`
`OPHTHALMOLOGY
`
`’—-—----—-——- WWW.ARCHOPHTHALMOL.COM
`
`NOVEMBER 2000
`
`
`
`
`
`
`Iris coloboma with iris heterochmmia. See page 1590.
`
`
`
`TOIMMUNE RETINOPATHY: PATIENTS WITH
`IIRECOVERIN IMMUNOREACTIVITY AND
`VRETINAL DEGENERATION
`
`VITAMIN SUPPLEMENT USE AND INCIDENT
`CATARACTS IN A POPULATION—BASED STUDY
`
`
`
`'ALYSIS OF TOPICAL CYCLOSPORINE
`EATMENT OF PATIENTS WITH DRY EYE
`NDROME: EFFECT ON CONJUNCTIVAL
`MPHOCYTES
`
`SUCCESSFUL AMBLYOPIA THERAPY INITIATED
`AFTER AGE 7 YEARS: COMPLIANCE CURES
`
`
`
`15 AND CILIARY BODY MELANOMAS:
`iIRASOUND BIOMICROSCOPY WITH
`ITOIATHOLOGIC CORRELATION
`
`A NEW BB FORCEPS
`
`
`
`COMPLETE TABLE OF CONTENTS 0
`
`Herican Medical Association
`
`imans Iedicated t0 the health of America
`
`#8::
`
`UNI
`35F:
`130E
`PHD]
`
`3';
`“I“.
`
`
`
`am!
`
`F:- I
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`
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`APOTEX1015,pg.1
`
`'
`'
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`APOTEX 1015, pg. 1
`
`

`

`ARCHIVES
`__-------------------------------------------------_ .1
`OPHTHALMOLOGY
`
`NOVEMBER 2000 VOLUME 118, NUMBER 11 PAGES 1481—1608
`
`
`
`Analysis of Topical Cyclosporine
`Treatment of Patients With Dry Eye
`Syndrome: Effect on Conjunctival
`Lymphocytes
`Kathleen S. Kunert, MD;
`Ann S. Tisdale, MS; Michael E. Stern, PhD;
`]. A. Smith; Ilene K. Gipson, PhD
`
`Conjunctival Melanoma: Risk Factors
`for Recurrence, Exenteration,
`Metastasis, and Death
`in 150 Consecutive Patients
`
`Carol L. Shields, MD; jerry A. Shields, MD;
`Kaan Gandaz, MD; Jacqueline Cater, PhD;
`Gary V. Mercado, MD; Nicole Gross, MD;
`Brian Lally, MD
`
`Baerveldt Drainage Implants in Eyes
`With a Preexisting Scleral Buckle
`Ingrid U. Scott, MD, MPH; Steven]. Gedde, MD;
`Donald L. Badenz, MD; David S. Greenfield, MD;
`Harry W. Flynn, jr, MD;
`William]. Fetter, MS;
`Mozart O. Mello,]r, MD;
`Rohit Krishna, MD;
`David G. Godfrey, MD
`
`Iris and Ciliary Body Melanomas:
`Ultrasound Biomicroscopy
`With Histopathologic Correlation
`Flavio A. Marigo, MD; Paul T. Finger, MD;
`Steven A. McCormick, MD;
`Raymond Iezzi, MD;
`K. Esahi, MD; H. Ishihawa, MD;
`jeffrey M. Liehmann, MD;
`Robert Ritch, MD
`
`1489
`
`1497
`
`Autoimmune Retinopathy: Patients
`With Antirecoverin Immunoreactivity
`and Panretinal Degeneration
`john R. Hechenlively, MD; Amani A. Fawzi, MD;
`jill Oversier, BS; Berry Ljordan, PhD;
`Nata Aptsiaari, MD, PhD
`
`Successful Amblyopia Therapy Initiated
`After Age 7 Years: Compliance Cures
`Helen A. Mintz-Hittner, MD;
`Kristina M. Fernandez, MA
`
`The Effect of Anterior Transposition
`of the Inferior Oblique Muscle on the
`Palpebral Fissure
`Burton]. Kashner, MD
`
`1525
`
`1535
`
`1542 l
`
` MBORATORY SCIENCES
`
`1509
`
`Subconjunctival Carboplatin
`in Retinoblastoma: Impact of Tumor
`Burden and Dose Schedule
`
`1549
`
`Brandy H. Hayden, BS; Timothy G. Murray, MD;
`Ingrid U. Scott, MD, MPH; Nicole Cicciarelli;
`Eleut Hernandez; William Fetter, MS;
`Lilia Fulton, BS;
`joan M. O’Brien, MD
`
`1515
`
`
`
`EPIDEMIOLOGY AND BIOS'I'A'I'IS'I'ICSE.
`
`Vitamin Supplement Use and Incident
`Cataracts in a Population-Based Study
`Julie A. Mares-Perlman, PhD;
`Barbara]. Lyle, PhD; Ronald Klein, MD;
`Alicia I. Fisher, MS; William E. Brady, MS;
`' Gina M. VandenLangenherg, PhD;
`jillian N. Trahulsi, BS; Mari Palta, PhD
`
`1556
`
`American Medical Association
`
`Physicians dedicated to the health of America
`
`Copyright 2000 by the American Medical Association. All rights reserved.
`Reproduction without permission is prohibited.
`All articles published, including editorials, letters, and book reviews, represent
`the opinions of the authors and do not reflect
`the policy of the American
`Medical Association, the Editorial Board, or the institution with which the
`author is affiliated, unless this is clearly specified.
`
`Catherine D. DeAngelis, MD, MPH
`Editor in Chief, Scientific Publications
`Sr Multimedia Applications
`Ruben L. Kennett
`Vice President, Publishing
`Peter L. Payerli
`Publisher
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`Cheryl Iversun
`Managing Editor
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`Roberl A. Musacehio, PhD
`Senior Vice President,
`Publishing 57: Business Scrvi ces
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`WWW. ARCHOPHTHALMOLCOM
`ARCH OPHTHALMOL/VOL 118, NOV 2000
`14-84
`
`APOTEX 1015, pg. 2
`
`APOTEX 1015, pg. 2
`
`

`

`Analysis of Topical Cyclosporine Treatment
`·of Patients With Dry Eye Syndrome
`
`Effect on Conjunctival Lymphocytes
`
`Kathleen S. Kunert, MD; Ann S. Tisdale, MS; Michael E. Stern, PhD;]. A. Smith; Ilene K. Gipson, PhD
`
`Obiective: To study the effect of topical cyclosporine
`on lymphocyte activation within the conjunctiva of
`patients with moderate to severe dry eye syndrome
`(Sjogren and non-Sjogren) .
`
`Me thods: Biopsy specimens were obtained at baseline
`and after 6 months of cyclosporine treatment from eyes
`of 32 patients with moderate to severe dry eye syn(cid:173)
`drome; 19 were cyclosporine treated (0.05% cyclospor(cid:173)
`ine, n= 13; 0.1% cyclosporine, n=6) and 13 were ve(cid:173)
`hicle treated. Within this group there were 12 with Sjogren
`syndrome and 20 with non-Sjogren syndrome. Biopsy
`tissue was analyzed using immunohistochemical local(cid:173)
`ization of binding of monoclonal antibodies to lympho(cid:173)
`cytic markers CD3 , CD4, and CDS as well as lympho(cid:173)
`cyte activation markers CD11a and HLA-DR.
`
`Re sults: In cyclosporine-treated eyes, biopsy results of
`conjunctivae showed decreases in the number of cells posi-
`
`tive for CD3, CD4, and CDS, while in vehicle-treated eyes,
`results showed increases in these markers, although these
`differences were not statistically significant. Following
`treatment with 0.05% cyclosporine, there was a signifi(cid:173)
`cant decrease in the number of cells expressing the lym(cid:173)
`phocyte activation markers CD11a (P<.05) and HLA-DR
`(P< .05), indicating less activation oflymphocytes as com(cid:173)
`pared with vehicle treatment. Within the Sjogren pa(cid:173)
`tient subgroup, those treated with 0.05% cyclosporine
`also showed a significant decrease in the number of cells
`positive for CD11a (P< .001) as well as CD3 (P< .03),
`indicating a reduction in number of activated lympho(cid:173)
`cytes.
`
`Co nclusion: Treatment of dry eye syndrome with topi(cid:173)
`cal cyclosporine significantly reduced the numbers of ac(cid:173)
`tivated lymphocytes within the conjunctiva.
`
`Arch Ophthalmol. 2000;118:1489-1496
`
`K ERATOCONJUNCTIVITISsicca
`
`(KCS) , or dry eye syn(cid:173)
`drome, is characterized by
`chronic dryness of the cor(cid:173)
`nea and conjunctiva. 1 Pa(cid:173)
`tients with KCS typically show symp-
`toms of ocular discomfort ranging from
`irritation to severe pain. Redness, burn(cid:173)
`ing, itching, foreign body sensation, con(cid:173)
`tact lens intolerance, photophobia, and
`blurred vision can occur. 2
`Although KCS can arise from vari(cid:173)
`ous types of diseases, common to all is the
`involvement of immune-mediated or in(cid:173)
`flammatory-mediated pathways. 3 Immu(cid:173)
`nopathologic studies of the lacrimal gland
`in patients with Sjogren syndrome show
`progressive lymphocytic infiltration, pri(cid:173)
`marily consisting of CD4+ T and B cells. 4
`5
`•
`This infiltration is believed to be respon(cid:173)
`sible for the destruction of normal secre(cid:173)
`tory function. 6 Lymphocytic infiltration of
`the lacrimal gland has also been de(cid:173)
`scribed in patients with non-Sjogren
`KCS. 7
`8 Although the immunopathologic
`•
`
`analysis of the lacrimal gland has re(cid:173)
`ceived considerable attention, less work
`has been done on pathological changes oc(cid:173)
`curring in the ocular surface. The chronic
`dryness of the ocular surface in Sjogren
`syndrome has been attributed to deterio(cid:173)
`ration oflacrimal gland function with de(cid:173)
`10 However, in
`creased tear production. 9
`·
`Sjogren syndrome, conjunctival epithe(cid:173)
`lial and stromal T -cell infiltration (pre(cid:173)
`dominantly CD3+ and CD4+ T lympho(cid:173)
`cytes) has also been shown to occur along
`with drying of the ocular surface. 9·11
`Supporting a role for an immuno(cid:173)
`pathogenesis of KCS are the reports of ac(cid:173)
`tivated lymphocytes as demonstrated by
`expression oflymphocyte activation mark(cid:173)
`ers such as HLA-DR (MHC class II) and
`ICAM-1 (intercellular adhesion mol(cid:173)
`ecule-1) in the conjunctiva of patients with
`13 To date , there is
`Sjogren syndrome. 12
`·
`little information on the effect of modu(cid:173)
`lating these molecules in the conjunctiva
`of patients with Sjogren and non(cid:173)
`Sjogren syndrome.
`
`ARCH OPHTHALMOL/VOL 118, NOV 2000
`1489
`
`WWW.ARCHOPHTHALMOL.COM
`
`From the Schepens Eye
`Research Institute and
`Department of Ophthalmology,
`Harvard Medical School,
`Boston, Mass (Drs Kunert and
`Gipson,· Ms Tisdale); Allergan,
`Inc, Irvine, Calif (Dr Stern);
`and the National Eye Institute,
`Bethesda, Md (Ms Smith).
`Dr Stern is an employee of
`Allergan Inc.
`
`APOTEX 1015, pg. 3
`
`

`

`SUBJECTS AND METHODS
`
`SUBJECTS
`
`Conjunctival biopsy specimens from 32 patients were ex(cid:173)
`amined; l3 patients were treated with 0.05% CsA, 6 with
`0.1% CsA, and 13 with vehicle alone. This subject group
`was randomly chosen from a double-masked, vehicle(cid:173)
`controlled clinical study designed by Allergan, Inc, Irvine,
`Calif, to investigate the efficacy and safety of topical CsA
`in the treatment of moderate to severe KCS.21 The study
`was conducted in compliance with Good Clinical Prac(cid:173)
`tices, investigational site institutional review board regu(cid:173)
`lations, sponsor and investigator obligations, informed con(cid:173)
`sent regulations, and the Declaration of Helsinki. Potential
`patients signed a prescreening informed consent form and
`a second written informed consent form prior to actual en(cid:173)
`rollment. 21 The protocol for this study is described briefly
`here. Adult patients of either sex were eligible for partid(cid:173)
`pation if they had a diagnosis of moderate to severe KCS
`at initial examination as defined by the following criteria:
`(l) Schirmer test results (without anesthesia) less than or
`equal to 5 mm/5 min in at least 1 eye (if Schirmer test re(cid:173)
`sults without anesthesia equaled 0 mm/5 min, then Schirmer
`test results with nasal stimulation had to be > 3 mm/5 min
`in the same eye); (2) sum of corneal and interpalpebral con(cid:173)
`junctival staining greater than or equal to +5 in the same
`eye where corneal staining was greater than or equal to +2;
`(3) a baseline Ocular Surface Disease Index22 score of 0.1
`with no more than 3 responses of "not applicable"; and ( 4)
`a score greater than or equal to 3 on the Subjective Facial
`Expression Scale. 21 Signs and symptoms must have been
`present despite conventional management.
`Patients were excluded from the study if they had par(cid:173)
`ticipated in an earlier clinical trial with CsA ophthalmic
`emulsion or had used systemic or topical ophthalmic CsA
`within 90 days prior to the study. Other exclusion criteria
`were the presence or history of any systemic or ocular dis(cid:173)
`order or condition (including ocular surgery, trauma, and
`disease); current or recent use of topical ophthalmic or sys(cid:173)
`temic medications that could affect a dry eye condition;
`known hypersensitivity to any component of the drug or
`procedural medications such as stains or anesthetics ;
`
`required contact lens wear during the study; recent (within
`1 month) or anticipated use of temporary punctal plugs dur(cid:173)
`ing the study; permanent occlusion oflacrimal puncta within
`3 months of the study; or if the patients were pregnant, lac(cid:173)
`tating, or planning a pregnancy. Patients were also ex(cid:173)
`cluded if they appeared to have end-stage lacrimal gland
`disease (Schirmer reading with nasal stimulation < 3 mm/~
`min) or if:their KCS was secondary to the destruction of
`conjunctival goblet cells or scarring.
`A retrospective diagnosis of Sjogren syndrome was used
`with modified criteria reported by Vitali et aF3 to ensure
`that a consistent definition of Sjogren syndrome was as(cid:173)
`signed to the patients enrolled. Diagnosis included pres(cid:173)
`ence of at least one of the following autoantibodies in sera:
`antinuclear antibody (ANA) , rheumatoid factor (RF) , and
`Sjogren syndrome autoantibodies class SS-A (Ro) and class
`SS-B (La) . In addition, oral and ocular symptoms were used
`to classify patients with Sjogren syndrome.
`Patients instilled 1 drop of 0.05% or 0.1% CsA oph(cid:173)
`thalmic emulsions or vehicle of CsA ophthalmic emulsion
`twice daily in each eye for 6 months; once on waking in
`the morning and once at bedtime. Patients were allowed
`to use assigned artificial tears (REFRESH Lubricant Eye
`Drops; Allergan Inc) as needed up to month 4.
`Full-thickness conjunctival biopsy specimens of a stan(cid:173)
`dard size (2-3 mm) were removed from the "worse" eye
`by surgeons following standard procedure. 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. At the baseline
`visit, the conjunctival biopsy specimen was obtained from
`the inferonasal quadrant close to midline. At the 6-month
`visit, the sample was removed from the same eye but from
`the inferotemporal quadrant, also close to midline.
`
`TISSUE PROCESSING FOR
`IMMUNOHISTOCHEMICAL ANALYSIS
`
`After removal, the baseline biopsy specimens were imme(cid:173)
`diately frozen in OCT embedding compound (Tissue-Tek;
`Miles Laboratories, Elkhart, Ind) in a cryomold (Miles
`Laboratories) and stored at -80°C until patient-matched
`
`Currently, administration of artificial tears is the most
`common therapy available for lubricating a dry ocular sur(cid:173)
`face. This palliative treatment gives only temporary and in(cid:173)
`complete symptomatic relief and does not address the cause
`of the symptoms, which may include immune-mediated
`inflammation of the ocular surface. Evidence of inflamma(cid:173)
`tory processes in the pathogenesis of KCS led to the de(cid:173)
`velopment of cyclosporine ( CsA) as a first attempt to treat
`this condition therapeutically. Cyclosporine is an immu(cid:173)
`nosuppressive agent commonly used systemically to treat
`inflammatory diseases such as psoriasis or rheumatoid ar(cid:173)
`thritis or to prevent organ transplant rejection. 14 Topical
`CsA has been used as treatment of ocular conditions such
`as vernal keratoconjunctivitis, 15 corneal transplants/ 6 cor(cid:173)
`neal ulcers/ 7 and herpetic stromal keratitis. 18 The effect of
`this drug on inflammatory diseases is due to its ability to
`
`inhibit T -cell-mediated inflammation by preventing the: ac(cid:173)
`tivation ofT cells (by antigen-presenting cells or
`20 Activated T cells are responsible for the pro(cid:173)
`cytokines) .19
`·
`duction of inflammatory substances such as cytokines,
`which lead to further tissue damage and, in turn, to the ac(cid:173)
`tivation of more T cells and the production of even more
`inflammatory substances.
`Clinical trials with this drug have shown improve(cid:173)
`ment in various objective measures of KCS such as cor(cid:173)
`neal staining and Schirmer test valuesY To attempt to
`find tissue correlates in these patients, conjunctival bi(cid:173)
`opsy specimens from patients with Sjogren and non(cid:173)
`Sjogren KCS treated with CsA or vehicle were evaluated
`immunohistochemically for the presence of activated T
`cells ( CD3+ [Pan-T cell], CD4+ [T helper cell], and CDS+
`[cytotoxic T cell]) and lymphocyte-activation markers
`
`ARCH OPHTHALMOL/VOL 118, NOV 2000
`1490
`
`WWW.ARCHOPHTHALMOL.COM
`
`APOTEX 1015, pg. 4
`
`

`

`6-month biopsy specimens were obtained and similarly fro(cid:173)
`zen. Six-micrometer sections were taken from each block,
`mounted on gelatin-coated slides, and processed for im(cid:173)
`munohistochemical analysis. Sectioning of tissue blocks and
`immunohistochemical experiments were performed as pairs
`of biopsies, pretreatment and posttreatment, to minimize
`differences due to experimental conditions.
`
`IMMUNOHISTOCHEMICAL ANALYSIS
`
`Immunohistochemical staining for lymphocytic markers as
`well as lymphocyte activation markers was conducted us(cid:173)
`ing monoclonal antibodies to CD3 (PharMingen, San Diego,
`CaliD, CD4 (Becton-Dickinson, Sanjose, CaliD, CDS (Bee(cid:173)
`torr-Dickinson, Sanjose) , CDlla (PharMingen, San Diego) ,
`and HLA-DR (PharMingen). Cryostat sections were fixed
`in cold acetone ( -20°C) for 3 minutes and air dried at room
`temperature for 30 to 45 minutes. They were then rinsed
`in 3 changes of phosphate-buffered saline (PBS) and incu(cid:173)
`bated in PBS with 1% bovine serum albumin (BSA) (Sigma
`Chemical Co, StLouis, Mo) for 10 minutes. Sections were
`incubated for 1 hour at room temperature in primary an(cid:173)
`tibodies at concentrations derived empirically: CD3, l.O
`pg/mL; CD4, 5.0 pg/mL; CDS , 2.5 pg/mL; CDlla, 10.0
`pg/mL; and HLA-DR, l.O pg!mL. Sections were rinsed in
`PBS alone, followed by 10 minutes in PBS with 1% BSA be(cid:173)
`fore incubation for 1 hour at room temperature in the sec(cid:173)
`ondary antibody, fluorescein isothiocyanate-conjugated Af(cid:173)
`finipure Donkey Anti-Mouse IgG Qackson Immunoresearch,
`West Grove, Pa) at a dilution of 1/50. Sections were then
`rinsed in PBS, mounted in Vectashield (Vector Labs, Bur(cid:173)
`lingame, CaliD, cover-slipped, and viewed under a micro(cid:173)
`scope (Eclipse ESOO; Nikon, Melville, NY) interfaced with
`a digital camera (Spot Digital Camera; Diagnostic Instru(cid:173)
`ments Inc, Micro Video Instruments, Avon, Mass). Sec(cid:173)
`ondary antibody controls omitting the primary antibody
`for all biopsy specimens for each immunohistochemical
`analysis were run.
`Three separate images were acquired for each anti(cid:173)
`body and biopsy specimen under a X 20 objective using a
`Spot acquisition program (Diagnostic Instruments Inc) . The
`first field selected for imaging was the field with the high(cid:173)
`est number of positive cells, followed by images to the left
`
`and right of that area. In this manner the entire biopsy area
`was usually captured.
`
`COUNTING PROCEDURE
`
`Measurement of the entire area of epithelium and stroma
`(substantia propria) was achieved by tracing the area us(cid:173)
`ing the lasso tool under the Adobe Photoshop computer
`program (Adobe Systems Inc, Sanjose, CaliD . The total data
`area, measured in pixels, was acquired through the "Im(cid:173)
`age: Histogram" command in Photoshop. Two indepen(cid:173)
`dent counts were recorded for cells positive for each anti(cid:173)
`body within the traced area. Cells per unit area of pixels
`were adjusted to real unit area or cells per millimeter squared
`of real tissue area, based on 2S.346 pixels per centimeter
`in Photoshop and the fact that 1 mm equals 67.S em equals
`1922 pixels at X20 magnification on the Nikon micro(cid:173)
`scope. Data were recorded as cells per millimeter squared
`for all markers, and statistical analysis was based on these
`measurements.
`
`STATISTICAL METHODS
`
`Baseline characteristics were tabulated and summarized by
`treatment groups. Overall differences among treatment
`groups were tested using a 2-way analysis of variance
`(AN OVA) for continuous variables and the Fisher exact test
`for categorical variables.
`Percent changes in the number of cells expressing
`lymphocytic and/or lymphocyte activation markers were
`summarized using descriptive statistics (ie, sample size,
`mean, SD, minimum, maximum, and median). A 1-way
`ANOVA with main effect for treatment was used to test
`for differences in percent change from baseline and
`ratios among treatment groups by visit. If the test for
`among-group differences in main effect was significant,
`then all 3 pairwise comparisons were made. Within(cid:173)
`group changes from baseline were analyze d by the
`paired t test method.
`The same analysis was performed on Sjogren and
`non-Sjogren subpopulations, excluding the 0.1% CsA
`treatment group in which there was only 1 patient in the
`Sjogren subset.
`
`(CD11a and HLA-DR) to further understand the under(cid:173)
`lying mechanism of CsA treatment.
`
`RESULTS
`
`PATIENT POPULATION
`
`The mean±SD age of our subjects was 59.0± 13.5 years
`(range, 2S.S-S4.2 years), including 27 women and 5 men.
`\Vithin this group, there were 12 Sjogren and 20 non(cid:173)
`Sjogren patients.
`
`LYMPHOCYTIC MARKERS
`
`In general, there was a decrease from baseline in the num(cid:173)
`ber of cells positive for CD3 , CD4, and CDS following
`
`treatment with either concentration of CsA. The only ex(cid:173)
`ception was that there was a mean increase from base(cid:173)
`line in the CD4-positive T helper cell population follow(cid:173)
`ing 0.05% CsA treatment. In comparison, all cells positive
`for the lymphocytic markers increased from baseline fol(cid:173)
`lowing vehicle treatment.
`Figure 1 shows the percent change from baseline
`for cells expressing the lymphocytic markers (CD3, CD4,
`and CDS) after 6 months of treatment for the overall pa(cid:173)
`tient population. Note that there was a reduction from
`baseline in the number of CD3-positive cells in the CsA(cid:173)
`treated groups, while there was an increase from base(cid:173)
`line in the vehicle-treated group. There was also an in(cid:173)
`crease from baseline in the numbers of CD4-positive cells
`in the vehicle group, with a smaller increase in the 0.05%
`CsA group and a slight decrease in the 0.1% CsA group.
`
`ARCH OPHTHALMOLIVOL 118, NOV 2000
`1491
`
`WWW. ARCHOPHTHALMOL .COM
`
`APOTEX 1015, pg. 5
`
`

`

`140
`
`120
`
`100
`
`80
`
`"' ! 60
`
`u
`40
`<f!.
`c
`"' 20
`~
`
`-20
`
`-40
`
`-60
`
`0 0.05% CsA
`• 0.1% CsA
`0 Vehicle
`
`250
`
`200
`
`w 0.05% CsA
`• 0.1 % CsA
`0 Vehicle
`
`150
`"' Ol
`~ 100
`u
`<f!.
`c 50
`"' ~
`
`-~
`
`-50
`
`CD3
`
`CD4
`
`COB
`
`CD11a
`
`HLA-DR
`
`-100
`
`Figure 1. Percent change for cells positive for the lymphocytic markers GD3,
`GD4, and GDB in the overall patient population. Values presented are mean
`percent change±SE from baseline at month 6. GsA indicates cyclosporine.
`
`Figure 3. Percent change for cells positive for the lymphocyte activation
`markers GD11a and HLA-DR in the overall patient population. Values
`presented are mean percent change±SE from baseline at month 6. The P
`values are relative to pairwise comparisons (P< .05) and within-group
`differences (P<. 03). GsA indicates cyclosporine.
`
`20
`
`10
`
`-10
`
`~ -20
`
`~ -30
`~ -40
`~ -50
`::2:
`-60
`
`-70
`
`-80
`
`-90
`
`-100
`
`P<.03
`
`I
`
`·~~""
`
`'"~~kw:N:~
`
`l_
`
`I D 0.05% C;;A
`0 Vehicle
`
`Sjogren Syndrome
`
`CD3
`
`_l_
`
`l
`
`Non-S)ogren Syndrome
`
`Figure 2. Percent change for GD3-positive cells from the Sjogren syndrome
`and non-Sjogren syndrome subpopulations. Values presented are mean
`percent change±SE from baseline at month 6. The P value is relative to
`pairwise comparisons from 1-way analysis of variance. GsA indicates
`cyclosporine.
`
`The CDS-positive cells exhibited the same pattern as CD3-
`positive cells but with less of a decrease from baseline
`following CsA and less of an increase from baseline fol(cid:173)
`lowing vehicle treatment. However, the change from base(cid:173)
`line in the number ofT lymphocytes (CD3+, CD4+, and
`CDS+) did not reach statistical significance, either among
`or within treatment groups (Figure 1).
`Within the Sjogren subgroup, 0.5% CsA treatment
`resulted in significantly greater (P< .03) decreases in CD3-
`positive cells than did vehicle. The CD3-positive cells de(cid:173)
`creased from baseline in all treatment groups among the
`non-Sjogren subgroup. However, this decrease was not
`statistically significant in either group (Figure 2).
`
`lYMPHOCYTE-ACTIVATION MARKERS
`
`In general, there was a decrease from baseline in the num(cid:173)
`ber of cells positive for lymphocyte activation markers
`CDlla and HLA-DR following CsA treatment com(cid:173)
`pared with an increase from baseline in these cells fol-
`
`40
`
`20
`
`f -20
`
`~ -40
`c "' ~ -60
`
`-80
`
`-100
`
`-120
`
`T
`
`I
`
`P<.b01
`
`I
`
`1
`
`I D 0.05% ,,,
`D Vehicle
`
`_L
`
`_L
`
`Sjogren Syndrome
`
`Non-Sji.igren Syndrome
`
`CD11a
`
`[·.
`
`f
`
`··- -
`
`Figure 4. Percent change for GD11a-positive cells from the Sjogren
`syndrome and non-Sjogren syndrome subsets. Values presented are m.::an
`percent change±SE from baseline at month 6. The P value is relative to
`pairwise comparisons from 1-way analysis of variance. GsA indicates
`cyclosporine.
`
`lowing vehicle treatment for the overall patient popula(cid:173)
`tion.
`Statistical analysis revealed a significant among(cid:173)
`group difference in change from baseline for cells ex(cid:173)
`pressing CDlla (P=.04) and HLA-DR (P= .02) for the
`overall patient population. Pairwise comparisons showed
`significant reductions with 0.05% CsA treatment com(cid:173)
`pared with treatment with vehicle in cells positive for both
`markers CDlla (P= .05) and HLA-DR (P=.O l 6)
`(Figure 3) . Furthermore, a comparison within indi(cid:173)
`vidual treatment groups, comparing pretreatment to post(cid:173)
`treatment results, revealed a statistically significant de(cid:173)
`crease from baseline for HLA-DR in the 0.05% CsA group
`(P= .03) (Figure 3).
`Within the Sjogren subgroup treated with 0.5% CsA,
`there were significantly greater (P< .OOl) decreases in cells
`positive for CD lla than in vehicle. There was a de(cid:173)
`crease from baseline in both treatment groups (CsA and
`vehicle) among the non-Sjogren subgroup (Figure 4 ).
`This decrease did not reach statistical significance.
`
`ARCH OPHTHALMOL!VOL 118, NOV 2000
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`

`

`Figure 5. Immunofluorescence micrographs demonstrating cells positive for the lymphocyte activation marker CD11a in conjunctival biopsy specimens of
`patients with non-Sjogren keratoconjunctivitis sicca pretreatment and posttreatment with (A and B) 0.05% cyclosporine and (C and D) vehicle. The number of
`positive cells within epithelium and substantia propria in the cyclosporine-treated group decreased, while the number in the vehicle-treated biopsy sample
`increased (bar=25 pm).
`
`Figure 5 and Figure 6 show a representative set
`of immunofluorescence micrographs for cells positive for
`the markers CD11a and HLA-DR from the non-Sjogren
`subgroup treated with 0.05% CsA or vehicle. Figure 7
`shows immunofluorescence micrographs for cells posi(cid:173)
`tive for the markers CD3 and CD 11a from patients with
`Sjogren KCS treated with 0.05% CsA.
`
`COMMENT
`
`In the present study, immunohistochemical analysis was
`used to evaluate changes in the presence of cells posi(cid:173)
`tive for lymphocytic and lymphocyte activation mark(cid:173)
`ers in conjunctival biopsy specimens of patients with mod(cid:173)
`erate to severe KCS, following treatment with 0.05% CsA,
`0.1% CsA, or vehicle. We found that CsA treatment re(cid:173)
`d11ced the number of activated T lymphocytes within the
`ocular surface of patients with and without Sjogren syn(cid:173)
`drome. After 6 months of treatment with 0.05% CsA, sta(cid:173)
`tistically significant decreases were seen in cells positive
`for CD11a and HLA-DR compared with those in vehicle
`for the overall patient population. Within the Sjogren pa(cid:173)
`tient subgroup treated with 0.05% CsA, there were also
`significantly greater decreases than with vehicle in the
`number of cells positive for CD3 and CD 11a.
`
`These findings provide additional evidence that in(cid:173)
`flammation plays a role in the pathogenesis of KCS and
`suggests that modulating the underlying immune re(cid:173)
`sponse may prove more efficacious in the treatment of
`KCS than the frequent use of artificial tears. Topical CsA
`has been successfully used for the treatment of canine
`dry eye for many years. Studies in the canine KCS model
`have demonstrated that CsA decreases the conjunctival
`26
`and lacrimal gland lymphocytic infiltrates. 24
`-
`However, there have been only a limited number of
`reports on the use of topical CsA in the treatment of dry
`29 with only 1 attempt to look
`eye syndrome in humans 27
`-
`, at the effect of the treatment at a cellular level.30 Power
`et aP0 reported a significant reduction in CD4-positive
`T lymphocytes in both the conjunctival epithelium and
`the substantia propria of patients with secondary Sjo(cid:173)
`gren syndrome compared with non-dry eye controls fol(cid:173)
`lowing treatment with CsA. The present study also dem(cid:173)
`onstrated a significant decrease in CD3-positive cells after
`6 months of 0.05% CsA treatment in patients with Sjo(cid:173)
`gren syndrome.
`Furthermore, the number of cells positive for CD 11a
`and HLA-DR, which are lymphocyte activation mark(cid:173)
`ers, decreased significantly in patient populations treated
`with CsA. HLA-DR is a class II major histocompatibility
`
`ARCH OPHTHALMOLIVOL 118, NOV 2000
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`

`Figure 6. Immunofluorescence micrographs demonstrating cells positive tor HLA-DR in conjunctival biopsy specimens of patients with non-Sjogren
`keratoconjunctivitis sicca pretreatment and posttreatment with (A and B) 0.05% cyclosporine and (C and D) vehicle. A decrease in the number of positive cells
`within epithelium and substantia propria in the 0.05% cyclosporine-treated group is apparent compared with an increase in number in the vehicle-treated biopsy
`sample. E and F, Example of a negative control tor a vehicle biopsy in which the primary antibody was omitted. Bar=25 Jlm (A-C).
`
`complex antigen that is expressed in inflamed regions and
`serves as a ligand for the T -cell receptor. CD4+ T lym(cid:173)
`phocytes are activated through a signal from HLA-DR mol(cid:173)
`ecules of antigen-presenting cells. 31 Immunopathologic
`studies show evidence of immune activation of the con(cid:173)
`junctival epithelium in Sjogren syndrome. Compared with
`control eyes, a significantly greater percentage of con(cid:173)
`junctival epithelial cells from patients with Sjogren syn(cid:173)
`drome express the HLA-DR moleculeY·32 Hingorani et
`aP3 report a decrease in HLA-DR expression on cells in
`the substantia propria of patients with atopic keratocon(cid:173)
`junctivitis following 3 months of treatment with CsA. In
`
`confirmation of these findings, the data presented here
`demonstrate a reduction in the number of cells positive
`for the lymphocyte activation marker HLA-DR after 6
`months of 0.05% CsA treatment.
`CD 11a/LFA-1 (lymphocyte function-associated an- ·
`tigen) is associated with adhesion of lymphocytes, mac(cid:173)
`rophages, and granulocytes and is a ligand of intercel(cid:173)
`lular adhesion molecule-1 (ICAM-1), which supports the
`binding of lymphocytes to antigen-presenting cells. 34
`CD11a is up-regulated during activation of human lym(cid:173)
`phocytes and, with its ligand ICAM-1, plays an impor(cid:173)
`tant role in cell-to-cell interactions and cell migration of
`
`ARCH OPHTHALMOL/VOL llS, NOV 2000
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`

`Figure 7. Immunofluorescence micrographs demonstrating cells positive for (A and B) CD3 and (C and D) CD11a in conjunctival biopsy specimens of patients
`with Sjogren keratoconjunctivitis sicca pretreatment and posttreatment with 0.05% cyclosporine. Note the decrease in number of positive cells within the
`epithelium and substantia propria in the posttreatment biopsy specimens (bar=25 pm).
`
`lymphocytes into the surrounding tissue such as the con(cid:173)
`37 Cyclo(cid:173)
`junctival epithelium and substantia propria. 35
`-
`sporine has been shown to regulate immune-based in(cid:173)
`flammation within epithelial tissues by inhibiting ICAM-1
`production.38 Our data support these results, showing re(cid:173)
`duced immune activation by means of a decrease in the
`number of cells positive for CD lla after a 6-month course
`of 0.05% topical CsA treatment.
`Part of the beneficial effect of CsA might be due to
`the reduction in T -cell activation as illustrated by a de(cid:173)
`crease of cells positive for HLA-DR. By preventing the
`migration of new lymphocytes into the conjunctiva, as
`suggested by the reduction in CD lla-positive cells, CsA
`may help to. reduce the inflammatory process. The fact
`that the data show a reduction in positive cells mainly
`fo r the lymphocyte activation markers CD lla and
`HLA-DR suggests that CsA is promoting lymphocytes to
`a more quiescent status rather than eliminating present
`lymphocytes. This might explain why the change from
`baseline in the number ofT lymphocytes (CD3+, CD4+,
`and CD8+) did not reach statistical significance for the
`overall patient population. However, another contribut(cid:173)
`ing factor may be the small patient number and high vari(cid:173)
`ability within each treatment group.
`
`These results provide further evidence that topical
`use of CsA may have a local immunoregulatory effect on
`inflammation in the conjunctiva-of patients with dry eye
`syndrome. This effect is evident in the reduction of the
`number of cells positive for lymphocyte activation mark(cid:173)
`ers. In preventing the