Ocular surface and tear functions after topical cyclosporine ... - Nature

Bone Marrow Transplantation (2008) 41, 293–302 & 2008 Nature Publishing Group All rights reserved 0268-3369/08 $30.00

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ORIGINAL ARTICLE

Ocular surface and tear functions after topical cyclosporine treatment in dry eye patients with chronic graft-versus-host disease Y Wang1,2,3, Y Ogawa1, M Dogru1,3,4, M Kawai1, Y Tatematsu1, M Uchino1, N Okada1, A Igarashi1, A Kujira1, H Fujishima1, S Okamoto5, J Shimazaki1,4 and K Tsubota1 1 Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan; 2Department of Ophthalmology, Eye and ENT Hospital of Fudan University School of Medicine, Shanghai, China; 3J & J Ocular Surface and Visual Optics Department, Keio University School of Medicine, Tokyo, Japan; 4Department of Ophthalmology, Tokyo Dental College, Chiba, Japan and 5Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan

We investigated the effect of 0.05% topical cyclosporine (Cys) on the ocular surface and tear functions in dry eye patients with chronic GVHD (cGVHD) in a prospective comparative study. Thirty eyes of 15 patients refractory to baseline treatment were recruited and the patients assigned for topical Cys treatment group (14 eyes of 7 patients) and control group (12 eyes of 6 patients) respectively. Two patients dropped out because of intolerable irritation while using topical Cys eye drops. Visual analog scale symptom scores, corneal sensitivity, Schirmer I test value, tear film break-up time (TBUT), tear evaporation rate and ocular surface vital staining scores were recorded at baseline and at the end of the following one month. Conjunctival impression and brush cytology were performed before and after the treatment. After topical Cys treatment, significant improvements were found in symptom scores, corneal sensitivity, tear evaporation rate, TBUT, vital staining scores, goblet cells density, conjunctival squamous metaplasia grade, inflammatory cell numbers and the MUC5AC expression. Our study suggests that 0.05% topical Cys may be an effective treatment for dry eye patients with cGVHD. The improvements in the ocular surface and tear functions resulted presumably from the decreased inflammation, increased goblet cell density and MUC5AC mRNA expression. Bone Marrow Transplantation (2008) 41, 293–302; doi:10.1038/sj.bmt.1705900; published online 5 November 2007 Keywords: cyclosporine; chronic GVHD; dry eye; MUC5AC

Correspondence: Dr Y Ogawa, Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: [email protected] Received 30 April 2007; revised 21 August 2007; accepted 20 September 2007; published online 5 November 2007

Introduction Hematopoietic stem cell transplantation (HSCT) is an established treatment for many hematological malignancies.1,2 Chronic GVHD (cGVHD) is a major cause of morbidity and mortality in patients who underwent allogeneic HSCT. GVHD causes several ocular manifestations in 45–60% of the patients, among which dry eye is the most frequent complication, occurring in nearly 50% of allogeneic HSCT recipients.3–6 Dry eye typically occurs 6 months after the transplantation and the severity has been reported to be correlated with the presence of cGVHD and meibomian gland disease.5–9 Several treatments have been reported to alleviate the dry eye syndrome and produce objective improvement in dry eye associated with cGVHD, including topical lubricants, topical retinoic acid, autologous serum drops, punctal occlusion, moist chambers, tarsorrhaphy as well as systemic immunosuppressive treatment with FK-506 and corticosteroids. Surgical treatment with an amniotic membrane graft and penetrating keratoplasty may also be necessary for more severe cases.3,10–13 Unfortunately, effective treatments are still limited in dry eye disease with cGVHD. Phase II and III clinical trials have shown topical cyclosporine (Cys) to be safe and effective in the treatment of moderate to severe dry eyes.14–16 At present, there are no detailed studies employing ocular surface tests such as conjunctival impression and brush cytology (BC), tear evaporation and corneal sensitivity to evaluate the effect of topical Cys on cGVHD related dry eye. We evaluated several parameters such as tear evaporation rate, goblet cell density and MUC5AC mRNA expression, before and after topical Cys use in dry eye patients with cGVHD for the first time.

Patients and methods This was a prospective comparative study and not randomized or vehicle control study. We assigned the patients for topical Cys treatment group and control group

Topical cyclosporine in dry eye in patients with cGVHD Y Wang et al

Bone Marrow Transplantation

Abbreviations: CS ¼ corneal sensitivity; Cys ¼ topical cyclosporine; FL ¼ fluorescein staining; GCD ¼ goblet cells density; RB ¼ rose bengal staining; SMG ¼ squamous metaplasia grade; TBUT ¼ tear break-up time; TER ¼ tear evaporation rate; Time ¼ the time of dry eye onset; VAS ¼ visual analog scale symptom scores.

8.8376.81 6.1075.41 0.11 2305.4071990.60 3099.2074529.60 0.46 1.5870.73 1.5970.63 0.43 5.7172.16 276.227230.69 5.2571.29 566.677506.14 0.45 0.20 6.2971.98 5.5871.56 0.34 3.3674.67 4.6774.46 0.19 3.4371.51 3.1771.60 0.66 6.0973.90 7.3672.88 0.18 7.1477.05 72.86715.41 52.6877.10 11.50715.35 56.67716.70 57.4672.90 0.24 0.01 0.09 49.71715.60 39.50710.10 0.13

MUC5AC mRNA expression SMG (grade) GCD (cells/mm2) RB (pts) FL (pts) Schirmer (mm) TBUT (s) TER ( 107 g/cm2/s) CS (mm) VAS (%) Time (months) Age (years)

Comparison of the baseline between the topical cyclosporine group and the control group

N (eyes)

14 12 Cys Control P-value

Visual analog scale symptom scores Absence of any symptoms constituted a score of 0 points on the visual analog symptom scales. Intense, unbearable

Table 1

alternatively depending on patients’ consent to use topical Cys eye drops. All the patients selected for this study had a stable ophthalmologic and systemic condition in relation to GVHD. Thirty eyes of 15 patients refractory to baseline treatment including nonpreserved artificial tears, autologous serum and punctal plug occlusion were recruited and alternatively assigned to a topical Cys treatment group and a control group. Eighteen eyes of 9 patients received 0.05% topical Cys, 4 times per eye per day, with baseline treatment as the Cys treatment group. Twelve eyes of 6 patients were alternatively assigned with the same baseline treatment as the control group. Informed consent was obtained from all subjects and an institutional ethic board review was also obtained. The study followed the guidelines of the tenets of the Declaration of Helsinki. The dry eye disease was diagnosed according to the diagnostic criteria reported previously.5 Briefly, patients with (1) dry eye related symptoms, (2) positive staining with fluorescein or rose bengal and (3) Schirmer’s I test results less than or equal 5 mm, or a tear film break-up time (TBUT) value less than or equal 5 s were diagnosed as having definite dry eyes. The degree of dry eye was determined based on the value of vital staining (rose bengal and fluorescein scores) and the status of tear dynamics (the values of Schirmer’s test and TBUT). The objective parameters of pre-treatment characteristics except subjective symptom were comparable between treatment and nontreatment groups (Table 1). Because of dry eye patients, it is very common that the symptoms are not parallel with the severity of dry eye condition. In different patients, their subjective reaction on the same level of ocular surface damage is much different. The 0.05% topical Cys was made by the pharmacy at the Keio University Hospital (Cys 0.05 g, a-Cyclodextrin 8.00 g, Sodium Chloride 0.695 g, sterilized water 100 ml). The frequency of topical artificial tears and autologous serum in both group were same and also no changes were done during the study. Patients used topical artificial tears 6 times per eye per day and autologous eye drops 10 times per eye per day throughout this study. Before and after 1 month at the end of the treatment, the visual analog scale symptom scores were recorded. Tear evaporimetry, TBUT, ocular surface vital staining scores, corneal sensitivity (CS), Schirmer’s I test, impression and BC examinations were performed in the respective order. All examinations were done prior to treatment and again at the end of 1 month after treatment by the same two researchers (YW and YO). Shimazaki’s grading was used for the assessment of the meibomian gland disease.17 Inclusion criteria included dry eye patients with cGVHD refractory to conventional dry eye treatment with nonpreserved artificial tears, autologous serum and punctal occlusion for at least 8 weeks. Exclusion criteria included history of Sjo¨gren’s syndrome, other systemic or ocular diseases, history of ocular surgery, contact lens use or drug use. No changes were allowed to the patients’ systemic immunosuppressive treatment during the 1 month follow-up.

Inflammatory cells (%)

294


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symptomology was given a full score of 100 points. Briefly, the visual analog symptom scales were prepared as 10-cm lines and the patients were requested to write down the worst symptom and to check a point on the line corresponding to the degree of their worst symptom.18

Tear function parameters Tear evaporation rate. The tear film evaporation rate was measured as described previously19,20 by using a tear evaporimeter (KAO Corporation, Tokyo, Japan) following the manufacturer’s instructions. We measured evaporation rates with the eye closed and the eye open (natural blinking) twice to avoid the contribution of evaporation from lid skin. We calculated the difference in the values obtained in the open and closed eye states as the tear evaporation rate. Tear film break-up time. The standard TBUT measurement was performed after instillation of 2 ml of preservative free solution containing 1% fluorescein and 1% rose bengal in the conjunctival sac with a micropipette.21 A TBUT value of less than 5 s was considered abnormal. Schirmer’s I test. The standard Schirmer’s test without topical anesthesia was performed. The sterilized strips of filter paper (Showa Yakuhin Kako Co. Ltd, Tokyo, Japan) were placed in the lateral canthus away from the cornea and left in place for 5 min. Readings were reported in millimeters of wetting for 5 min.

Corneal sensitivity CS was measured using a Cochet-Bonnet aesthesiometer. The measurements were begun with the fully extended nylon filament (60 mm). The tip of the nylon filament was applied perpendicularly to the surface of the cornea, making certain not to touch the eyelashes, and was pushed until the fiber’s first visible bending. The length of fiber was gradually decreased until a blink reflex was observed. The length was recorded in units of millimeter. Measurements were taken from the central cornea and the mean of three serial measurements was recorded as the corneal sensitivity reading of that eye.22 An esthesiometry reading of 60 mm was regarded as normal corneal sensitivity. Ocular vital staining scores A preservative free solution (2 ml) consisting of 1% fluorescein and 1% rose bengal dye were applied to the conjunctival sac with a micropipette. The fluorescein staining score of the cornea ranged from 0 to 9 points. The rose bengal staining score of the cornea and conjunctiva also ranged from 0 to 9 points. Any score above 3 points was regarded as abnormal.21 Impression cytology parameters The impression cytology specimens were obtained after administration of topical anesthesia with 0.4% oxybuprocaine as described previously. The specimens from the nasal interpalpebral bulbar conjunctiva were stained with periodic acid–Schiff (PAS), dehydrated in ascending grades of ethanol and then with xylol, and finally coverslipped. The

specimens from temporal conjunctiva underwent immunohistochemical staining with Mucin 5AC (CLH2) monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The quantitative studies of conjunctival goblet cells and squamous metaplasia (SM) of conjunctival epithelial cells were conducted by counting the cells in three randomly selected nonoverlapping areas using a light microscope at a magnification of 400. The results were then averaged to obtain a single sample score. The goblet cell densities were reported as cells per square millimeter. The specimens were also assigned a grade of conjunctival epithelial SM according to the Nelson’s grading scheme.22 The same researcher (YW) who was masked to whom the samples came from evaluated the specimens for goblet cell counts, SM grades, and mucin pickup. A SM grading of 0 (abnormal 1–3) was regarded as normal condition.22 The impression cytology specimens underwent immunohistochemistry staining as reported previously.23 Briefly, the primary mouse monoclonal antibody to MUC5AC at a dilution of 1:100 was recommended with PBS wash afterward. Samples were then processed by VECTASTAIN ABC kit (Vector Laboratories Inc., Burlingame, CA, USA) protocol (DAB-peroxide staining) and treated with biotinylated anti-mouse secondary antibodies for 30 min. Finally, the specimens were counterstained with hematoxylin, dehydrated in ascending grades of ethanol and then with xylol and coverslipped for light microscopic examination. Negative controls were comprised of specimens incubated with the corresponding pre-immune serum replacing the primary antibody. The evaluation of specimens under light microscopy for the presence of positive immunohistochemical staining for MUC5AC was also performed in a masked fashion.

Brush cytology specimens and inflammatory cell counting BC specimens were obtained after administration of topical 0.4% oxybuprocaine. Two adjacent areas of the central upper palpebral conjunctiva were used for sampling. The conjunctiva was scraped with dental brushes (Dentalpro, Jacks Co., Osaka, Japan). After sampling, brushes were immediately placed in Hank’s Balanced Salt Solution (HBSS; Invitrogen-Gibco, Grand Island, NY, USA). One of the samples was centrifuged at 4 1C, 15 000 r.p.m. for 5 min (Tomy MX-300, Japan). The supernatants were discarded and replaced with 1 ml of Isogen solution (Nippon Gene Co., Tokyo, Japan) and stored at 70 1C for real-time quantitative RT-PCR. The other sample was centrifuged at room temperature for 10 min using a cytocentrifuge at 700 r.p.m. to create a slide sample containing monolayer of cells. The slides were then stained using Diff-Quik staining (Sysmex Co., Koube, Japan). The same researcher (YW) who was masked to whom the samples came from counted 500 inflammatory and epithelial cells in nonoverlapping fields under microscopic observation (magnification, 400). The inflammation was reported as the percentage of inflammatory cells present in the 500 cells counted.22,24 Quantitative real-time RT-PCR for MUC5AC Quantitative real-time RT-PCR was performed as described previously.23 Briefly, RNA was extracted from Bone Marrow Transplantation


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Isogen samples and quantitative real-time PCR was performed according to the manufacturer’s instructions (Applied Biosystems, Weiterstadt, Germany). cDNA (10 ng) was amplified in 25 ml final volume in the presence of 1.25 ml of the following ‘Assay by Design’ oligonucleotides (MUC5AC and GAPDH, Applied Biosystems). Test gene primer and probe sets were optimized for concentration, amplification efficiency, and faithful co-amplification with housekeeper gene primer and probe sets, the latter including GAPDH. Quantitative real-time RT-PCR was set up in 96 well plates using the above reagents and Taq Man master mix and as indicated by optimization data and it was run on 7700 ABI thermal cyclers (Applied Biosystems). The thermal profile consisted of 50 1C for 2 min, 95 1C for 10 min, followed by 40 cycles of 94 1C for 15 s and 60 1C for 1 min. Real time data were acquired and analyzed using Sequence Detection System Software (Applied Biosystems) with manual adjustment of the baseline and threshold parameters. The expression levels of mRNA were normalized by the median expression of a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH).

Statistical analysis For statistical comparisons, Wilcoxon matched-pairs signed-ranks test was used for comparison of the changes in the visual analog scale symptom scores, TBUT, Schirmer’s I test values, ocular vital staining scores, conjunctival goblet cell densities and SM grades, MUC5AC mRNA expression levels and inflammatory cell percentage in each group before and after 1 month of treatment.

Results Patients’ demographic characteristics were summarized in Table 2. Twenty-six eyes of 13 patients finished the current study. Topical Cys treatment was stopped in two patients at 1 week due to intolerable irritation. The data from them were not used to evaluate the treatment effects of Cys, they were just used to evaluate the complications. Besides intolerable irritation, another two patients complained of mild irritation and itchy sensation, which lasted for about 5 min after topical Cys instillation. However, both of them agreed to continue with the topical Cys treatment. Detailed baseline parameters were shown in Table 1. The degree of dry eye was determined based on the value of vital staining (rose bengal and fluorescein scores) and the status of tear dynamics (the value of Schirmer’s test and TBUT). The objective parameters of pre-treatment characteristics were comparable between treatment and nontreatment group except the visual analog scale symptom scores. The treatment group (14 eyes of 7 patients; 3 males, 4 females; mean age, 49.7715.6 years), received 0.05% topical Cys 4 times per eye per day for 1 month

Patient characteristics

Table 2 Case no

Mann–Whitney Test was used to compare the difference in values found at baseline and at 1 month between Cys treatment group and control group. A P-value less than 0.05 was considered statistically significant. Statistical analysis was performed using the In Stat programme (GraphPad Software, San Diego, CA, USA).

Age

Gender

Diagnosis

Topical treatment

Time (Mo)

cGVHD other than the eyes

From HSCT to dry eye

From dry eye to recruit

Systemic immunosuppressant (IS) at the time of treatment

1a 2a 3a

52 63 64

M M M

MDS MDS MM

AT, AS, PP, VitAb AT, AS, PP, VitA, Cys AT, AS, PP, VitA Cys

Lung, skin, mouth Liver, skin, mouth Skin, mouth

7 6 7

22 59 23

4a 5a 6a 7a 8a 9a 10c 11c 12c 13c

35 30 50 36 66 54 34 46 31 40

F F M M F F F F F M

CML AML AML CML MM AML ALL AML ALL CML

Mouth Mouth Mouth, skin, liver Mouth Mouth, skin None None Lung, skin, mouth Skin None

7 7 3 7 11 5 3 6 42 12

54 86 66 81 47 87 144 67 50 90

14c 15c

56 30

M M

CML AML

AT, AS, PP, VitA Cys AT, AS, PP, VitA Cys AT, AS, PP, VitAb AT, AS, PP, VitA Cys AT, AS, PP, VitA, Cys AT, AS, PP, VitA Cys AT, AS, PP, VitA AT, AS, PP, VitA AT, AS, PP, VitA AT, AS, VitA laser puctum occlusion AT, AS, PP, VitA AT, AS, PP, VitA

PSL 30 mg per day Cys A 150 mg per day Cys A 25 mg per morning 25 mg per night Cys A 100 mg per day None None None PSL 10 mg per day None None None None None

3 3

165 55

None None

None Liver

Abbreviations: ALL ¼ acute lymphoblastic leukemia; AML ¼ acute myelogenous leukaemia; AS ¼ autologous serum eye drops; AT ¼ artificial tear; CML ¼ chronic myelogenous leukaemia; Cys ¼ 0.05% topical cyclosporine eye drops; Cys A ¼ Cyclosporine A; F ¼ female; HSCT ¼ hematopoietic cell transplantation; M ¼ male; MDS ¼ myelodysplastic syndrome; MM ¼ multiple myeloma; Mo ¼ month; PP ¼ punctal plug; PSL ¼ prednisolone; VitA ¼ VitA eye drops. a 0.05% Cyclosporine treatment group. b The topical cyclosporine treatment was stopped because of irritation. c Control group.



Abbreviations: CS ¼ corneal sensitivity; FL ¼ fluorescein scores; GCD ¼ goblet cells density; RB ¼ rose bengal scores; SMG ¼ squamous metaplasia grade; TBUT ¼ tear break-up time; TER ¼ tear evaporation rate; VAS ¼ visual analog scale symptom scores. *Po0.05, compared with the scores before topical Cys treatment, Wilcoxon matched-pairs signed-ranks test. **Po0.05, compared with the scores after one month topical Cys treatment, Mann–Whitney Test.

2305.471990.6 2766.872048.3* 3099.274529.6 2543.973858.7 1.5870.73 0.9970.44* 1.5970.63 1.6570.45** 276.227230.69 886.257602.37* 566.677506.14 473.237355.62** 5.7172.16 3.5771.40* 5.2571.29 5.8371.27** 6.2971.98 3.7171.734* 5.5871.56 5.7571.42** 3.3674.67 2.9372.34 4.6774.46 4.3373.60 3.4371.51 4.7971.48* 3.1771.60 2.2570.75** 6.0973.90 4.2472.35* 7.3672.88 6.3772.63** 52.6877.10 56.4673.93* 57.4672.90 57.9272.09 72.86715.41 52.86724.63* 56.67716.70 45.83733.15 14 14 12 12

Schirmer (mm) TBUT (s) TER ( 107g/cm2/s) CS (mm) VAS (%) N (eyes)

Ocular surface vital staining scores Both the mean fluorescein and rose bengal scores improved significantly with 1 month of topical Cys treatment (Po0.05). The mean vital staining scores didn’t show significant changes at the end of the trial in the control group (P40.05). The mean vital staining scores at the end of the 1 month trial were significantly lower in the topical Cys treatment group compared to the control group (Po0.05). TBUT improved in 12 of 14 eyes (85%) in the Cys group, whereas none of the patients in the control group improved. As for fluorescein and rose bengal score, 13 of

Comparison of the scores of baseline and at the end of the trial

Schirmer’s test All patients had severe aqueous deficiency dry eyes in this study. There were no significant changes in the mean Schirmer values at the end of the trial both in the treatment and control group (P40.05).

Table 3

Tear film break-up time The mean TBUT improved significantly with 1 month of 0.05% topical Cys treatment whereas a significant worsening of the TBUT was observed in the control group (Po0.05). The mean TBUT in the treatment group at the end of 1 month was significantly higher than the mean TBUT in the control group (Po0.05).

FL (pts)

Tear evaporation rate One month of topical Cys treatment was associated with a significant reduction in tear evaporation (Po0.05). Although a slight decrease in the control group was observed at the end of the trial, the difference was not statistically significant (P40.05). The mean tear evaporation rate in the treatment group at the end of 1 month was significantly less than the mean tear evaporation rate in the control group (Po0.05).

Cys baseline Cys one month Control baseline Control one month

Inflammatory cells (%) MUC5AC mRNA expression SMG (grade) RB (pts)

GCD (cells/mm2)

Visual analog scale symptom scores The most frequent and worst symptoms reported by the patients were pain, dryness, foreign body sensation and irritation. The mean worst visual analog scale symptom score in the Cys group improved from 72.86% to 52.86% with 1 month of treatment (Po0.05). There was a slight decrease in the mean visual analog scale symptom score in the control group from the baseline score of 56.67% in the beginning of the trial to 45.83% at the end of the study. The difference was not statistically significant (P40.05).

8.8376.81 4.4172.94* 6.1075.41 5.6774.80

297

while continuing the baseline treatment. In the control group (12 eyes of 6 patients; 3 males, 3 females; mean age, 39.7710.1 years), the baseline dry eye treatments were maintained and the subjects were followed for 1 month. The onset time of dry eye in the Cys group and control group were 7.1477.05 months and 11.5075.35 months respectively. There was no significant difference between two groups (P40.05). The changes of the scores of the parameters before and after treatment were summarized in Table 3 and are described below.



298

14 eyes (92.6%) improved in the Cys treatment group, whereas 4 of 12 eyes (33.3%) improved in the control group. Figures 1a–d showed the marked improvements in fluorescein and rose bengal staining patterns from a representative 36-years-old male dry eye patient with cGVHD.

Corneal sensitivity The mean corneal sensitivity showed a significant improvement at the end of the trial in the topical Cys treatment group (Po0.05), whereas no significant changes were observed in the control group (P40.05). Impression cytology parameters All imprints showed sheets of conjunctival epithelial cells, variable numbers of goblet cells and variable degrees of mucin pickup. Squamous metaplasia grades. The mean SM showed a significant improvement with 1 month of topical Cys treatment (Po0.05), whereas no significant change was observed in the control group at the end of the trial (P40.05). The mean SM grade in the Cys treatment group at the end of the trial was also significantly better compared with the control group (Po0.05).

dramatic increase in goblet cell numbers with 1 month of 0.05% topical Cys in the treatment group, increased mucin pickup and improvement of epithelial SM grade. Figures 2c and d confirm with the immunohistochemical staining for MUC5AC, the increase in MUC5AC positive goblet cells and mucin membrane pickup on the epithelial cells.

Brush cytology parameters Real-time quantitative RT-PCR for MUC5AC mRNA expression. The mean relative MUC5AC mRNA expression improved significantly with 1 month of topical Cys treatment (Po0.05), whereas a slight nonsignificant decrease in MUC5AC mRNA expression was observed in the control group (P40.05). Inflammatory cell numbers. The mean inflammatory cell number decreased significantly with 1 month of 0.05% topical Cys treatment (Po0.05), whereas no significant change was observed in the control group (P40.05). Figures 3a and b show BC specimens before and after topical Cys treatment in a 35-year-old female dry eye patient with cGVHD. The improvement in the epithelial phenotype and marked decrease in the inflammatory infiltrates were observed.

Discussion Goblet cell density. The mean goblet cell density showed a dramatic and significant increase in the topical Cys treatment group (Po0.05) with no significant change in the control group (P40.05). Figures 2a and b show the

Figure 1

This is the first detailed study evaluating the efficacy of topical Cys by focusing on the tear functions and ocular surface status including corneal sensitivity, tear evapora-

Representative slit lamp photographs from a 36-year-old male patient before and after 1 month of 0.05% cyclosporine (Cys) eye drop treatment. (a) Baseline fluorescein staining. The green staining showed the area of positive fluorescein staining. Fluorescein score was 7 points. (b) After 1 month of 0.05% topical Cys treatment. Note obvious decrease in the fluorescein staining area. Fluorescein score was 3 points. (c) Baseline rose bengal staining. The red staining on the cornea and conjunctiva was the positive rose bengal staining (7 points). Note obvious ocular surface inflammation. (d) After 1 month of 0.05% topical Cys treatment. Note the decreased inflammation of ocular surface and rose bengal score decreased to 5 points.



299

Figure 2

Representative conjunctival impression cytology specimens before and after 1 month of 0.05% cyclosporine (Cys) eye drop treatment. (a) Baseline specimen. Note the high grade squamous metaplasia (SM) and without any clear goblet cells. White arrow indicates the keratinized conjunctival epithelial cells. Periodic acid–Schiff (PAS) staining, magnification, 400. (b) After 1 month of 0.05% topical Cys treatment. Note obvious improvement of SM and dramatic increase in the number of goblet cells (black arrows). PAS staining, magnification, 400. (c) Baseline specimen. Brown color indicates the positive MUC5AC staining. Note the high grade SM and few goblet cells with MUC5AC positive immunohistochemical staining (black arrow). Magnification, 400. (d) After 1 month of 0.05% topical Cys treatment. Note the significant increase in the number of the MUC5AC positive goblet cells (black arrows) and the MUC5AC membrane pickup (yellow arrows). Magnification, 400.

Figure 3 Representative brush cytology (BC) specimens before and after 1 month of topical 0.05% cyclosporine (Cys) treatment in a 35-year-old female patient. (a) Baseline BC specimen. Note the obvious inflammatory cells infiltration (yellow arrows) and the high grade squamous metaplasia (SM; black arrows). Diff-Quick staining, magnification, 400. (b) After 1 month of 0.05% topical Cys treatment. Note the marked decrease in the number of inflammatory cells and the lower grade SM. Diff-Quick staining, magnification, 400.

tion rate, conjunctival MUC5AC mRNA expression and goblet cell density in severe cGVHD dry eye patients who were refractory to maximal medical treatment with nonpreserved artificial tears, autologous serum eye drops and superoinferior punctal plug occlusion who also consented to treatment with topical Cys, with a nonrandomized control group. Although this was not a double blinded or vehicle control study, our findings showed in detail that dry eye status improved after using topical Cys treatment compared with the control group which had the same dry

eye status with maximal baseline treatment. In this study, we found that 0.05% topical Cys treatment 4 times per eye per day for 1 month was observed to be associated with a significant improvement of TBUT, vital staining scores, impression and BC parameters. The improvement in TBUT was also associated with a concomitant improvement in the tear evaporation rate and symptom scores in the 0.05% topical Cys treatment group. The marked recovery of the ocular surface seen in this study may have resulted from the improvement in the Bone Marrow Transplantation


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mucin layer and was supported by the increased level of the goblet cell density and protein and mRNA level of MUC5AC expression as supported by the findings from BC and impression cytology specimens in this study. We think that the improvement of the ocular surface epithelial health also resulted from decreased ocular surface inflammation25 as shown by BC and from improvement of corneal sensitivity.26 Indeed, corneal nerves are known to exert trophic influences on ocular surface epithelia, and seem to maintain the epithelial integrity and cellular milieu.26 A recent study has also reported that the density of the conjunctival goblet cells increased significantly after 6 months of treatment with 0.05% Cys A compared with the baseline density or with the treatment with vehicle in dry eye patients.27 The efficiency of 0.05% and 0.1% topical Cys drugs in the treatment of moderate to severe dry eye patients with Sjo¨gren’s syndrome or other autoimmune disease have also been shown previously.28,29 A previous noncontrolled trial in 8 patients with cGVHD dry eyes showed significant improvement of Schirmer basal secretion scores, TBUT and tear lysozyme levels after 3 months of treatment with 0.05% topical Cys.30 Another retrospective noncontrolled study in 16 patients with cGVHD dry eye disease treated with 0.05% topical Cys showed significant improvements of symptom scores and vital staining after a mean follow up of 90 days.31 The two studies showed increased Schirmer values, but the baseline Schirmer value in these two studies were much higher (from 7.274.0 mm to 11.372.2 mm after 3 months of treatment, from 8.0 mm to 8.5 mm after a mean follow up of 90 days) than the values reported in our study (from 3.473.1 mm to 4.774.5 mm after 1 month of treatment), suggesting a much more severe disease in our case compared to these two previous reports. Previous reports seem to involve mild dry eye associated with cGVHD in the majority of the patients. In contrast, we chose severe patients who were refractory to the maximal conventional treatments. We think that the absence of improvement in the Schirmer’s test value in our case may be explained by extensive fibrosis and destruction of the lacrimal gland.32,33 In a previous case report by us,13 dry eye associated with cGVHD had observed marked improvement in terms of symptoms and the findings of ocular surface within 44 days of systemic administration of FK506, an alternative immunosuppressant for GVHD. However, the results of the Schirmer’s tests were not normalized in this patient. These findings were consistent with the results of our present study, validating the specificity of the results in our present study. Although the pathogenesis of cGVHD related dry eye disease is still under debate, one of the pathophysiological events is considered to be the inflammation in addition to fibrosis in the conjunctiva and lacrimal glands.13,32 The conjunctiva in patients with cGVHD also shows infiltration with lymphocytes, predominantly T cells13 suggesting that the conjunctiva is targeted in cGVHD. Pathological changes in the lacrimal glands include infiltration of T cells in the periductal area and the ductal epithelia and excessive fibrosis in the extracellular matrix of ducts, lobules and vessels. It has been suggested that the sequence of events leading to dry eye involves the breakdown of the blood vessel basal laminae leading to migration of T cells Bone Marrow Transplantation

into the conjunctiva and lacrimal gland. The activated T cells proliferate and migrate into the periductal areas and around vessels and contribute to the destruction of the ductal and conjunctival epithelia. Concurrently, CD34 þ stromal fibroblasts are likely to be activated by cytokines released by inflammatory cells such as T cells.13,32,33 Cys, an immunosuppressant known to inhibit the activity of transcription factors of the nuclear factors of the activated T cells, promotes induction of cytokine production and blocks the activation of antigen-specific T cells.34 Previous study using animal models showed that the mechanisms of action of topical Cys was considered to be prevention of lymphocyte infiltration and reducing expression of Fas-L on T cells.35 Another study showed downregulation of CD44, adhesion molecule on CD4 þ T cells, and decreased the expression of mRNA of IL-2 and T-cell receptor-constant of b-chain.36 Therefore, we speculate that topical Cys might have been effective in reducing the amount of T-cell infiltration, activation and proliferation, in the conjunctiva as well as lacrimal gland, leading to improvement in the density of the goblet cells which secrete the mucinous component of the tear film layer. The half-life of T cells is thought to be 120 days. Therefore, there is another possibility that the response in this patient group in 30 days may be related to another reason outside of the inflammatory component. Increased goblet cells density and MUC5AC expression were found after 1-month topical Cys treatment. We thought this was one of the reasons of improvement in ocular surface and tear functions. In cGVHD, T cells target the conjunctival epithelia. Turn over of conjunctival epithelia including goblet cells is thought to be 5–7 days. Therefore, we think that 1 month is sufficient to regenerate the damaged conjunctival epithelia by reducing infiltration of the number of T cells. After tapering systemic immunosuppressant, prominent T-cell infiltration and interstitial fibrosis were noted as we have previously reported.32,33 Even in the early phase of dry eye in patients with cGVHD, newly synthesized collagen fibrils and activated fibroblasts were observed in the lacrimal gland in these reports. These previous findings, taken together with the clinical findings of the cases in this study suggest that the lacrimal gland in patients with severe dry eye associated with cGVHD already had irreversible damage due to early fibrosis before starting the topical Cys, and address the importance of earlier treatment to prevent a permanent decrease in aqueous tear components. Although systemic Cys and prednisolone have remained a standard therapy for systemic cGVHD, the efficacy of this systemic therapy has not always been sufficient for cGVHD related dry eye patients. Prolonged treatment with systemic Cys with prednisolone for prophylaxis of GVHD increases the risk of infections that can lead to mortality.13 Moreover, long-term systemic immunosuppression may also increase the probability of leukemic relapse. To escape the side effects of systemic immunosuppressants, topical eye drops containing Cys would be a better therapy for preventing the development of dry eye with cGVHD. Therefore, appropriate timing and institution of immunosuppressive drugs may limit the progression of the dry eye disease in this relentless disease and it is necessary to have


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communication and interaction in detail between the internist and the ophthalmologist related to tapering and cessation of the systemic immunosuppressant and the timing for the commencement of topical Cys. The preliminary results of this study suggested that 0.05% topical Cys treatment might be superior to nontreatment in the control group of dry eye patients with cGVHD who received the same baseline treatment and remained refractory to it. Further prospective randomized controlled studies on a larger population employing vehicle/placebo solutions are definitely essential. Although major adverse events were not observed in the current study, two patients discontinued the drops due to severe irritation. Other two patients complain mild irritation and itch. The oil base of topical Cys may be the reason for the unpleasantness, therefore the formula of topical Cys still needs to be improved. On the other hand, current study just followed 1 month, the long-term safety and efficacy of topical Cys on cGVHD related dry eye need further study. Topical Cys treatment did not improve the tear quantity in this series of patients who might have had extensive lacrimal gland inflammation, and destruction. Therefore, appropriate timing and earlier institution of topical therapy with detailed information on tapering of the systemic immunosuppressant and the onset of dry eye provided by both internist and ophthalmologist might have improved the tear quantity in the patients of the current trial. Future studies looking into these issues and comparing the efficacies of topical Cys with other immunosuppressant and vehicle/placebo groups will add invaluable information in this field.

Acknowledgements The authors have no proprietary interest in any of the products mentioned in the manuscript. Presented in part at the 60th Japan Congress of Clinical Ophthalmology, 5–8 October 2006, Kyoto, Japan in the same name as this manuscript, ‘’Ocular Surface and Tear Functions after Topical Cyclosporine Treatment in Dry Eye Patients with Chronic Graft-Versus-Host Disease’. Work supported by a grant from the Japanese Ministry of Education, Science, Sports and Culture #18591932. Dr Wang and Dr Ogawa contributed equally to the current work and should be considered of double first authors.

References 1 Horowitz M. Uses and growth of hematopoietic cell transplantation immunology. In: Blume KG, Forman SJ, Applebaum FR (eds). Thomas’ Hematopoietic Cell Transplantation, 3rd edn. Blackwell: Malden, MA, 2004, pp 9–15. 2 Martin PJ. Overview of hematopoietic cell transplantation. In: Blume KG, Forman SJ, Applebaum FR (eds). Thomas’ Hematopoietic Cell Transplantation, 3rd edn. Blackwell: Malden, MA, 2004, pp 16–30. 3 Ogawa Y, Kuwana M. Dry eye as a major complication associated with chronic graft-versus-host disease after hematopoietic stem cell transplantation. Cornea 2003; 22: S19–S27. 4 Franklin RM, Kenyon KR, Tutschka PJ, Saral R, Green WR, Santos GW. Ocular manifestations of graft-vs-host disease. Ophthalmology 1983; 90: 4–13.

5 Ogawa Y, Okamoto S, Wakui M, Watanabe R, Yamada M, Yoshino M et al. Dry eye after hematopoietic stem cell transplantation. Br J Ophthalmol 1999; 83: 1125–1130. 6 Hirst LW, Jabs DA, Tutschka PJ, Green WR, Santos GW. The eye in bone marrow transplantation. I. Clinical study. Arch Ophthalmol 1983; 101: 580–584. 7 Shulman HM, Sullivan KM, Weiden PL, McDonald GB, Striker GE, Sale GE et al. Chronic graft-versus-host syndrome in man. A long-term clinicopathologic study of 20 Seattle patients. Am J Med 1980; 69: 204–217. 8 Mencucci R, Rossi Ferrini C, Bosi A, Volpe R, Guidi S, Salvi G. Ophthalmological aspects in allogenic bone marrow transplantation: Sjogren-like syndrome in graft-versus-host disease. Eur J Ophthalmol 1997; 7: 13–18. 9 Tichelli A, Duell T, Weiss M, Socie G, Ljungman P, Cohen A et al. Late-onset keratoconjunctivitis sicca syndrome after bone marrow transplantation: incidence and risk factors. European group for blood and marrow transplantation (EBMT) Working party on late effects. Bone Marrow Transplant 1996; 17: 1105–1111. 10 Ogawa Y, Okamoto S, Mori T, Yamada M, Mashima Y, Watanabe R et al. Autologous serum eye drops for the treatment of severe dry eye in patients with chronic graftversus-host disease. Bone Marrow Transplant 2003; 31: 579–583. 11 Murphy PT, Sivakumaran M, Fahy G, Hutchinson RM. Successful use of topical retinoic acid in severe dry eye due to chronic graft-versus-host disease. Bone Marrow Transplant 1996; 18: 641–642. 12 Rocha EM, Pelegrino FS, de Paiva CS, Vigorito AC, de Souza CA. GVHD dry eyes treated with autologous serum tears. Bone Marrow Transplant 2000; 25: 1101–1103. 13 Ogawa Y, Okamoto S, Kuwana M, Mori T, Watanabe R, Nakajima T et al. Successful treatment of dry eye in two patients with chronic graft versus host disease with systemic administration of FK506 and corticosteroids. Cornea 2001; 20: 430–434. 14 Sall K, Stevenson OD, Mundorf TK, Reis BL. Two multicenter, randomized studies of the efficacy and safety of cyclosporine ophthalmic emulsion in moderate to severe dry eye disease. CsA Phase 3 Study Group. Ophthalmology 2000; 107: 631–639. 15 Stevenson D, Tauber J, Reis BL. Efficacy and safety of cyclosporin A ophthalmic emulsion in the treatment of moderate-to-severe dry eye disease: a dose-ranging, randomized trial. The cyclosporin A phase 2 study group. Ophthalmology 2000; 107: 967–974. 16 Barber LD, Pflugfelder SC, Tauber J, Foulks GN. Phase III safety evaluation of cyclosporine 0.1% ophthalmic emulsion administered twice daily to dry eye disease patients for up to 3 years. Ophthalmology 2005; 112: 1790–1794. 17 Shimazaki J, Goto E, Ono M, Shimmura S, Tsubota K. Meibomian gland dysfunction in patients with Sjogren syndrome. Ophthalmology 1998; 105: 1485–1488. 18 Kojima T, Ishida R, Dogru M, Goto E, Matsumoto Y, Kaido M et al. The effect of autologous serum eyedrops in the treatment of severe dry eye disease: a prospective randomized case-control study. Am J Ophthalmol 2005; 139: 242–246. 19 Tsubota K, Yamada M. Tear evaporation from the ocular surface. Invest Ophthalmol Vis Sci 1992; 33: 2942–2950. 20 Goto E, Endo K, Suzuki A, Fujikura Y, Matsumoto Y, Tsubota K et al. Tear evaporation dynamics in normal subjects and subjects with obstructive meibomian gland dysfunction. Invest Ophthalmol Vis Sci 2003; 44: 533–539. 21 Toda I, Tsubota K. Practical double vital staining for ocular surface evaluation. Cornea 1993; 12: 366–367. 22 Nelson JD. Impression cytology. Cornea 1988; 7: 71–81. 23 Dogru M, Asano-Kato N, Tanaka M, Igarashi A, Shimmura S, Shimazaki J et al. Ocular surface and MUC5AC alterations Bone Marrow Transplantation


302

24

25 26 27

28

29 30

in atopic patients with corneal shield ulcers. Curr Eye Res 2005; 30: 897–908. Miyoshi T, Fukagawa K, Shimmura S, Fujishima H, Takano Y, Takamura E et al. Interleukin-8 concentrations in conjunctival epithelium brush cytology samples correlate with neutrophil, eosinophil infiltration, and corneal damage. Cornea 2001; 20: 743–747. Pflugfelder SC. Anti-inflammatory therapy of dry eye. Ocul Surf 2003; 1: 31–36. Nishida T. Neurotrophic mediators and corneal wound healing. Ocul Surf 2005; 3: 194–202. Kunert KS, Tisdale AS, Gipson IK. Goblet cell numbers and epithelial proliferation in the conjunctiva of patients with dry eye syndrome treated with cyclosporine. Arch Ophthalmol 2002; 120: 330–337. Wilson SE, Perry HD. Long-term resolution of chronic dry eye symptoms and signs after topical cyclosporine treatment. Ophthalmology 2007; 114: 76–79. Tatlipinar S, Akpek EK. Topical cyclosporin in the treatment of ocular surface disorders. Br J Ophthalmol 2005; 89: 1363–1367. Rao SN, Rao RD. Efficacy of topical cyclosporine 0.05% in the treatment of dry eye associated with graft versus host disease. Cornea 2006; 25: 674–678.


31 Lelli Jr GJ, Musch DC, Gupta A, Farjo QA, Nairus TM, Mian SI et al. Ophthalmic cyclosporine use in ocular GVHD. Cornea 2006; 25: 635–638. 32 Ogawa Y, Kuwana M, Yamazaki K, Mashima Y, Yamada M, Mori T et al. Periductal area as the primary site for T-cell activation in lacrimal gland chronic graft-versushost disease. Invest Ophthalmol Vis Sci 2003; 44: 1888– 1896. 33 Ogawa Y, Yamazaki K, Kuwana M, Mashima Y, Nakamura Y, Ishida S et al. A significant role of stromal fibroblasts in rapidly progressive dry eye in patients with chronic GVHD. Invest Ophthalmol Vis Sci 2001; 42: 111–119. 34 Duncan N, Craddock C. Optimizing the use of cyclosporin in allogeneic stem cell transplantation. Bone Marrow Transplant 2006; 38: 169–174. 35 Tsubota K, Fujita H, Tadano K, Takeuchi T, Murakami T, Saito I et al. Improvement of lacrimal function by topical application of CyA in murine models of Sjo¨gren’s syndrome. Invest Ophthalmol Vis Sci 2001; 42: 101–110. 36 Tsubota K, Saito I, Ishimaru N, Hayashi Y. Use of topical cyclosporin A in a primary Sjo¨gren’s syndrome mouse model. Invest Ophthalmol Vis Sci 1998; 39: 1551–1559.