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`ƒîððð ß³»®·½¿² Ó»¼·½¿´ ß­­±½·¿¬·±²ò ß´´ ®·¹¸¬­ ®»­»®ª»¼ò
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`
`6-month biopsy specimens were obtained and similarly fro-
`êó³±²¬¸ ¾·±°­§ ­°»½·³»²­ ©»®» ±¾¬¿·²»¼ ¿²¼ ­·³·´¿®´§ º®±ó
`zen. Six-micrometer sections were taken from each block,
`¦»²ò Í·¨ó³·½®±³»¬»® ­»½¬·±²­ ©»®» ¬¿µ»² º®±³ »¿½¸ ¾´±½µô
`mounted on gelatin-coated slides, and processed for im-
`³±«²¬»¼ ±² ¹»´¿¬·²ó½±¿¬»¼ ­´·¼»­ô ¿²¼ °®±½»­­»¼ º±® ·³ó
`munohistochemical analysis. Sectioning of tissue blocks and
`³«²±¸·­¬±½¸»³·½¿´ ¿²¿´§­·­ò Í»½¬·±²·²¹ ±º ¬·­­«» ¾´±½µ­ ¿²¼
`immunohistochemical experiments were performed as pairs
`·³³«²±¸·­¬±½¸»³·½¿´ »¨°»®·³»²¬­ ©»®» °»®º±®³»¼ ¿­ °¿·®­
`of biopsies, pretreatment and posttreatment, to minimize
`±º ¾·±°­·»­ô °®»¬®»¿¬³»²¬ ¿²¼ °±­¬¬®»¿¬³»²¬ô ¬± ³·²·³·¦»
`differences due to experimental conditions.
`¼·ºº»®»²½»­ ¼«» ¬± »¨°»®·³»²¬¿´ ½±²¼·¬·±²­ò
`
`IMMUNOHISTOCHEMICAL ANALYSIS
`×ÓÓËÒÑØ×ÍÌÑÝØÛÓ×ÝßÔ ßÒßÔÇÍ×Í
`
`Immunohistochemical staining for lymphocytic markers as
`׳³«²±¸·­¬±½¸»³·½¿´ ­¬¿·²·²¹ º±® ´§³°¸±½§¬·½ ³¿®µ»®­ ¿­
`well as lymphocyte activation markers was conducted us-
`©»´´ ¿­ ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µ»®­ ©¿­ ½±²¼«½¬»¼ «­ó
`ing monoclonal antibodies to CD3 (PharMingen, San Diego,
`·²¹ ³±²±½´±²¿´ ¿²¬·¾±¼·»­ ¬± ÝÜí øи¿®Ó·²¹»²ô Í¿² Ü·»¹±ô
`Calif), CD4 (Becton—Dickinson, Sanjose, Calif), CD8 (Bec-
`Ý¿´·º÷ô ÝÜì øÞ»½¬±²óÜ·½µ·²­±²ô Í¿² Ö±­»ô Ý¿´·º÷ô ÝÜè øÞ»½ó
`ton—Dickinson, Sanjose), CD1 1a (PharMingen, San Diego),
`¬±²óÜ·½µ·²­±²ô Í¿² Ö±­»÷ô ÝÜïï¿ øи¿®Ó·²¹»²ô Í¿² Ü·»¹±÷ô
`and HLA—DR (PharMingen). Cryostat sections were fixed
`¿²¼ ØÔßóÜÎ øи¿®Ó·²¹»²÷ò Ý®§±­¬¿¬ ­»½¬·±²­ ©»®» º·¨»¼
`in cold acetone (—20°C) for 3 minutes and air dried at room
`·² ½±´¼ ¿½»¬±²» ø"îðWÝ÷ º±® í ³·²«¬»­ ¿²¼ ¿·® ¼®·»¼ ¿¬ ®±±³
`temperature for 30 to 45 minutes. They were then rinsed
`¬»³°»®¿¬«®» º±® í𠬱 ìë ³·²«¬»­ò ̸»§ ©»®» ¬¸»² ®·²­»¼
`in 3 changes of phosphate-buffered saline (PBS) and incu-
`·² í ½¸¿²¹»­ ±º °¸±­°¸¿¬»ó¾«ºº»®»¼ ­¿´·²» øÐÞÍ÷ ¿²¼ ·²½«ó
`bated in PBS with 1% bovine serum albumin (BSA) (Sigma
`¾¿¬»¼ ·² ÐÞÍ ©·¬¸ ïû ¾±ª·²» ­»®«³ ¿´¾«³·² øÞÍß÷ øÍ·¹³¿
`Chemical Co, St Louis, Mo) for 10 minutes. Sections were
`ݸ»³·½¿´ ݱô ͬ Ô±«·­ô Ó±÷ º±® ïð ³·²«¬»­ò Í»½¬·±²­ ©»®»
`incubated for 1 hour at room temperature in primary an-
`·²½«¾¿¬»¼ º±® ï ¸±«® ¿¬ ®±±³ ¬»³°»®¿¬«®» ·² °®·³¿®§ ¿²ó
`tibodies at concentrations derived empirically: CD3, 1.0
`¬·¾±¼·»­ ¿¬ ½±²½»²¬®¿¬·±²­ ¼»®·ª»¼ »³°·®·½¿´´§æ ÝÜíô ïòð
`ug/mL; CD4, 5.0 ug/mL; CD8, 2.5 ug/mL; CD11a, 10.0
`¡¹ñ³Ôå ÝÜìô ëòð ¡¹ñ³Ôå ÝÜèô îòë ¡¹ñ³Ôå ÝÜïï¿ô ïðòð
`ug/mL; and HLA—DR, 1.0 ug/mL. Sections were rinsed in
`¡¹ñ³Ôå ¿²¼ ØÔßóÜÎô ïòð ¡¹ñ³Ôò Í»½¬·±²­ ©»®» ®·²­»¼ ·²
`PBS alone, followed by 10 minutes in PBS with 1% BSA be-
`ÐÞÍ ¿´±²»ô º±´´±©»¼ ¾§ ïð ³·²«¬»­ ·² ÐÞÍ ©·¬¸ ïû ÞÍß ¾»ó
`fore incubation for 1 hour at room temperature in the sec-
`º±®» ·²½«¾¿¬·±² º±® ï ¸±«® ¿¬ ®±±³ ¬»³°»®¿¬«®» ·² ¬¸» ­»½ó
`ondary antibody, fluorescein isothiocyanate—conjugated Af-
`±²¼¿®§ ¿²¬·¾±¼§ô º´«±®»­½»·² ·­±¬¸·±½§¿²¿¬»o½±²¶«¹¿¬»¼ ߺó
`finipure Donkey Anti-Mouse IgG (]ackson Immunoresearch,
`º·²·°«®» ܱ²µ»§ ß²¬·óÓ±«­» ×¹Ù øÖ¿½µ­±² ׳³«²±®»­»¿®½¸ô
`West Grove, Pa) at a dilution of 1/50. Sections were then
`É»­¬ Ù®±ª»ô п÷ ¿¬ ¿ ¼·´«¬·±² ±º ïñëðò Í»½¬·±²­ ©»®» ¬¸»²
`rinsed in PBS, mounted in Vectashield (Vector Labs, Bur-
`®·²­»¼ ·² ÐÞÍô ³±«²¬»¼ ·² Ê»½¬¿­¸·»´¼ øÊ»½¬±® Ô¿¾­ô Þ«®ó
`lingame, Calif), cover-slipped, and viewed under a micro-
`´·²¹¿³»ô Ý¿´·º÷ô ½±ª»®ó­´·°°»¼ô ¿²¼ ª·»©»¼ «²¼»® ¿ ³·½®±ó
`scope (Eclipse E800; Nikon, Melville, NY) interfaced with
`­½±°» øÛ½´·°­» Ûèððå Ò·µ±²ô Ó»´ª·´´»ô ÒÇ÷ ·²¬»®º¿½»¼ ©·¬¸
`a digital camera (Spot Digital Camera; Diagnostic Instru-
`¿ ¼·¹·¬¿´ ½¿³»®¿ øÍ°±¬ Ü·¹·¬¿´ Ý¿³»®¿å Ü·¿¹²±­¬·½ ײ­¬®«ó
`ments Inc, Micro Video Instruments, Avon, Mass). Sec-
`³»²¬­ ײ½ô Ó·½®± Ê·¼»± ײ­¬®«³»²¬­ô ߪ±²ô Ó¿­­÷ò Í»½ó
`ondary antibody controls omitting the primary antibody
`±²¼¿®§ ¿²¬·¾±¼§ ½±²¬®±´­ ±³·¬¬·²¹ ¬¸» °®·³¿®§ ¿²¬·¾±¼§
`for all biopsy specimens for each immunohistochemical
`º±® ¿´´ ¾·±°­§ ­°»½·³»²­ º±® »¿½¸ ·³³«²±¸·­¬±½¸»³·½¿´
`analysis were run.
`¿²¿´§­·­ ©»®» ®«²ò
`Three separate images were acquired for each anti-
`̸®»» ­»°¿®¿¬» ·³¿¹»­ ©»®» ¿½¯«·®»¼ º±® »¿½¸ ¿²¬·ó
`body and biopsy specimen under a X20 objective using a
`¾±¼§ ¿²¼ ¾·±°­§ ­°»½·³»² «²¼»® ¿ íîð ±¾¶»½¬·ª» «­·²¹ ¿
`Spot acquisition program (Diagnostic Instruments Inc). The
`Í°±¬ ¿½¯«·­·¬·±² °®±¹®¿³ øÜ·¿¹²±­¬·½ ײ­¬®«³»²¬­ ײ½÷ò ̸»
`first field selected for imaging was the field with the high-
`º·®­¬ º·»´¼ ­»´»½¬»¼ º±® ·³¿¹·²¹ ©¿­ ¬¸» º·»´¼ ©·¬¸ ¬¸» ¸·¹¸ó
`est number of positive cells, followed by images to the left
`»­¬ ²«³¾»® ±º °±­·¬·ª» ½»´´­ô º±´´±©»¼ ¾§ ·³¿¹»­ ¬± ¬¸» ´»º¬
`
`and right of that area. In this manner the entire biopsy area
`¿²¼ ®·¹¸¬ ±º ¬¸¿¬ ¿®»¿ò ײ ¬¸·­ ³¿²²»® ¬¸» »²¬·®» ¾·±°­§ ¿®»¿
`was usually captured.
`©¿­ «­«¿´´§ ½¿°¬«®»¼ò
`
`COUNTING PROCEDURE
`ÝÑËÒÌ×ÒÙ ÐÎÑÝÛÜËÎÛ
`
`Measurement of the entire area of epithelium and stroma
`Ó»¿­«®»³»²¬ ±º ¬¸» »²¬·®» ¿®»¿ ±º »°·¬¸»´·«³ ¿²¼ ­¬®±³¿
`(substantia propria) was achieved by tracing the area us-
`ø­«¾­¬¿²¬·¿ °®±°®·¿÷ ©¿­ ¿½¸·»ª»¼ ¾§ ¬®¿½·²¹ ¬¸» ¿®»¿ «­ó
`ing the lasso tool under the Adobe Photoshop computer
`·²¹ ¬¸» ´¿­­± ¬±±´ «²¼»® ¬¸» ß¼±¾» 豬±­¸±° ½±³°«¬»®
`program (Adobe Systems Inc, Sanjose, Calif). The total data
`°®±¹®¿³ øß¼±¾» ͧ­¬»³­ ײ½ô Í¿² Ö±­»ô Ý¿´·º÷ò ̸» ¬±¬¿´ ¼¿¬¿
`area, measured in pixels, was acquired through the “Im-
`¿®»¿ô ³»¿­«®»¼ ·² °·¨»´­ô ©¿­ ¿½¯«·®»¼ ¬¸®±«¹¸ ¬¸» v׳ó
`age: Histogram” command in Photoshop. Two indepen-
`¿¹»æ Ø·­¬±¹®¿³f ½±³³¿²¼ ·² 豬±­¸±°ò Ì©± ·²¼»°»²ó
`dent counts were recorded for cells positive for each anti-
`¼»²¬ ½±«²¬­ ©»®» ®»½±®¼»¼ º±® ½»´´­ °±­·¬·ª» º±® »¿½¸ ¿²¬·ó
`body within the traced area. Cells per unit area of pixels
`¾±¼§ ©·¬¸·² ¬¸» ¬®¿½»¼ ¿®»¿ò Ý»´´­ °»® «²·¬ ¿®»¿ ±º °·¨»´­
`were adjusted to real unit area or cells per millimeter squared
`©»®» ¿¼¶«­¬»¼ ¬± ®»¿´ «²·¬ ¿®»¿ ±® ½»´´­ °»® ³·´´·³»¬»® ­¯«¿®»¼
`of real tissue area, based on 28.346 pixels per centimeter
`±º ®»¿´ ¬·­­«» ¿®»¿ô ¾¿­»¼ ±² îèòíìê °·¨»´­ °»® ½»²¬·³»¬»®
`in Photoshop and the fact that 1 mm equals 67.8 cm equals
`·² 豬±­¸±° ¿²¼ ¬¸» º¿½¬ ¬¸¿¬ ï ³³ »¯«¿´­ êéòè ½³ »¯«¿´­
`1922 pixels at X20 magnification on the Nikon micro-
`ïçîî °·¨»´­ ¿¬ íî𠳿¹²·º·½¿¬·±² ±² ¬¸» Ò·µ±² ³·½®±ó
`scope. Data were recorded as cells per millimeter squared
`­½±°»ò Ü¿¬¿ ©»®» ®»½±®¼»¼ ¿­ ½»´´­ °»® ³·´´·³»¬»® ­¯«¿®»¼
`for all markers, and statistical analysis was based on these
`º±® ¿´´ ³¿®µ»®­ô ¿²¼ ­¬¿¬·­¬·½¿´ ¿²¿´§­·­ ©¿­ ¾¿­»¼ ±² ¬¸»­»
`measurements.
`³»¿­«®»³»²¬­ò
`
`STATISTICAL METHODS
`ÍÌßÌ×ÍÌ×ÝßÔ ÓÛÌØÑÜÍ
`
`Baseline characteristics were tabulated and summarized by
`Þ¿­»´·²» ½¸¿®¿½¬»®·­¬·½­ ©»®» ¬¿¾«´¿¬»¼ ¿²¼ ­«³³¿®·¦»¼ ¾§
`treatment groups. Overall differences among treatment
`¬®»¿¬³»²¬ ¹®±«°­ò Ѫ»®¿´´ ¼·ºº»®»²½»­ ¿³±²¹ ¬®»¿¬³»²¬
`groups were tested using a 2-way analysis of variance
`¹®±«°­ ©»®» ¬»­¬»¼ «­·²¹ ¿ î󩿧 ¿²¿´§­·­ ±º ª¿®·¿²½»
`(ANOVA) for continuous variables and the Fisher exact test
`øßÒÑÊß÷ º±® ½±²¬·²«±«­ ª¿®·¿¾´»­ ¿²¼ ¬¸» Ú·­¸»® »¨¿½¬ ¬»­¬
`for categorical variables.
`º±® ½¿¬»¹±®·½¿´ ª¿®·¿¾´»­ò
`Percent changes in the number of cells expressing
`л®½»²¬ ½¸¿²¹»­ ·² ¬¸» ²«³¾»® ±º ½»´´­ »¨°®»­­·²¹
`lymphocytic and/or lymphocyte activation markers were
`´§³°¸±½§¬·½ ¿²¼ñ±® ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µ»®­ ©»®»
`summarized using descriptive statistics (ie, sample size,
`­«³³¿®·¦»¼ «­·²¹ ¼»­½®·°¬·ª» ­¬¿¬·­¬·½­ ø·»ô ­¿³°´» ­·¦»ô
`mean, SD, minimum, maximum, and median). A 1-way
`³»¿²ô ÍÜô ³·²·³«³ô ³¿¨·³«³ô ¿²¼ ³»¼·¿²÷ò ß ï󩿧
`ANOVA with main effect for treatment was used to test
`ßÒÑÊß ©·¬¸ ³¿·² »ºº»½¬ º±® ¬®»¿¬³»²¬ ©¿­ «­»¼ ¬± ¬»­¬
`for differences in percent change from baseline and
`º±® ¼·ºº»®»²½»­ ·² °»®½»²¬ ½¸¿²¹» º®±³ ¾¿­»´·²» ¿²¼
`ratios among treatment groups by visit. If the test for
`®¿¬·±­ ¿³±²¹ ¬®»¿¬³»²¬ ¹®±«°­ ¾§ ª·­·¬ò ׺ ¬¸» ¬»­¬ º±®
`among-group differences in main effect was significant,
`¿³±²¹ó¹®±«° ¼·ºº»®»²½»­ ·² ³¿·² »ºº»½¬ ©¿­ ­·¹²·º·½¿²¬ô
`then all 3 pairwise comparisons were made. Within-
`¬¸»² ¿´´ í °¿·®©·­» ½±³°¿®·­±²­ ©»®» ³¿¼»ò É·¬¸·²ó
`group changes from baseline were analyzed by the
`¹®±«° ½¸¿²¹»­ º®±³ ¾¿­»´·²» ©»®» ¿²¿´§¦»¼ ¾§ ¬¸»
`paired t test method.
`°¿·®»¼ ¬ ¬»­¬ ³»¬¸±¼ò
`The same analysis was performed on Sjogren and
`̸» ­¿³» ¿²¿´§­·­ ©¿­ °»®º±®³»¼ ±² Ͷ±X ¹®»² ¿²¼
`non-Sjogren subpopulations, excluding the 0.1% CsA
`²±²óͶ±X ¹®»² ­«¾°±°«´¿¬·±²­ô »¨½´«¼·²¹ ¬¸» ðòïû Ý­ß
`treatment group in which there was only 1 patient in the
`¬®»¿¬³»²¬ ¹®±«° ·² ©¸·½¸ ¬¸»®» ©¿­ ±²´§ ï °¿¬·»²¬ ·² ¬¸»
`Sjogren subset.
`Ͷ±X ¹®»² ­«¾­»¬ò
`
`(CD1 1a and HLA—DR) to further understand the under-
`øÝÜïï¿ ¿²¼ ØÔßóÜÎ÷ ¬± º«®¬¸»® «²¼»®­¬¿²¼ ¬¸» «²¼»®ó
`´§·²¹ ³»½¸¿²·­³ ±º Ý­ß ¬®»¿¬³»²¬ò
`lying mechanism of CsA treatment.
`
`ÎÛÍËÔÌÍ
`
`¬®»¿¬³»²¬ ©·¬¸ »·¬¸»® ½±²½»²¬®¿¬·±² ±º Ý­ßò ̸» ±²´§ »¨ó
`treatment with either concentration of CsA. The only ex-
`½»°¬·±² ©¿­ ¬¸¿¬ ¬¸»®» ©¿­ ¿ ³»¿² ·²½®»¿­» º®±³ ¾¿­»ó
`ception was that there was a mean increase from base-
`´·²» ·² ¬¸» ÝÜìó°±­·¬·ª» Ì ¸»´°»® ½»´´ °±°«´¿¬·±² º±´´±©ó
`line in the CD4-positive T helper cell population follow-
`·²¹ ðòðëû Ý­ß ¬®»¿¬³»²¬ò ײ ½±³°¿®·­±²ô ¿´´ ½»´´­ °±­·¬·ª»
`ing 0.05% CsA treatment. In comparison, all cells positive
`º±® ¬¸» ´§³°¸±½§¬·½ ³¿®µ»®­ ·²½®»¿­»¼ º®±³ ¾¿­»´·²» º±´ó
`for the lymphocytic markers increased from baseline fol-
`´±©·²¹ ª»¸·½´» ¬®»¿¬³»²¬ò
`lowing vehicle treatment.
`Ú·¹«®» ï ­¸±©­ ¬¸» °»®½»²¬ ½¸¿²¹» º®±³ ¾¿­»´·²»
`Figure I shows the percent change from baseline
`º±® ½»´´­ »¨°®»­­·²¹ ¬¸» ´§³°¸±½§¬·½ ³¿®µ»®­ øÝÜíô ÝÜìô
`for cells expressing the lymphocytic markers (CD3, CD4,
`¿²¼ ÝÜè÷ ¿º¬»® ê ³±²¬¸­ ±º ¬®»¿¬³»²¬ º±® ¬¸» ±ª»®¿´´ °¿ó
`and CD8) after 6 months of treatment for the overall pa-
`¬·»²¬ °±°«´¿¬·±²ò Ò±¬» ¬¸¿¬ ¬¸»®» ©¿­ ¿ ®»¼«½¬·±² º®±³
`tient population. Note that there was a reduction from
`¾¿­»´·²» ·² ¬¸» ²«³¾»® ±º ÝÜíó°±­·¬·ª» ½»´´­ ·² ¬¸» Ý­ßó
`baseline in the number of CD3-positive cells in the CsA-
`¬®»¿¬»¼ ¹®±«°­ô ©¸·´» ¬¸»®» ©¿­ ¿² ·²½®»¿­» º®±³ ¾¿­»ó
`treated groups, while there was an increase from base-
`´·²» ·² ¬¸» ª»¸·½´»ó¬®»¿¬»¼ ¹®±«°ò ̸»®» ©¿­ ¿´­± ¿² ·²ó
`line in the vehicle-treated group. There was also an in-
`½®»¿­» º®±³ ¾¿­»´·²» ·² ¬¸» ²«³¾»®­ ±º ÝÜìó°±­·¬·ª» ½»´´­
`crease from baseline in the numbers of CD4-positive cells
`·² ¬¸» ª»¸·½´» ¹®±«°ô ©·¬¸ ¿ ­³¿´´»® ·²½®»¿­» ·² ¬¸» ðòðëû
`ײ ¹»²»®¿´ô ¬¸»®» ©¿­ ¿ ¼»½®»¿­» º®±³ ¾¿­»´·²» ·² ¬¸» ²«³ó
`in the vehicle group, with a smaller increase in the 0.05%
`In general, there was a decrease from baseline in the num-
`Ý­ß ¹®±«° ¿²¼ ¿ ­´·¹¸¬ ¼»½®»¿­» ·² ¬¸» ðòïû Ý­ß ¹®±«°ò
`¾»® ±º ½»´´­ °±­·¬·ª» º±® ÝÜíô ÝÜìô ¿²¼ ÝÜè º±´´±©·²¹
`CsA group and a slight decrease in the 0.1% CsA group.
`ber of cells positive for CD3, CD4, and CD8 following
`
`WWW.ARCHOPHTHALMOL.COM
`(REPRINTED) ARCH OPHTHALMOLIVOL 118, NOV 2000
`1491
`ÉÉÉòßÎÝØÑÐØÌØßÔÓÑÔòÝÑÓ
`øÎÛÐÎ×ÒÌÛÜ÷ ßÎÝØ ÑÐØÌØßÔÓÑÔ ñ ÊÑÔ ïïèô ÒÑÊ îððð
`ïìçï
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`J P
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`ATIENT POPULATION
`ÐßÌ×ÛÒÌ ÐÑÐËÔßÌ×ÑÒ
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`̸» ³»¿²€ ÍÜ ¿¹» ±º ±«® ­«¾¶»½¬­ ©¿­ ëçòð €ïíòë §»¿®­
`The mean=SD age of our subjects was 59.0 2 13.5 years
`ø®¿²¹»ô îèòèóèìòî §»¿®­÷ô ·²½´«¼·²¹ îé ©±³»² ¿²¼ ë ³»²ò
`(range, 28.8-84.2 years), including 27 women and 5 men.
`É·¬¸·² ¬¸·­ ¹®±«°ô ¬¸»®» ©»®» ïî Ͷ±X ¹®»² ¿²¼ îð ²±²ó
`Within this group, there were 12 Sjogren and 20 non-
`Sj ogren patients.
`Ͷ±X ¹®»² °¿¬·»²¬­ò
`
`LYMPHOCYTIC MARKERS
`ÔÇÓÐØÑÝÇÌ×Ý ÓßÎÕÛÎÍ
`
`©2000 American Medical Association. All rights reserved.
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`Ю»¬®»¿¬³»²¬
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`б­¬¬®»¿¬³»²¬
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`Figure 5. Immunofluorescence micrographs demonstrating cells positive for the lymphocyte activation marker 0D11a in conjunctival biopsy specimens of
`Ú·¹«®» ëò
`patients with non—Sj6gren keratoconjunctivitis sicca pretreatment and posttreatment with (A and B) 0.05% cyclosporine and (C and 0) 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).
`
`1&4
`ÝÑÓÓÛÒÌ
`
`Ú·¹«®» ë ¿²¼ Ú·¹«®» ê ­¸±© ¿ ®»°®»­»²¬¿¬·ª» ­»¬
`Figure 5 and Figure 6 show a representative set
`±º ·³³«²±º´«±®»­½»²½» ³·½®±¹®¿°¸­ º±® ½»´´­ °±­·¬·ª» º±®
`of immunofluorescenee micrographs for cells positive for
`¬¸» ³¿®µ»®­ ÝÜïï¿ ¿²¼ ØÔßóÜÎ º®±³ ¬¸» ²±²óͶ±X ¹®»²
`the markers CD1 1a and HLA-DR from the non-Sjogren
`­«¾¹®±«° ¬®»¿¬»¼ ©·¬¸ ðòðëû Ý­ß ±® ª»¸·½´»ò Ú·¹«®» é
`subgroup treated with 0.05% CsA or vehicle. Figure 1
`­¸±©­ ·³³«²±º´«±®»­½»²½» ³·½®±¹®¿°¸­ º±® ½»´´­ °±­·ó
`shows immunofluorescenee micrographs for cells posi-
`¬·ª» º±® ¬¸» ³¿®µ»®­ ÝÜí ¿²¼ ÝÜïï¿ º®±³ °¿¬·»²¬­ ©·¬¸
`tive for the markers CD3 and CD1 1a from patients with
`Ͷ±X ¹®»² ÕÝÍ ¬®»¿¬»¼ ©·¬¸ ðòðëû Ý­ßò
`Sjogren KCS treated with 0.05% CsA.
`
`̸»­» º·²¼·²¹­ °®±ª·¼» ¿¼¼·¬·±²¿´ »ª·¼»²½» ¬¸¿¬ ·²ó
`These findings provide additional evidence that in-
`º´¿³³¿¬·±² °´¿§­ ¿ ®±´» ·² ¬¸» °¿¬¸±¹»²»­·­ ±º ÕÝÍ ¿²¼
`flammation plays a role in the pathogenesis of KCS and
`­«¹¹»­¬­ ¬¸¿¬ ³±¼«´¿¬·²¹ ¬¸» «²¼»®´§·²¹ ·³³«²» ®»ó
`suggests that modulating the underlying immune re-
`­°±²­» ³¿§ °®±ª» ³±®» »ºº·½¿½·±«­ ·² ¬¸» ¬®»¿¬³»²¬ ±º
`sponse may prove more efficacious in the treatment of
`ÕÝÍ ¬¸¿² ¬¸» º®»¯«»²¬ «­» ±º ¿®¬·º·½·¿´ ¬»¿®­ò ̱°·½¿´ Ý­ß
`KCS than the frequent use of artificial tears. Topical CsA
`¸¿­ ¾»»² ­«½½»­­º«´´§ «­»¼ º±® ¬¸» ¬®»¿¬³»²¬ ±º ½¿²·²»
`has been successfully used for the treatment of canine
`¼®§ »§» º±® ³¿²§ §»¿®­ò ͬ«¼·»­ ·² ¬¸» ½¿²·²» ÕÝÍ ³±¼»´
`dry eye for many years. Studies in the canine KCS model
`¸¿ª» ¼»³±²­¬®¿¬»¼ ¬¸¿¬ Ý­ß ¼»½®»¿­»­ ¬¸» ½±²¶«²½¬·ª¿´
`have demonstrated that CsA decreases the conjunctival
`¿²¼ ´¿½®·³¿´ ¹´¿²¼ ´§³°¸±½§¬·½ ·²º·´¬®¿¬»­òîìóîê
`and lacrimal gland lymphocytic infiltrates.2“'25
`ر©»ª»®ô ¬¸»®» ¸¿ª» ¾»»² ±²´§ ¿ ´·³·¬»¼ ²«³¾»® ±º
`However, there have been only a limited number of
`®»°±®¬­ ±² ¬¸» «­» ±º ¬±°·½¿´ Ý­ß ·² ¬¸» ¬®»¿¬³»²¬ ±º ¼®§
`ײ ¬¸» °®»­»²¬ ­¬«¼§ô ·³³«²±¸·­¬±½¸»³·½¿´ ¿²¿´§­·­ ©¿­
`reports on the use of topical CsA in the treatment of dry
`In the present study, immunohistochemical analysis was
`»§» ­§²¼®±³» ·² ¸«³¿²­îéóîç ©·¬¸ ±²´§ ï ¿¬¬»³°¬ ¬± ´±±µ
`«­»¼ ¬± »ª¿´«¿¬» ½¸¿²¹»­ ·² ¬¸» °®»­»²½» ±º ½»´´­ °±­·ó
`used to evaluate changes in the presence of cells posi-
`eye syndrome in humans27'29 with only 1 attempt to look
`at the effect of the treatment at a cellular level.” Power
`¿¬ ¬¸» »ºº»½¬ ±º ¬¸» ¬®»¿¬³»²¬ ¿¬ ¿ ½»´´«´¿® ´»ª»´òíð б©»®
`¬·ª» º±® ´§³°¸±½§¬·½ ¿²¼ ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µó
`tive for lymphocytic and lymphocyte activation mark-
`»¬ ¿´íð ®»°±®¬»¼ ¿ ­·¹²·º·½¿²¬ ®»¼«½¬·±² ·² ÝÜìó°±­·¬·ª»
`»®­ ·² ½±²¶«²½¬·ª¿´ ¾·±°­§ ­°»½·³»²­ ±º °¿¬·»²¬­ ©·¬¸ ³±¼ó
`ers in conjunctival biopsy specimens of patients with mod-
`et al3° reported a significant reduction in CD4-positive
`Ì ´§³°¸±½§¬»­ ·² ¾±¬¸ ¬¸» ½±²¶«²½¬·ª¿´ »°·¬¸»´·«³ ¿²¼
`»®¿¬» ¬± ­»ª»®» ÕÝÍô º±´´±©·²¹ ¬®»¿¬³»²¬ ©·¬¸ ðòðëû Ý­ßô
`T lymphocytes in both the conjunctival epithelium and
`erate to severe KCS, following treatment with 0.05% CsA,
`0.1% CsA, or vehicle. We found that CsA treatment re-
`¬¸» ­«¾­¬¿²¬·¿ °®±°®·¿ ±º °¿¬·»²¬­ ©·¬¸ ­»½±²¼¿®§ Ͷ±X ó
`ðòïû Ý­ßô ±® ª»¸·½´»ò É» º±«²¼ ¬¸¿¬ Ý­ß ¬®»¿¬³»²¬ ®»ó
`the substantia propria of patients with secondary Sjo-
`¹®»² ­§²¼®±³» ½±³°¿®»¼ ©·¬¸ ²±²o¼®§ »§» ½±²¬®±´­ º±´ó
`¼«½»¼ ¬¸» ²«³¾»® ±º ¿½¬·ª¿¬»¼ Ì ´§³°¸±½§¬»­ ©·¬¸·² ¬¸»
`gren syndrome compared with non—dry eye controls fol-
`duced the number of activated T lymphocytes within the
`´±©·²¹ ¬®»¿¬³»²¬ ©·¬¸ Ý­ßò ̸» °®»­»²¬ ­¬«¼§ ¿´­± ¼»³ó
`±½«´¿® ­«®º¿½» ±º °¿¬·»²¬­ ©·¬¸ ¿²¼ ©·¬¸±«¬ Ͷ±X ¹®»² ­§²ó
`lowing treatment with CsA. The present study also dem-
`ocular surface of patients with and without Sj ogren syn-
`drome. After 6 months of treatment with 0.05% CsA, sta-
`±²­¬®¿¬»¼ ¿ ­·¹²·º·½¿²¬ ¼»½®»¿­» ·² ÝÜíó°±­·¬·ª» ½»´´­ ¿º¬»®
`¼®±³»ò ߺ¬»® ê ³±²¬¸­ ±º ¬®»¿¬³»²¬ ©·¬¸ ðòðëû Ý­ßô ­¬¿ó
`onstrated a significant decrease in CD3-positive cells after
`ê ³±²¬¸­ ±º ðòðëû Ý­ß ¬®»¿¬³»²¬ ·² °¿¬·»²¬­ ©·¬¸ Ͷ±X ó
`¬·­¬·½¿´´§ ­·¹²·º·½¿²¬ ¼»½®»¿­»­ ©»®» ­»»² ·² ½»´´­ °±­·¬·ª»
`6 months of 0.05% CsA treatment in patients with Sjo-
`tistically significant decreases were seen in cells positive
`¹®»² ­§²¼®±³»ò
`º±® ÝÜïï¿ ¿²¼ ØÔßóÜÎ ½±³°¿®»¼ ©·¬¸ ¬¸±­» ·² ª»¸·½´»
`gren syndrome.
`for CD1 1a and HLA-DR compared with those in vehicle
`Ú«®¬¸»®³±®»ô ¬¸» ²«³¾»® ±º ½»´´­ °±­·¬·ª» º±® ÝÜïï¿
`º±® ¬¸» ±ª»®¿´´ °¿¬·»²¬ °±°«´¿¬·±²ò É·¬¸·² ¬¸» Ͷ±X ¹®»² °¿ó
`Furthermore, the number of cells positive for CD1 1a
`for the overall patient population. Within the Sjogren pa-
`¿²¼ ØÔßóÜÎô ©¸·½¸ ¿®» ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µó
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`and HLA-DR, which are lymphocyte activation mark-
`tient subgroup treated with 0.05% CsA, there were also
`»®­ô ¼»½®»¿­»¼ ­·¹²·º·½¿²¬´§ ·² °¿¬·»²¬ °±°«´¿¬·±²­ ¬®»¿¬»¼
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`ers, decreased significantly in patient populations treated
`significantly greater decreases than with vehicle in the
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`²«³¾»® ±º ½»´´­ °±­·¬·ª» º±® ÝÜí ¿²¼ ÝÜïï¿ò
`with CsA. HLA-DR is a class 11 major histocompatibility
`number of cells positive for CD3 and CD11a.
`
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`©2000 American Medical Association. All rights reserved.
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`Figure 6. lmmunofluorescence micrographs demonstrating cells positive for HLA-DR in con/unct/val biopsy specimens of patients with non—S/tigren
`Ú·¹«®» êò
`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 fora vehicle biopsy in which the primary antibody was omitted. Bar=25 pm (A-0).
`
`½±³°´»¨ ¿²¬·¹»² ¬¸¿¬ ·­ »¨°®»­­»¼ ·² ·²º´¿³»¼ ®»¹·±²­ ¿²¼
`complex antigen that is expressed in inflamed regions and
`­»®ª»­ ¿­ ¿ ´·¹¿²¼ º±® ¬¸» Ìó½»´´ ®»½»°¬±®ò ÝÜìõ Ì ´§³ó
`serves as a ligand for the T—cell receptor. CD4+ T lym-
`°¸±½§¬»­ ¿®» ¿½¬·ª¿¬»¼ ¬¸®±«¹¸ ¿ ­·¹²¿´ º®±³ ØÔßóÜÎ ³±´ó
`phocytes are activated through a signal from HLA—DR mol-
`»½«´»­ ±º ¿²¬·¹»²ó°®»­»²¬·²¹ ½»´´­òíï ׳³«²±°¿¬¸±´±¹·½
`ecules of antigen—presenting cells.” Immunopathologic
`studies show evidence of immune activation of the con-
`­¬«¼·»­ ­¸±© »ª·¼»²½» ±º ·³³«²» ¿½¬·ª¿¬·±² ±º ¬¸» ½±²ó
`junctival epithelium in Sj ogren syndrome. Compared with
`¶«²½¬·ª¿´ »°·¬¸»´·«³ ·² Ͷ±X ¹®»² ­§²¼®±³»ò ݱ³°¿®»¼ ©·¬¸
`½±²¬®±´ »§»­ô ¿ ­·¹²·º·½¿²¬´§ ¹®»¿¬»® °»®½»²¬¿¹» ±º ½±²ó
`control eyes, a significantly greater percentage of con-
`¶«²½¬·ª¿´ »°·¬¸»´·¿´ ½»´´­ º®±³ °¿¬·»²¬­ ©·¬¸ Ͷ±X ¹®»² ­§²ó
`junctival epithelial cells from patients with Sjog