bromine tablets (1,3-dimethylol-5,5-dimethyl hydantoin). Patch tests were performed with Spanish standard series (GEIDC) and also with bromine tablets (water solution at 0.03, 0.3 and 3 mg/L and petrolatum at 1%, 5% and 10%) and dimethylol dimethyl hydantoin 2% water solution commercial extract. Patch tests with bromine tablets (water solution at 0.03, 0.3 and 3 mg/L and petrolatum at 1%, 5% and 10%) were perfomed on ten healthy controls (five atopic). Results: The patch tests performed with Spanish standard series (GEIDC) were positive in eight patients: nickel (5), cobalt (4), chrome (2), formaldehyde (1), quaternium (1), paraphenylenediamine (PPD) mix (1), thiomerosal (1), carba mix (1). The patch tests performed with bromine tablets were positive in eight patients for the 1%, 5% and 10% preparation of petrolatum, in five for the 3 mg/L water solution and in two for the 0.3 mg/dL water solution. All healthy controls were positive to the 10% petrolatum preparation and five of them were also positive to the 5% preparation. Conclusions: We report eight cases of allergic contact dermatitis as a result of exposure to 1,3-dimethylol-5,5-dimethyl hydantoin when used as a disinfectant for swimming


pools. One of the cases presents contact dermatitis for formaldehyde and quaternium because these products are released and/or cross-react with 1,3-dimethylol-5,5dimethyl hydantoin. Petrolatum preparations of this product at 5% and 10% were irritative. In view of the increasing use of bromide products to disinfect water, we think that these sensitisations should be taken into account in users of swimming pools and thermal centers (spa).

381 Nickel biomonitoring and nickel contact dermatitis Fedorov, M1; Darsow, U1; Schwegler, U2; Twardella, D2; Schaller, K3; Habernegg, R2; Fromme, H2; Ring, J1; Behrendt, H4 1 Technical University Munich, Department of Dermatology and Allergy Biederstein, Munich, Germany, 2 Bavarian National Office for Health and Food Security (LGL), Oberschleissheim, Germany, 3Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine, Erlangen, Germany, 4Technical University Munich, Division of Environmental Dermatology and Allergy, Munich, Germany

Background: Nickel is the most frequently detected cause of allergic contact dermatitis. An ingestion of nickel (500ı` g/day) may lead to flares of nickel contact dermatitis. Methods: We examined the systemic nickel load by determination of nickel in the urine of 164 female volunteers (age 18 to 46 years) with and without nickel contact dermatitis diagnosed in a university allergy unit. The influence of age, nutrition (e.g. food additives), dermatologic factors (atopic dermatitis, nickel contact dermatitis) and nickel exposure by patch test were recorded prospectively using questionnaires and hospital files. Nickel was measured using atomic absorption spectrometry (AAS) with two different standardized methods (IPASUM and LGL). Results: Comparing the methods, the IPASUM assay was established as standard. The limit of determination of 0.2 mg nickel/L was exceeded by all samples. The 95%-percentiles of urine nickel content were 3.77 mg/L (age group 18–30 years) and 3.98 mg/L (31– 46 years). Bivariate analyses pointed to a significantly increased nickel load with increasing age, ingestion of food additives, drinking stagnation water and consumption of nickel-rich food. In the multivariate analysis stagnation water and nickel-rich food were no longer significant. Nickel

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patch tests increased temporarily the urine nickel level (not significant). Conclusions: We showed that the 95%percentile of the nickel load of our collective markedly exceeded the 3 mg/L reference value of the German human biomonitoring commission. Age and consumption of food additives are the most significant factors of influence. Use of stagnation tap water can contribute to the total load. Patients with atopic eczema show nickel concentrations not different from non-atopic controls. Nickel sensitisation does not affect the current nickel content in the urine.

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Abstract moved to 891b.

383 Associations of TNF-alpha gene polymorphisms and mastocytosis. A study of the European Competence Network in Mastocytosis (ECNM) Nedoszytko, B1; Niedoszytko, M2; Lange, M1; van Doormaal, J3; Glen˜, J1; Zab3otna, M1; Renke, J4; Varkonyi, J5; Jassem, E2; Roszkiewicz, J1; Hidvegi, B5; Valent, P6 1 Medical University of Gdansk, Department of Dermatology, Gdansk, Poland, 2Medical University of Gdansk, Department of Allergology, Gdansk, Poland, 3 University Medical Center, Department of Allergology, Groningen, the Netherlands, 4Medical University of Gdansk, Department of Pediatrics, Gdansk, Poland, 5 Semmelweis University, Department of Internal Medicine, Budapest, Hungary, 6Medical University of Vienna, Department of Internal Medicine I, Vienna, Austria

Background: Mastocytosis (MC) is a heterogenous disease involving mast cells and their progenitors with still unknown pathogenesis. Among the predisposing factors may be TNF-a one of the major mast cell derived mediator. Since certain allelic variants of TNF-a gene are associated with increased or decreased production of the TNF-a, the disturbed cytokine balance may be under genetic control. Aim: In our study we evaluated the frequency of TNF-a-238 G/A and -308 G/ A promoter polymorphisms in mastocytosis patients and in healthy controls. Materials and methods: Out of 114 patients with mastocytosis, 64 (55%) were from Poland, 26 (23%) from the Netherlands, 19 (17%) from Austria, and 5 (5%) from Hungary. The study group included 85 adults including 57 (67%) women and 28 (33%) men with a mean age of 44 years (range 18–80 years); and 29 children including 14 (48%) women and 15 (52%) men, with a mean age of 5 years (range 0.5–17 years). The control group consisted of 59 (44%) women and 77 (56%) men with a mean age 32 years (range 20–60). The most

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prevalent diagnosis was indolent systemic mastocytosis (ISM), found in 58 (51%), followed by cutaneus mastocytosis (CM) in 44 (39%), monoclonal mast cell activation syndrome (MMAS) in 3 (2%), and aggressive systemic mastocytosis (ASM) in 8 (8%). Diagnoses were established according to WHO criteria. The polymorphism-238 G/A and 308 G/A in the promoter region of TNF-a gene was determined by ARMSPCR method. Results: The TNF-a-238 A allele was associated with a significantly increased risk of mastocytosis (OR 5 3.3; 95%CI 5 1.4–7.2; P 5 0.002) compared to the TNF-a 238 G allele. The frequency of the TNF-a 238 A allele was significantly increased in mastocytosis patients compared with the controls (19.3% in adults MC patients and 25% in children MC vs. 7.4% in the controls, P 5 0.008). No association was found between TNF-a-308A allele frequency and mastocytosis. Conclusion: The results showed the strong association between-238 G/A TNF-a promoter gene polymorphism and mastocytosis.

384 The relationship between posttraumatic stress disorder (PTSD), psychiatric comorbidity and personality traits among patients with chronic idiopathic urticaria Symons, C1; Chung, M2; Gilliam, J3; Kaminski, E1 Derriford Hospital, Department of Clinical Immunology and Allergy, Plymouth, United Kingdom, 2University of Plymouth, Clinical Psychology Teaching Unit, Plymouth, United Kingdom, 3Derriford Hospital, Department of Clinical Immunology and Allergy, Plymouth, United Kingdom

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Background: Several studies have highlighted a link between trauma exposure and dermatological disorders, but no empirical studies have established a relationship between posttraumatic stress disorder (PTSD) and chronic idiopathic urticaria (CIU). Aims: To investigate 1) whether CIU patients are affected by PTSD, 2) whether CIU patients with PTSD experience more severe psychiatric co-morbidity than those without PTSD and 3) the relationship between personality traits, PTSD, the severity of CIU and psychiatric co-morbidity among CIU patients. Methods: One hundred patients with CIU and 60 with allergy were recruited for the experimental and control groups respectively from an Immunology clinic in the UK. They completed surveys and the severity of CIU was assessed using a local variation of a scoring system devised by EAACI in 1994. Results: 1) 34% of patients with CIU and 18% with allergy met the diagnostic criteria for PTSD. Patients with CIU were 2.31 Journal Compilation

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times more likely to have a current diagnosis of PTSD resulting from past traumas than allergy patients. 2) Controlling for life event stress and perceived stress, there were significant differences between groups (CIU PTSD, CIU no-PTSD, allergy PTSD, allergy no-PTSD) in somatic problems, anxiety, and social dysfunction. The level of depression was similar between groups. 3) Regression analyses showed that, controlling for perceived and life event stress, no association was found between personality traits, PTSD diagnosis and the severity of CIU. Perceived stress in model 1 contributed to predicting co-morbidity. After controlling for perceived stress, model 2, with PTSD diagnosis entered, improved significantly the prediction of co-morbidity. After adjusting for the stress variables in model 1 and PTSD diagnosis in model 2, model 3, with personality variables entered, did not significantly improve the prediction. Regression coefficients showed that neuroticism contributed to predicting co-morbidity. A moderator effect found that neuroticism interacted with PTSD diagnosis in predicting co-morbidity: CIU patients’ neuroticism moderated the impact of PTSD on co-morbidity. Conclusion: Patients with CIU can have concurrent PTSD resulting from past traumas implying that traumatic memories or emotions could be manifested through skin as CIU. They also tend to develop psychiatric co-morbidity related to their different degrees of PTSD severity and neuroticism.

385 A usefulness of atopy patch tests in children suffering from atopic eczema as compared to the tests of immediate hypersensitivity Liska, M; Gutova, V; Hess, Z; Panzner, P University Hospital Pilsen, Department of Immunology and Allergology, Pilsen, Czech Republic

Background: In addition to common used testing of immediate hypersensitivity (skin prick-tests, specific IgE), atopy patch tests (APT) are increasingly carried out in patients suffering from atopic eczema to detect delayed type hypersensitivity reactions. Methods: We tested a group of 32 children with atopic eczema for hypersensitivity to five common airborn allergens (birch and grass pollen, cat dander, house-dust mites) using skin prick test (SPT), specific IgE and APT. Results of these tests were then compared with patients’ personal history of eczema exacerbations. Results: Delayed type hypersensitivity to tested allergens (positive APT) was proven in 21 children (66%), immediate type hypersensitivity (positive SPT and/or specific IgE) in 17 children (53%). APTs alone



were positive in 7 patients (22%), whereas SPT and/or specific IgE alone were positive in 5 patients (16%). Both types of hypersensitivity were found out in 15 patients (47%). The results of APT correlated better (13 patients; 41%) with personal history of eczema exacerbations as compared to SPT and/or specific IgE (8 patients; 25%). Conclusion: Atopy patch testing could elucidate the mechanisms contributing to atopic eczema exacerbations. It represents a perspective diagnostic tool with a great potential for improving the care for atopic eczema patients.

386 Evaluation of the photopatch test. Experience of 3 years (2004–2007) Blanco Alberca, C; Garcıá Ortiz, J; Hernandez Arauzo, N; Sanchez Palla, P; Vega Gutierrez, J; Sanchı´s Merino, M; Fernańdez Corte´s, S Hospital Rio Hortega, Allergy Department, Valladolid, Spain

Background: The main aim of this study was to identify the causing photoallergens of photoallergic dermatitis in the population in Allergy Department. Moreover, we wanted to know the current situation of the photoallergy in our area and verify if the photoallergens we use are the most suitable and if it would be useful to add new substances. Material and methods: A total of 44 patients were studied with the photopatch test during 3 years. We gathered the information of the following variables: age, sex, personal medical history, location of the injuries, implied substance and route of administration, time of latency, results of the diagnostic tests and clinical relevancy. We use the standard battery of the Spanish Group of Photopatch (GEF) except for the solar filters mix that was replaced with a specific solar filters battery. We apply these photoallergens in the back of the patients for duplicate, radiating one of the series at 48 h with 5 J/ cm2 of UVA, the readings were taken at 48 and 96 h. Results: Fifteen out of 44 patients (34.09%) presented one or more positive photopatch test, the ages between 21 and 79 years (average: 43.29). Twelve patients were males and 32 female patients (relation 2.66 : 1). No significant statistical difference was observed between the two sexes and the positivity of the photopatch.The most frequent detected photoallergens were nonsteroidal antiinflammatory drugs (45.16% of the positive photopatch tests), particularly the ketoprofen, followed by the promethazine (16.13%) and chlorpromazine (16.13%). We only obtained 2 positive cases to solar filters (6.45%).The route of administration of the suspicious substances was in most cases topic, and the most frequent location r 2008 The Authors Journal Compilation

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of the injuries were photoexposed areas. Of the 31 positive photopatch tests, we consider positive relevancy in 16 (51.6%). Conclusions: 1. Our results confirms that nonsteroidal antiinflammatory are the substances more frequently implied in reactions of photoallergy in our population, and particularly the ketoprofen, which is in concordant with the information exposed in the medical literature. 2. The number of positive tests to solar filters has been low in our population, as a result we believe it is suitably to extend the battery of solar filters to test our patients. 3. The statistical analysis of the relation between the sex and the positivity of the photopatch indicates that there is no significant difference.

387 Latex allergy: new insights to explain different sensitisation profiles in different risk groups Peixinho, C1; Tavares-Ratado, P2; Toma´s, M3; TabordaBarata, L1; Tomaz, C1 1 University of Beira Interior, CICS – Centro de Investigacaõ em Cieˆncias da Sau´de, Covilha˜, Portugal, 2Sousa Martins Hospital, Laboratory of Clinical Pathology, Guarda, Portugal, 3Guarda Healthcare Centre, Guarda, Portugal

Background: Latex sensitisation is recognized as a serious health problem since latex products are increasingly used, especially among health care workers (HCW) and patients undergoing multiple surgical interventions, such as spina bifida patients. Differences in latex allergen sensitisation profiles have been described between both groups. Major allergens for spina bifida patients are Hev b 1, 3 and 7, while for HCW, major allergens are Hev b 2, 5, 6.01 and 13. The reason for these differential sensitisation profiles is currently unknown. The aim of this study was to investigate latex allergen profiles on internal and external surfaces of natural rubber latex gloves. Methods: Eighty-two samples of commonly used surgical gloves (41 glove brands), were used for analysis. Specific allergen levels of Hev b 1, 3, 5 and 6.02 on both surfaces of the gloves were quantified using an enzyme immunometric assay, a FITkits. Results: Differences in allergen levels were observed between internal and external surfaces of all glove types. Concentrations of Hev b 1 and Hev b 3 were significantly higher on external surfaces, while internal surfaces had higher allergen levels of Hev b 5 and Hev b 6.02 (Po0.001, in both cases). Analysis of surgical and examination, powdered and non powdered gloves also showed that the content of Hev b 5 and Hev b 6.02 was significantly higher on internal surfaces while that of Hev b 1 and Hev b 3 was higher on external surfaces.


Conclusion: Our study showed different allergen profiles on internal and external surfaces of natural rubber latex gloves. These results may suggest a relationship between latex allergen localization and sensitisation routes in different risk groups.

388 Association of diamine oxidase polymorphisms with low diamine oxidase serum activity and histamine intolerance Maintz, L1; Rodriguez, E2; BuXmann, C1; Fimmers, R3; Bieber, T1; Weidinger, S4; Novak, N5 1 University of Bonn, Department of Dermatology and Allergy, Bonn, Germany, 2GSF-National Research Center for Environment and Health, Institute of Epidemiology, Neuherberg, Germany, 3University of Bonn, Department of Medical Biometry and Epidemiology, Bonn, Germany, 4 Technical University Munich and GSF-National Research Center for Environment and Health, Department of Dermatology and Allergy, ZAUM, Munich, Germany, 5GSF-National Research Center for Environment and Health, Neuherberg and University of Bonn, Department of Dermatology and Allergy, Bonn, Germany

Background: Histamine intolerance (HIT) results from a disequilibrium of accumulated histamine and the capacity for histamine degradation. Diamine oxidase (DAO) represents the main enzyme for the metabolism of extracellular histamine whereas intracellular histamine seems to be catabolised by histamine-N-methyltransferase (HNMT). An impaired histamine degradation based on a reduced DAO activity and the resulting excess of histamine may cause numerous symptoms mimicking an allergic reaction. However, the genetic background of HIT has not been investigated yet. Objectives: To evaluate the association of DAO and HNMT single nucleotide polymorphisms (SNP) with low DAO serum levels and clinical symptoms of HIT in a total of 372 individuals including patients with clinical symptoms of HIT, low-DAO activity as well as respective controls. Methods: The phenotype of the subjects has been characterized with a standardized questionnaire evaluating HIT symptoms. DAO serum activity has been determined with radioextractionassays using [3 H]-labelled putrescine-dihydrochloride as a substrate. Genomic DNA was extracted from leukocytes. Assays for SNP genotyping were developed for 13 DAO and 1 HNMT tagging SNPs and genotyping was carried out using the MassARRAY system. Results: The clinical phenotype HIT was significantly associated with 3 SNPs in the DAO gene; DAO 1 in the promoter region (P 5 0.026, OR 3.24 [95% CI 1.02, 10.35]), DAO 2 in Intron 1 (P 5 0.005, OR 3.78 [95% CI 1.35, 10.6] and DAO 3 in Exon 2 (P 5 0.006, OR 3.75 [95% CI 1.34, 10.5]). Moreover, an impaired enzymatic function with reduced DAO activities was also

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associated with these SNPS: DAO 1: P 5 0.017, OR 3.21 [95% CI 1.12, 9.15], DAO 2 P 5 0.001, OR 4.31 [95% CI 1.69, 11.03]), DAO 3 P 5 0.001, OR 4.26 [95% CI 1.67, 10.88] and with another SNP, DAO 4 in Exon 2 (P 5 0.004). Conclusion: SNP in the DAO gene are associated with an impaired DAO activity and symptoms of HIT. These data provide the first evidence for a genetic predisposition for low DAO serum levels and impaired histamine degrading capacity.

389 Comparison between autologous serum skin test and autologous plasma skin test as a diagnostic method in patients with chronic urticaria Nunez-Acevedo, B; Rubio-Perez, M; Garcia-Sifuentes, L; Gonzalez-Gutierrez, M; Martinez-Cocera, C; FernandezRivas, M Hospital Clinico San Carlos, Allergy Department, Madrid, Spain

Background: The prevalence of chronic urticaria in Spain is 2.9%, and 60% is considered idiopathic. The intradermal test with autologous serum is the most useful diagnostic method obtaining positive results between 25–50%. This percentage is increased until 95% if autologous plasma test is used. Objective: To compare the intradermal test with autologous serum (ASST) and with autologous plasma (APST) in patients diagnosed of chronic urticaria. Material and methods: Thirty one patients diagnosed of chronic urticaria were included. Chronic urticaria was diagnosed on the basis of the appearance of continuous or recurrent hives with or without angioedema for more than 6 weeks and with symptoms in the last 6 months. Blood test, thyroid hormones, hepatitis serology, Immunoglobulins (IgA, IgE, IgG, IgM), complement, parasites in stools, autoantibodies and specific IgE for Anisakis, tested in all patients were negative or within normal limits in the great mayority. All the patients underwent intradermal test with 0.05 mL of autologous serum and plasma and with saline and histamine as negative and positive controls respectively. The appearance of a weal redness with a diameter greater than 1.5 mm over the saline-induced response at 30 min was considered as positive. Results: 2/31 (6.5%) patiens scored positive on ASST and 12/31 (38.7%) patients scored positive on APST (Po0.002). – 8/11 patients with active urticaria (72.7%), scored positive on APST and only 4/20 patients with inactive uritcaria (20%), scored positive on APST (P 5 0.007). – 8/9 patients who had an associated angiedema (88.9%) scored positive on APST and only

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4/22 patients without angioedema (18.2%) scored positive on APST (Po0.001). Conclusion: In our patients, the APST has a higher sensitivity than ASST and therefore it is more useful to evaluate autoimmunity in patients with chronic urticaria. Positive results in APST seem to be associated with the activity of the urticaria and the presence of associated angioedema.

390 Validation of the Chronic Urticaria Quality of Life questionnaire (CU-Q2oL) in a Spanish patient population Valero, A1; Herdman, M2; Ferrer, M3; Jaúregui, I4; Bartra, J1; Da´vila, I5; del Cuvillo, A6; Montoro, J7; Mulllol, J8; Sastre, J9 1 Hospital Clinic, Unidad de Alergia, Barcelona, Spain, 2 IMIM-Hospital del Mar, Unitat de Recerca en Serveis Sanitaris, Barcelona, Spain, 3Clinica Universitaria de Navarra, Departamento de Alergia, Pamplona, Spain, 4 Hospital de Basurto, Servicio de Alergologıá, Bilbao, Spain, 5Hospital Clıńico, Servicio de Inmuno-Alergia, Salamanca, Spain, 6Clıńica Dr. Lobatoń, Alergia, Cadiz, Spain, 7Hospital La Plana de Villarreal, Unidad de Alergia, Castelloń, Spain, 8Hospital Clinic, Unidad de Rinologia, Barcelona, Spain, 9Fundacioń Jimeńez Dıáz, Servicio de Alergia, Madrid, Spain

Background: The aim of this study was to test the psychometric properties of a Spanish version of the Chronic Urticaria Quality of Life questionnaire (CU-Q2oL). Methods: After translation into Spanish, the CU-Q2oL was administered to patients with chronic urticaria aged 18 years and over. Feasibility was assessed by analyzing missing responses and ceiling and floor effects. Reliability was tested by examining internal consistency (Cronbach’s alpha). Construct validity was analyzed by examining convergent and discriminant validity with the Skindex-29 and by evaluating the CUQ2oL’s ability to discriminate between patients classified on clinical measures of according to a clinical classification of severity. Sensitivity to change was analyzed in a sub-group of patients who completed a second visit 4 weeks after baseline. Results: A total of 695 patients were included for analysis. Mean age (SD) was 42.4 (SD 15.0) years and 62.1% of the sample was female. 91.9% of the sample answered all of the items on the CU-Q2oL. Over 15% of patients scored at the floor (best possible health) on 5 of the 6 dimensions. Cronbach’s alpha coefficients were 40.80 for all dimensions of the CU-Q2oL, and 0.86 for the overall score. The CU-Q2oL correlated as expected with the Skindex-29, and discriminated between groups with different clinician-rated severity of wheals and itching (mean [SD] overall scores of 18.6 [13.5] and 30.4 [19.9] for mild vs. severe wheals, and 14.8 [11.1] and 26.6 [19.1] for mild vs. severe itching, respectively). The questionnaire was sensitive to change, with an effect size of 1.0

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for the overall score in patients reporting an improvement on a health transition scale. Conclusions: The Spanish version of the CU-Q2oL has shown satisfactory reliability, validity, and sensitivity to change and can be used as an outcome measure for chronic urticaria patients in clinical and research settings.

391 Increased secretion of platelet specific mediators in patients with delayed pressure urticaria Kasperska-Zajac, A; Brzoza, Z; Rogala, B Medical University of Silesia, Internal Diseases, Allergology and Clinical Immuno, Zabrze, Poland

Background: Little is known about inflammatory mediators involved in delayed pressure urticaria (DPU). Objective: To investigate secretion of platelet specific chemokines, platelet factor 4 (PF-4) and beta-thromboglobulin (beta-TG) in the course of DPU. Methods: Plasma concentration of PF-4 and beta-TG was measured in 8 adult patients with DPU and age and sex-matched 15 healthy controls. Results: Plasma PF-4 and beta-TG concentration was significantly increased in the DPU group compared with the control subjects. Conclusion: The present study as well as the earlier contribution, suggest that distinct platelet activity may be identified in different types of urticaria. Contrary to chronic idiopathic urticaria, chronic urticaria with positive response to autologous serum skin test and acute urticaria, delayed pressure urticaria may be associated with increased secretion of platelet chemokines, similar to that observed in cold urticaria.

392 Expression of tissue factor by eosinophils in patients with chronic urticaria Cugno, M1; Marzano, A2; Tedeschi, A3; Fanoni, D2; Venegoni, L3; Asero, R4 1 IRCCS Foundation Maggiore Policlinico Hospital, Mangiagalli, Regina Elena, Department of Internal Medicine, Milan, Italy, 2IRCCS Foundation Maggiore Policlinico Hospital, Mangiagalli, Regina Elena, Institute of Dermatological Sciences, Milan, Italy, 3IRCCS Foundation Maggiore Policlinico Hospital, Mangiagalli, Regina Elena, Allergy and Clinical Immunology Unit, Milan, Italy, 4Clinica San Carlo, Ambulatorio di Allergologia, Paderno Dugnano, Italy

Background: Although chronic urticaria (CU) is regarded as an autoimmune disorder mediated by histamine-releasing autoantibodies, an activation of blood coagulation via tissue factor (TF) and a strong expression of TF in lesional skin have been described. Eosinophils, which are involved in CU skin lesions, have been recently



demonstrated as the major source of TF in human blood. In this study we assessed whether eosinphils are the cellular source of TF in CU skin lesions. Methods: Ten patients with severe CU were studied. Skin biopsy specimens were taken from wheals of more recent onset. The control group consisted of 10 biopsy specimens of perilesional normal skin from different types of skin tumours. TF expression was evaluated by immunohistochemical methods using an anti-TF monoclonal antibody. Co-localization of TF and eosinophil cationic protein (ECP), a classic cell marker of eosinophils, was investigated by double staining studies using two specific monoclonal antibodies in the 4 specimens showing the highest TF reactivity scores. Results: All specimens from patients with CU clearly showed tissue factor expression that was absent in all normal control specimens (P 5 0.0001). The double staining experiments for TF and ECP clearly showed that the TF-positive cells were eosinophils. Conclusions: Eosinophils are the main source of TF in CU lesional skin. This finding highlights the role of these cells in the pathophysiology of CU and might pave the way for new therapeutic strategies.

393 Prognosis of mastocytosis: a long-term study of the Spanish network on mastocytosis (REMA) in a series of 186 Patients Escribano, L1; A´lvarez-Twose, I1; Sańchez-Munõz, L1; Jara, M2; Nuń˜ez, R3; Garcıá-Montero, A2; Teodosio, C2; Garcıá-Cosıó, M4; Bellas, C5; Orfao, A2 1 Hospital Virgen del Valle, Centro de Estudios de Mastocitosis de Castilla La, Toledo, Spain, 2Universidad de Salamanca, Department of Medicine and Cytometry, Salamanca, Spain, 3Hospital Ramoń y Cajal, Servicio de Hematologıá, Madrid, Spain, 4Hospital Ramoń y Cajal, Anatomıá Patolo´gica, Madrid, Spain, 5Hospital Puerta de Hierro, Departamento de Anatomıá Patolo´gica, Madrid, Spain

Background: Mastocytosis are ‘‘Rare Diseases’’ for which reliable predictors of progression have not been identified, information on its prognosis being limited. Methods: We analyzed 186 patients who were directly followed at the Spanish Network on Mastocytosis from January 1984 through April 2007. Primary end points of the study were clinical progression and overall survival as defined by transformation into a more aggressive form of the disease and death, respectively. Results: Multivariate analysis of prognostic factors for clinical progression showed that


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among good prognosis patients (cutaneous mastocytosis ‘‘CM – and indolent systemic mastocytosis ‘‘ISM’’) and ISM in particular, Beta2-microglobulin serum levels together with the bone marrow (BM) mast cell (MC) burden (41% and 30% of BM MC as assessed by flow cytometry- and histopathology, respectively), was the best combination of independent parameters for predicting progression into a more advanced form of the disease; the cumulative probability of clinical progression in good-prognosis was of 0% at 5 years, 0.9% at 20 years and 7.1% at 25 years. Interestingly, we found evolution of ISM to smoldering systemic mastocytosis (SSM) to be also rather infrequent with no clear impact on the prognosis of the disease. Thus, our results indicate that in ISM, more attention should be paid to B2 M serum levels and the number of BM MC. Regarding overall survival, the best predictive model for the whole patient series included the diagnostic subtype of the disease and the BM MC burden with a cumulative probability of death of 2% versus 61% at 10 years and of 2% and 100% at 15 years in the goodversus poor-prognosis categories, respectively. Till the moment of closing this study, 6 good-prognosis patients (4%) had died, all corresponding to ISM patients. Among these patients, death was directly related to the MC disorder in only one case, corresponding to a female who developed a chemorefractory CD117-positive AML when she was 37 years old, 21 years after disease onset. Of note, in this patient somatic D816 V KIT mutation was found in all hematopoietic cell lineages, including MC, as well as in oral mucosal cells. Conclusions: Our results clearly show that current classification of mastocytosis provides criteria for the prognostic stratification of the disease, the good-prognosis forms of mastocytosis having a long life expectancy, which should prevent the use of cytoreductive or novel targeted drugs in these cases.

data documenting their immunological profile and clinical status during the first three years of life are available. The increase of immunoglobulin free light chains was recently described in several pathological conditions including asthma. Free immunoglobulin light chains pathology is classically associated with monoclonal gammapathies. Its association with allergic disorders is surprising and unexplained. We therefore tested the above-mentioned cohort for the level of free kappa and lambda chains and correlated the results with clinical status and relevant laboratory markers. Methods: One hundred patients with severe form of AD, all children from birth to 3 years of age, and 102 healthy age matched controls were included in a prospective study. Ninety serum samples from each group were available for analysis. Light chains in sera were tested by Freelite assay (Binding Site, UK). Results: There was highly significant difference in both kappa (3.22 and 7.05 mg/L) and lambda (9.8 and 10.99 mg/L) serum levels between controls and patients, respectively (Po0.0001). The kappa/lambda ratio in allergic patients (0.64) was significantly higher than in controls (0.33) (Po0.0001). We could not confirm any association with age, IgE levels or severity of atopic dermatitis. Conclusion: The excess of free light chains is usually connected with plasma cell disorders and also with renal pathology. There are several recent reports documenting the increase of light chains in autoimmune and also allergic diseases. Here we document the increase of both kappa and lambda light chains and their ratio in children with severe atopic dermatitis. As no correlation with laboratory or clinical markers was found so far, the significance of such plasma cells activity in atopic dermatitis remains to be elucidated.

393b Increase of serum immunoglobulin free light chains in children with atopic dermatitis Sediva, A; Kayserova, J; Vernerova, E University Hospital Motol, Institute of Immunology, Prague, Czech Republic

Background: In a prospective study we follow a cohort of children with severe atopic dermatitis (SCORAD 50–80). The


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