11/4/2014
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`Ovid: Interleukin-6 Levels in the Conjunctival Epithelium of Patients with Dry Eye Disease Treated with Cyclosporine Ophthalmic Emulsion.
`
`Cornea
`Issue: Volume 19(4), July 2000, pp 492-496
`Copyright: © 2000 Lippincott Williams & Wilkins, Inc.
`Publication Type: [Clinical Sciences]
`
`[Clinical Sciences]
`Interleukin-6 Levels in the Conjunctival Epithelium of Patients with Dry Eye Disease Treated with Cyclosporine
`Ophthalmic Emulsion
`
`Turner, Kathleen D.V.M.; Pflugfelder, Stephen C. M.D.; Ji, Zhonghua M.D.; Feuer, William J. M.S.; Stern, Michael PhD; Reis, Brenda L. PhD
`
`Author Information
`From the Bascom Palmer Eye Institute (K.T., S.C.P., Z.J., W.J.F.), Department of Ophthalmology, University of Miami School of Medicine, Miami, Florida; and Allergan,
`Inc. (M.S., B.L.R.), Irvine, California, U.S.A.
`Submitted September 13, 1999.
`Revision received January 8, 2000.
`Accepted January 15, 2000.
`Supported by Allergan Inc., 2525 Dupont Drive, Irvine, CA 92713-9534, U.S.A.
`Address correspondence and reprint requests to S.C. Pflugfelder, M.D. Bascom Palmer Eye Institute, 900 N.W. 17th Street, Miami, FL 33136, U.S.A.
`Abstract
`
`
`
`Purpose. To evaluate interleukin-6 (IL-6) levels in the conjunctival epithelium of patients with moderate to
`severe dry eye disease before and after treatment with cyclosporin A ophthalmic emulsion (CsA) or its vehicle.
`
`
`Methods. Conjunctival cytology specimens were obtained from a subset of patients enrolled in a 6-month
`randomized, double-masked clinical trial of the efficacy and safety of topical CsA at baseline and after 3 and 6
`months of B.I.D. treatment with 0.05% cyclosporine emulsion (n = 13), 0.1% cyclosporine emulsion (n = 8), or vehicle
`(n = 10). RNA was extracted and a competitive reverse transcriptase polymerase chain reaction (RT-PCR) was used
`to evaluate the levels of mRNA encoding the inflammatory cytokine IL-6 and a housekeeping gene, G3PDH. Levels
`of IL-6 and G3PDH were measured and compared.
`
`
`Results. There was no change from baseline in the level of G3PDH after 3 or 6 months in any group. IL-6
`normalized for G3PDH (IL-6/G3PDH ratio) was not different from baseline at 3 months but showed a significant
`decrease from baseline in the group treated with 0.05% CsA (p = 0.048) at 6 months. No significant between-group
`differences were noted and no correlation was observed between the change in IL-6/G3PDH and corneal
`fluorescein staining.
`
`
`Conclusions. This preliminary, small-cohort study showed a decrease in IL-6 in the conjunctival epithelium of
`moderate to severe dry eye patients treated with 0.05% CsA for 6 months. The observed decrease suggests that
`dry eye disease involves immune-mediated inflammatory processes that may be decreased by treatment with
`topical ophthalmic cyclosporine.
`
`
`
`
`Specific evidence of chronic immune activation of the conjunctival epithelium in patients with dry eye
`suggests that cell-mediated inflammatory processes may play an important role in the pathogenesis of dry eye
`disease. 1–4 These immunopathologic findings include increased expression of immunologic adhesion molecules
`(i.e., HLA class II and ICAM-1 antigens) and inflammatory cytokines (IL-1, IL-6, IL-8, and TNF-[alpha]) in these
`patients. 5–7 Among the inflammatory cytokines evaluated, levels of interleukin-6 (IL-6) showed the greatest
`elevation in eyes with dry eye disease when compared to normal eyes. 6
`
`
`
`Further evidence that immune-mediated inflammatory processes are involved in the pathogenesis of dry eye
`disease comes from reports that topical use of the immunomodulatory agent cyclosporine can improve the signs
`and symptoms of dry eye. 8–10 However, it has not been determined whether the use of topical cyclosporine can
`cause measurable decreases in any of the inflammatory markers that are associated with dry eye disease.
`
`
`
`The purpose of this study was to evaluate the level of the inflammatory cytokine IL-6 in the conjunctiva of
`patients with keratoconjunctivitis sicca enrolled in a randomized trial of two different concentrations of
`cyclosporin A ophthalmic emulsion (CsA) and a castor oil emulsion vehicle. IL-6 was chosen because previous
`studies have shown that the level of IL-6 mRNA is elevated in the conjunctival epithelium of patients with
`Sjögren's syndrome keratoconjunctivitis sicca. 5,6 Furthermore, the level of IL-6 mRNA was found to decrease in
`bronchoalveolar lavage cells obtained from patients with acute lung transplant rejection who were treated with
`aerosolized cyclosporine. 11
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`Ovid: Interleukin-6 Levels in the Conjunctival Epithelium of Patients with Dry Eye Disease Treated with Cyclosporine Ophthalmic Emulsion.
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`PATIENTS AND METHODS
`
`Patients
`
`
`Evaluation of the level of IL-6 mRNA in the conjunctiva was performed on conjunctival cytology specimens
`from a subset of patients enrolled in a prospective, double-masked, vehicle-controlled, multi-center clinical trial
`of topical cyclosporine emulsion (CsA) for the treatment of dry eye disease. 9 Adult patients of either sex were
`eligible for participation if they presented with a diagnosis of moderate to severe dry eye disease as defined by
`the following criteria:
`
`
`1) Schirmer test without anesthesia of <=5 mm/5 minutes in at least one eye (If Schirmer test without anesthesia =
`0 mm/5 minutes, then Schirmer with nasal stimulation had to be > 3 mm/5 minutes in the same eye.);
`
`2) sum of corneal and interpalpebral conjunctival staining of >= +5 in the same eye where corneal staining was >=
`+2;
`
`3) a baseline Ocular Surface Disease Index© (OSDI©) score of 0.1 with no more than three responses of “not
`applicable;” and
`
`4) a score of >= 3 on the Subjective Facial Expression Scale.
`
`
`The OSDI© is a questionnaire consisting of 12 questions (each rated from 0 = none of the time to 4 = all of the
`time) for the evaluation of the impact of a patient's dry eye disease on his or her vision-related functioning. The
`overall score was calculated by dividing the sum of the responses for all questions answered by the total possible
`score and then multiplying by 100. Thus, overall scores ranged from 0 = no disability to 100 = complete disability.
`
`
`
`To be eligible for enrollment, signs and symptoms of dry eye disease must have presented despite
`conventional management, which may have included artificial tear drops, gels and ointments, sympathomimetic
`agents, parasympathomimetic agents, and punctal occlusion. Eligible patients were enrolled if they were deemed
`capable of following the study protocol and were considered likely to complete the treatment period and to
`return for all scheduled visits, if they had normal lid position and closure, and if they had a best-corrected early
`treatment of diabetic retinopathy study visual acuity score of +0.7 logmar or better in each eye.
`
`
`
`Patients were excluded from the study if they had used systemic or topical ophthalmic cyclosporine within 90
`days before the study. Other exclusion criteria included the presence or history of any systemic or ocular disorder
`or condition (including ocular surgery, trauma, and disease) that could possibly interfere with the interpretation of
`the study results; current or recent use of topical ophthalmic or systemic medications that could affect a dry eye
`condition; known hypersensitivity to any component of the study or procedural medications; required contact lens
`wear during the study; recent (within 1 month) or anticipated use of temporary punctal plugs during the study;
`permanent occlusion of lacrimal puncta within 3 months of the study; or if they were pregnant, lactating, or
`planning a pregnancy. Patients were also excluded if they appeared to have end stage lacrimal gland disease
`(Schirmer reading with nasal stimulation of < 3 mm/5 minutes) or if their dry eye disease was secondary to the
`destruction of conjunctival goblet cells or scarring. Any patient who no longer met the criteria for moderate to
`severe dry eye (as defined above) after completing the 2-week run-in phase was excluded from enrollment in the
`treatment phase of the study.
`
`
`
`Conjunctival epithelial samples were obtained at baseline, at 3 months, and at 6 months from four
`participating sites in the United States. Informed consent was obtained from all patients before the study. The
`study complied with the Tenets of the Declaration of Helsinki. Conjunctival epithelial samples were obtained by
`impression debridement with a nitrocellulose membrane from the temporal bulbar conjunctiva of the “worse”
`eye. The “worse” eye was defined as the eye with the worse Schirmer tear test value (without anesthesia) and
`the worse sum of corneal and interpalpebral conjunctival staining. If both eyes were comparable, then the right
`eye was used. The membranes were placed in Trizol RNA lysis buffer (Life Technologies, Rockville, MD, U.S.A.) and
`immediately frozen at -80°C. Samples were shipped in dry ice by a commercial overnight delivery service to the
`central laboratory at the University of Miami School of Medicine. Upon arrival, samples were placed in a -80°C
`freezer until the RNA was extracted.
`
`
`RNA Extraction and cDNA Synthesis
`
`
`RNA was extracted from the conjunctival epithelial samples using a previously reported protocol. 5
`Complementary DNA (cDNA) was synthesized with a first strand cDNA synthesis kit (Boehringer, Indianapolis, IN,
`U.S.A.) using 1 µg of total RNA. Reverse transcriptase negative controls were performed for each RNA sample. The
`efficiency of cDNA synthesis was assessed by evaluating the level of cyclophilin mRNA in each sample by
`polymerase chain reaction (PCR) amplification using 35 cycles. We added 2 µL of cDNA to the PCR reaction mixture
`containing 5 µL of 10× reaction buffer, 1 µL deoxy-nucleotide triphosphates (10 mmol/L deoxyadenosine
`triphosphate, 10 mmol/L deoxycytidine triphosphate, 10 mmol/L deoxyguanosine triphosphate, and 10 mmol/L
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`Ovid: Interleukin-6 Levels in the Conjunctival Epithelium of Patients with Dry Eye Disease Treated with Cyclosporine Ophthalmic Emulsion.
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`deoxythymidine triphosphate), 39.7 µL sterile water, 0.3 µL Taq DNA polymerase (5 units/µL), and 2 µL of primers
`specific for cyclophilin. 12 The upstream cyclophilin primer sequence is 5´ ATG GTT AAC CCC ACC GTG TTC GAC 3´,
`and the downstream primer sequence is 5´ CTG GAT TGC AGA GTT AAG TTT 3´. Cyclophilin is a constitutively
`expressed gene in all human cells that serves as the cyclosporine-binding protein. 13 The PCR products were
`separated by 1.6% agarose gel electrophoresis and the intensity of the band was visually graded from 0 (no band)
`to 4+ (strong). cDNA samples that yielded 2+, 3+, or 4+ bands were used for competitive reverse transcriptase
`polymerase chain reaction (RT-PCR) analysis of IL-6 and G3PDH, a housekeeping gene, levels. cDNA samples that
`yielded 0 or 1+ bands were resynthesized.
`
`
`Semiquantitative Analysis of mRNA Levels by Competitive RT-PCR
`
`
`The relative levels of RNA transcripts encoding the inflammatory cytokine IL-6 or the housekeeping protein
`G3PDH were evaluated by a competitive PCR technique. This technique was performed by adding 2 µL of sample
`cDNA to the PCR mixture described above, containing 2 µL of primers specific for IL-6 or G3PDH. 6 Both the IL-6 and
`G3PDH PCR reactions were run for 35 cycles. The primer sequences for IL-6 and G3PDH were chosen to span an
`intron to confirm that the PCR product was due to amplification of cDNA, not genomic DNA. The size of the
`amplified IL-6 cDNA product was 627 base pairs, and the size of the amplified G3PDH cDNA product was 983 base
`pairs. Each reaction tube used to amplify the IL-6 cDNA was spiked with a specifically designed “mimic” template
`(Clontech, Palo Alto, CA, U.S.A.) that contained DNA sequences that were complimentary to the IL-6 PCR primers.
`These primer-specific sequences were ligated to the 5´ and 3´ ends of a nonspecific DNA fragment (in this case, a
`portion of the v-erbB gene). The mimic templates compete with the IL-6-specific cDNA sequences in the
`conjunctival epithelial specimens for the reaction primers and they have a different length (in base pairs) after
`PCR amplification than the cytokine amplification product. The added mimics also served as internal controls in
`each reaction tube. Two concentrations of mimic (10-5 and 10-6 attomole/µL) were used for each sample. PCR
`reactions for G3PDH were also spiked with two concentrations (10-3 and 10-4 attomole/µL) of a mimic with G3PDH
`primer-specific sequences. PCR reactions for IL-6 and G3PDH were performed on baseline (month 0), month 3, and
`month 6 samples, simultaneously for each subject. The levels of PCR products were evaluated by 1.6% agarose gel
`electrophoresis. Gels were stained with ethidium bromide (10 mg/mL) and were photographed with a Polaroid
`camera (Cambridge, MA, U.S.A.). Baseline, month 3, and month 6 PCR products for IL-6 and G3PDH (and their
`mimics) from each subject were run together on one agarose gel (Fig. 1). Two final gels (one for the lower and the
`other for the higher mimic concentration) were run for each subject (Fig. 2). The mimic concentration yielding
`measurable bands for IL-6 and G3PDH that allowed comparisons between at least two time points (baseline to
`month 3, or baseline to month 6, or month 3 to month 6) was used for the final analysis.
`
`
`
`FIG. 1. Ethidium bromide-stained agarose gel. Initial PCR was performed using high and low concentrations of
`mimics for G3PDH (10-3 and 10-4 attomole/µL), and IL-6 (10-5 and 10-6 attomole/µL). The percentage of product
`(G3PDH or IL-6) per total amplified product (gene + mimic) in the lane is given at the bottom. amol = attomole.
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`Ovid: Interleukin-6 Levels in the Conjunctival Epithelium of Patients with Dry Eye Disease Treated with Cyclosporine Ophthalmic Emulsion.
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`
`
`FIG. 2. Example of final gels for each patient RNA sample. G3PDH primers were reacted with 10-4 attomole/µL
`mimic, and IL-6 with 10-6 attomole/µL mimic. The upper band in each lane is the amplified product and the lower
`band is the amplified mimic. Lanes 1, 3, and 5 are G3PDH and the G3PDH mimic; lanes 2, 4, and 6 are IL-6 and the
`IL-6 mimic. Lanes 1 and 2 are baseline, lanes 3 and 4 are month 3, lanes 5 and 6 are month 6, lane 7 is cDNA-(PCR
`reaction mixture without cDNA), and lane 8 is the DNA marker IX (Boehringer, Indianapolis, IN, U.S.A.).
`
`Photographs of each gel were scanned with a Hewlett Packard ScanJet (Palo Alto, CA, U.S.A.). The integrated
`optical density of the IL-6, G3PDH, and their mimic bands in the scanned images was measured. Levels of IL-6 and
`G3PDH in each lane were expressed as a fraction of total amplified product in each lane (e.g., the percentage of
`IL-6 = IL-6/[IL-6 + IL-6 mimic]). Levels of G3PDH in each treatment group were compared over time to determine
`whether the amount of G3PDH changed significantly with CsA treatment. Levels of IL-6 were evaluated at each
`time point (baseline, month 3, and month 6) in relation to the level of G3PDH in each sample by dividing the
`percentage of IL-6 by the G3PHD (normalized IL-6).
`
`
`Statistical Analysis
`
`
`A two-tailed paired Student t test was used to confirm that IL-6 and G3PDH, as a percentage of the product
`plus mimic total, decreased with sample dilution for each group at each follow-up period. Within group
`comparisons of G3PDH and IL-6, changes were performed with the two-tailed paired Student t test. Changes in the
`normalized IL-6 percentage from baseline to 3-and 6-month follow-up were calculated for each subject and
`between-group differences in the extent of change were examined with one-way analysis of variance.
`
`
`
`RESULTS
`
`Mimic as a Competitive Internal Control
`
`
`Mimics were added to the PCR reactions to act both as a positive internal control for the PCR reaction and as
`a competitive template used for calculating the percentage of amplified cytokine or housekeeping protein. To
`confirm the competitive nature of the mimic, the percentage of IL-6 and G3PDH in each lane was compared using
`high and low mimic concentrations. For each treatment group and time point, the percentage of IL-6 and G3PDH
`increased as the concentration of added mimic decreased (Table 1).
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`Ovid: Interleukin-6 Levels in the Conjunctival Epithelium of Patients with Dry Eye Disease Treated with Cyclosporine Ophthalmic Emulsion.
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`
`
`TABLE 1. Average percent of product
`
`Changes from Baseline
`
`
`There were no significant differences between G3PDH levels at months 0, 3, or 6 in any treatment group
`(Table 2). This demonstrates that the levels of the housekeeping protein (G3PDH) are not changed by CsA
`treatment.
`
`
`
`TABLE 2. Change in percent G3PDH from baseline
`
`The levels of IL-6 were then normalized to the G3PDH levels to identify treatment-related changes in IL-6
`mRNA levels. At 6 months posttreatment, a significant decrease in the ratio of the percentage of IL-6 to G3PDH
`was observed in the group treated with 0.05% CsA, but not in the vehicle or 0.10% CsA treatment groups (Table 3).
`The ratio of the percentage of IL-6 to G3PDH was not significantly different from baseline at month 3 in any
`treatment group. There were no significant differences in the between-group ratios of the percentage of IL-6 to
`the percentage of G3PDH.
`
`
`
`TABLE 3. Change in %IL-6/%G3PDH ratio from baselinea Among-group p values from one-way analysis of variance.b
`Values are mimic corrected ratios of IL-6 to G3PDH.c Within-group p value from paired-t-test.Note. CsA =
`cyclosporine ophthalmic emulsion. Values shown are mean ± standard deviation. A negative value indicates a
`decrease from baseline.
`
`DISCUSSION
`
`
`The most important finding of the present study was that normalized IL-6 levels significantly decreased from
`baseline after 6 months of treatment with 0.05% CsA, whereas the decreases seen after treatment with either
`0.1% CsA or vehicle were not statistically significant. The decrease in this inflammatory factor by cyclosporine is
`consistent with the results of another study conducted on conjunctival biopsies taken from a separate cohort of
`patients that participated in this multi-centered clinical trial. Kunert et al. 14 demonstrated that there was a
`significant decrease in the immune activation markers HLA-DR and CD11a after 6 months of treatment with 0.05%
`CsA, whereas these same markers increased in the vehicle group. Other studies have demonstrated that
`increased levels of IL-6, and other inflammatory markers, are associated with dry eye disease. 5–7,15 For example,
`a study by Pflugfelder et al. 6 used the same assay as the present study to demonstrate that the levels of IL-
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`Ovid: Interleukin-6 Levels in the Conjunctival Epithelium of Patients with Dry Eye Disease Treated with Cyclosporine Ophthalmic Emulsion.
`
`1[alpha], IL-6, IL-8, TNF-[alpha], and TNF-[beta]1 RNA found in the conjunctival epithelium of Sjögren's syndrome
`patients were significantly greater than those found in control patients. However, the present study and the
`other study performed on this patient population 14 are the first to demonstrate that a topical ophthalmic
`treatment for dry eye disease (cyclosporin A) can significantly decrease several markers of inflammation.
`
`
`
`In the present study, no statistically significant between-group differences were seen in IL-6 levels. Thus, it is
`not possible to rule out regression to the mean as the source of the IL-6 decrease. 16 However, a more likely
`explanation may be a large therapeutic effect observed in the vehicle group. Disruption of epidermal barrier
`function results in increased levels of several inflammatory cytokines, including TNF-[alpha], IL-1, and IL-6, in the
`epidermis. 17 A therapeutic effect of the oil-in-water vehicle of this cyclosporine emulsion might be expected, as
`topical application of certain lipid mixtures can accelerate epidermal barrier recovery after defined barrier insults
`in mice. 18
`
`
`
`A very small amount of RNA was obtained from the samples provided for this study. Consequently, a highly
`sensitive competitive RT-PCR assay was used to evaluate the levels of IL-6 and a constituently expressed gene,
`G3PDH. The assay was designed to determine the relative amount of each of these mRNAs in the sample when
`compared to a competitive template (mimic) that was added to each of the PCR reaction tubes. The validity of this
`approach is supported by the fact that the levels of G3PDH were not affected by either the study medication or
`the vehicle.
`
`
`
`In conclusion, the findings of the present study demonstrate that topical CsA decreases the levels of IL-6, an
`inflammatory cytokine that is upregulated in the conjunctiva of patients suffering from dry eye disease.
`
`
`
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`11. Iacono AT, Smaldone GC, Keenan RJ, et al. Dose-related reversal of acute lung rejection by aerosolized
`cyclosporine. Am J Respir Crit Care Med 1997; 155:1690–8. [Context Link]
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`13. Ryffel B, Woerly G, Greiner B, Haendler B, Mihatsch MJ, Foxwell BM. Distribution of the cyclosporin binding
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`14. Kunert KS, Tisdale AS, Stern ME, Smith JA, Gipson IK. Effect of cyclosporine ophthalmic emulsion on the
`number of inflammatory cells and goblet cells in conjunctival biopsies of patients with dry eye [ARVO abstract].
`Invest Ophthalmol Vis Sci 1999; 40 (4):S771. [Context Link]
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`15. Tsubota K, Fujihara T, Saito K, Takeuchi T. Conjunctival epithelium expression of HLA-DR in dry eye patients.
`Ophthalmologica 1999; 21316–19. [Context Link]
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`16. Fleiss JL. The design and analysis of clinical experiments. New York: Wiley, 1986. [Context Link]
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`17. Wood LC, Stadler K, Liou A, et al. Barrier disruption increases gene expression of cytokines and the 55kD TNF
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`18. Man MQ M, Feingold KR, Thorfeldt CR, Elias PM. Optimization of physiological lipid mixtures for barrier repair. J
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`
`Key Words: Conjunctiva; Cyclosporin A; Dry eye disease; Interleukin-6; Keratoconjunctivitis sicca
`
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