EE
`Analysis of Topical Cyclosporine Treatment
`ß²¿´§­·­ ±º ̱°·½¿´ ݧ½´±­°±®·²» Ì®»¿¬³»²¬
`of Patients With Dry Eye Syndrome
`±º п¬·»²¬­ É·¬¸ Ü®§ Û§» ͧ²¼®±³»
`
`ÝÔ×Ò×ÝßÔ ÍÝ×ÛÒÝÛÍ
`
`Effect on Conjunctival Lymphocytes
`Ûºº»½¬ ±² ݱ²¶«²½¬·ª¿´ Ô§³°¸±½§¬»­
`
`Kathleen S. Kunert, MD; AnnS. Tisdale, MS; Michael E. Stern, PhD; J. A. Smith; Ilene K. Gipson, PhD
`Õ¿¬¸´»»² Íò Õ«²»®¬ô ÓÜå ß²² Íò Ì·­¼¿´»ô ÓÍå Ó·½¸¿»´ Ûò ͬ»®²ô иÜå Öò ßò ͳ·¬¸å ×´»²» Õò Ù·°­±²ô иÜ
`
`Objective: To study the effect of topical cyclosporine
`Ѿ¶»½¬·ª»æ ̱ ­¬«¼§ ¬¸» »ºº»½¬ ±º ¬±°·½¿´ ½§½´±­°±®·²»
`on lymphocyte activation within the conjunctiva of
`±² ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ©·¬¸·² ¬¸» ½±²¶«²½¬·ª¿ ±º
`patients with moderate to severe dry eye syndrome
`°¿¬·»²¬­ ©·¬¸ ³±¼»®¿¬» ¬± ­»ª»®» ¼®§ »§» ­§²¼®±³»
`(Sjogren and non-Sjégren).
`øͶ±X ¹®»² ¿²¼ ²±²óͶ±X ¹®»²÷ò
`
`Methods: Biopsy specimens were obtainedat baseline
`Ó»¬¸±¼­æ Þ·±°­§ ­°»½·³»²­ ©»®» ±¾¬¿·²»¼ ¿¬ ¾¿­»´·²»
`and after 6 monthsof cyclosporine treatment from eyes
`¿²¼ ¿º¬»® ê ³±²¬¸­ ±º ½§½´±­°±®·²» ¬®»¿¬³»²¬ º®±³ »§»­
`of 32 patients with moderate to severe dry eye syn-
`±º íî °¿¬·»²¬­ ©·¬¸ ³±¼»®¿¬» ¬± ­»ª»®» ¼®§ »§» ­§²ó
`drome; 19 were cyclosporine treated (0.05% cyclospor-
`¼®±³»å ïç ©»®» ½§½´±­°±®·²» ¬®»¿¬»¼ øðòðëû ½§½´±­°±®ó
`ine, n=13; 0.1% cyclosporine, n=6) and 13 were ve-
`·²»ô ² ã ïíå ðòïû ½§½´±­°±®·²»ô ² ã ê÷ ¿²¼ ïí ©»®» ª»ó
`hicle treated. Within this group there were 12 with Sjogren
`¸·½´» ¬®»¿¬»¼ò É·¬¸·² ¬¸·­ ¹®±«° ¬¸»®» ©»®» ïî ©·¬¸ Ͷ±X ¹®»²
`syndrome and 20 with non—Sjégren syndrome. Biopsy
`­§²¼®±³» ¿²¼ îð ©·¬¸ ²±²oͶ±X ¹®»² ­§²¼®±³»ò Þ·±°­§
`tissue was analyzed using immunohistochemical local-
`¬·­­«» ©¿­ ¿²¿´§¦»¼ «­·²¹ ·³³«²±¸·­¬±½¸»³·½¿´ ´±½¿´ó
`ization of binding of monoclonal antibodies to lympho-
`·¦¿¬·±² ±º ¾·²¼·²¹ ±º ³±²±½´±²¿´ ¿²¬·¾±¼·»­ ¬± ´§³°¸±ó
`cytic markers CD3, CD4, and CD8as well as lympho-
`½§¬·½ ³¿®µ»®­ ÝÜíô ÝÜìô ¿²¼ ÝÜè ¿­ ©»´´ ¿­ ´§³°¸±ó
`cyte activation markers CD1la and HLA-DR.
`½§¬» ¿½¬·ª¿¬·±² ³¿®µ»®­ ÝÜïï¿ ¿²¼ ØÔßóÜÎò
`
`tive for CD3, CD4, and CD8,while in vehicle-treated eyes,
`¬·ª» º±® ÝÜíô ÝÜìô ¿²¼ ÝÜèô ©¸·´» ·² ª»¸·½´»ó¬®»¿¬»¼ »§»­ô
`results showedincreases in these markers, although these
`®»­«´¬­ ­¸±©»¼ ·²½®»¿­»­ ·² ¬¸»­» ³¿®µ»®­ô ¿´¬¸±«¹¸ ¬¸»­»
`differences were notstatistically significant. Following
`¼·ºº»®»²½»­ ©»®» ²±¬ ­¬¿¬·­¬·½¿´´§ ­·¹²·º·½¿²¬ò Ú±´´±©·²¹
`treatment with 0.05% cyclosporine, there wasa signifi-
`¬®»¿¬³»²¬ ©·¬¸ ðòðëû ½§½´±­°±®·²»ô ¬¸»®» ©¿­ ¿ ­·¹²·º·ó
`cant decrease in the numberofcells expressing the lym-
`½¿²¬ ¼»½®»¿­» ·² ¬¸» ²«³¾»® ±º ½»´´­ »¨°®»­­·²¹ ¬¸» ´§³ó
`phocyte activation markers CD11a (P<.05) and HLA-DR
`°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µ»®­ ÝÜïï¿ øÐôòðë÷ ¿²¼ ØÔßóÜÎ
`(P<.05), indicating less activation of lymphocytes as com-
`øÐôòðë÷ô ·²¼·½¿¬·²¹ ´»­­ ¿½¬·ª¿¬·±² ±º ´§³°¸±½§¬»­ ¿­ ½±³ó
`pared with vehicle treatment. Within the Sjogren pa-
`°¿®»¼ ©·¬¸ ª»¸·½´» ¬®»¿¬³»²¬ò É·¬¸·² ¬¸» Ͷ±X ¹®»² °¿ó
`tient subgroup, those treated with 0.05% cyclosporine
`¬·»²¬ ­«¾¹®±«°ô ¬¸±­» ¬®»¿¬»¼ ©·¬¸ ðòðëû ½§½´±­°±®·²»
`also showeda significant decrease in the numberofcells
`¿´­± ­¸±©»¼ ¿ ­·¹²·º·½¿²¬ ¼»½®»¿­» ·² ¬¸» ²«³¾»® ±º ½»´´­
`positive for CD1la (P<.001) as well as CD3 (P<.03),
`°±­·¬·ª» º±® ÝÜïï¿ øÐôòððï÷ ¿­ ©»´´ ¿­ ÝÜí øÐôòðí÷ô
`indicating a reduction in numberofactivated lympho-
`·²¼·½¿¬·²¹ ¿ ®»¼«½¬·±² ·² ²«³¾»® ±º ¿½¬·ª¿¬»¼ ´§³°¸±ó
`½§¬»­ò
`cytes.
`
`Conclusion: Treatmentof dry eye syndromewith topi-
`ݱ²½´«­·±²æ Ì®»¿¬³»²¬ ±º ¼®§ »§» ­§²¼®±³» ©·¬¸ ¬±°·ó
`cal cyclosporine significantly reduced the numbersof ac-
`½¿´ ½§½´±­°±®·²» ­·¹²·º·½¿²¬´§ ®»¼«½»¼ ¬¸» ²«³¾»®­ ±º ¿½ó
`tivated lymphocytes within the conjunctiva.
`¬·ª¿¬»¼ ´§³°¸±½§¬»­ ©·¬¸·² ¬¸» ½±²¶«²½¬·ª¿ò
`
`Results: In cyclosporine-treated eyes, biopsy results of
`λ­«´¬­æ ײ ½§½´±­°±®·²»ó¬®»¿¬»¼ »§»­ô ¾·±°­§ ®»­«´¬­ ±º
`Arch Ophthalmol. 2000;118:1489-1496
`conjunctivae showeddecreases in the numberofcells posi-
`ß®½¸ Ñ°¸¬¸¿´³±´ò îðððåïïèæïìèçóïìçê
`½±²¶«²½¬·ª¿» ­¸±©»¼ ¼»½®»¿­»­ ·² ¬¸» ²«³¾»® ±º ½»´´­ °±­·ó
`
`
`Õ ÛÎßÌÑÝÑÒÖËÒÝÌ×Ê×Ì×Í ­·½½¿
`
`analysis of the lacrimal gland has re-
`ERATOCONJUNCTIVITIS sicca
`¿²¿´§­·­ ±º ¬¸» ´¿½®·³¿´ ¹´¿²¼ ¸¿­ ®»ó
`(KCS), or dry eye syn-
`ceived considerable attention, less work
`øÕÝÍ÷ô ±® ¼®§ »§» ­§²ó
`½»·ª»¼ ½±²­·¼»®¿¾´» ¿¬¬»²¬·±²ô ´»­­ ©±®µ
`has been done on pathological changes oc-
`drome,is characterized by
`¼®±³»ô ·­ ½¸¿®¿½¬»®·¦»¼ ¾§
`¸¿­ ¾»»² ¼±²» ±² °¿¬¸±´±¹·½¿´ ½¸¿²¹»­ ±½ó
`curring in the ocular surface. The chronic
`chronic dryness ofthe cor-
`½¸®±²·½ ¼®§²»­­ ±º ¬¸» ½±®ó
`½«®®·²¹ ·² ¬¸» ±½«´¿® ­«®º¿½»ò ̸» ½¸®±²·½
`nea and conjunctiva.’ Pa-
`dryness of the ocular surface in Sjogren
`²»¿ ¿²¼ ½±²¶«²½¬·ª¿òï пó
`¼®§²»­­ ±º ¬¸» ±½«´¿® ­«®º¿½» ·² Ͷ±X ¹®»²
`syndromehasbeenattributed to deterio-
`tients with KCS typically show symp-
`¬·»²¬­ ©·¬¸ ÕÝÍ ¬§°·½¿´´§ ­¸±© ­§³°ó
`­§²¼®±³» ¸¿­ ¾»»² ¿¬¬®·¾«¬»¼ ¬± ¼»¬»®·±ó
`ration oflacrimal gland function with de-
`toms of ocular discomfort ranging from
`¬±³­ ±º ±½«´¿® ¼·­½±³º±®¬ ®¿²¹·²¹ º®±³
`®¿¬·±² ±º ´¿½®·³¿´ ¹´¿²¼ º«²½¬·±² ©·¬¸ ¼»ó
`creased tear production.®’° However, in
`irritation to severe pain. Redness, burn-
`½®»¿­»¼ ¬»¿® °®±¼«½¬·±²òçôïð ر©»ª»®ô ·²
`·®®·¬¿¬·±² ¬± ­»ª»®» °¿·²ò λ¼²»­­ô ¾«®²ó
`ing, itching, foreign body sensation, con-
`Sjogren syndrome, conjunctival epithe-
`·²¹ô ·¬½¸·²¹ô º±®»·¹² ¾±¼§ ­»²­¿¬·±²ô ½±²ó
`Ͷ±X ¹®»² ­§²¼®±³»ô ½±²¶«²½¬·ª¿´ »°·¬¸»ó
`lial and stromal T-cell infiltration (pre-
`tact lens intolerance, photophobia, and
`¬¿½¬ ´»²­ ·²¬±´»®¿²½»ô °¸±¬±°¸±¾·¿ô ¿²¼
`´·¿´ ¿²¼ ­¬®±³¿´ Ìó½»´´ ·²º·´¬®¿¬·±² ø°®»ó
`blurred vision can occur.*
`dominantly CD3+ and CD4+ T lympho-
`¾´«®®»¼ ª·­·±² ½¿² ±½½«®òî
`¼±³·²¿²¬´§ ÝÜíõ ¿²¼ ÝÜìõ Ì ´§³°¸±ó
`cytes) has also been shownto occur along
`Although KCS can arise from vari-
`ß´¬¸±«¹¸ ÕÝÍ ½¿² ¿®·­» º®±³ ª¿®·ó
`½§¬»­÷ ¸¿­ ¿´­± ¾»»² ­¸±©² ¬± ±½½«® ¿´±²¹
`with drying of the ocular surface.®"
`ous types of diseases, commonto all is the
`©·¬¸ ¼®§·²¹ ±º ¬¸» ±½«´¿® ­«®º¿½»òçôïï
`±«­ ¬§°»­ ±º ¼·­»¿­»­ô ½±³³±² ¬± ¿´´ ·­ ¬¸»
`involvement of immune-mediated or in-
`Supporting a role for an immuno-
`·²ª±´ª»³»²¬ ±º ·³³«²»ó³»¼·¿¬»¼ ±® ·²ó
`Í«°°±®¬·²¹ ¿ ®±´» º±® ¿² ·³³«²±ó
`flammatory-mediated pathways. Immu-
`pathogenesis of KCS arethe reports of ac-
`º´¿³³¿¬±®§ó³»¼·¿¬»¼ °¿¬¸©¿§­òí ׳³«ó
`°¿¬¸±¹»²»­·­ ±º ÕÝÍ ¿®» ¬¸» ®»°±®¬­ ±º ¿½ó
`nopathologic studiesof the lacrimal gland
`tivated lymphocytes as demonstrated by
`From the Schepens Eye
`²±°¿¬¸±´±¹·½ ­¬«¼·»­ ±º ¬¸» ´¿½®·³¿´ ¹´¿²¼
`¬·ª¿¬»¼ ´§³°¸±½§¬»­ ¿­ ¼»³±²­¬®¿¬»¼ ¾§
`Ú®±³ ¬¸» ͽ¸»°»²­ Û§»
`Research Institute and
`expression of lymphocyte activation mark-
`in patients with Sjogren syndrome show
`λ­»¿®½¸ ײ­¬·¬«¬» ¿²¼
`·² °¿¬·»²¬­ ©·¬¸ Ͷ±X ¹®»² ­§²¼®±³» ­¸±©
`»¨°®»­­·±² ±º ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µó
`Department of Ophthalmology,
`Ü»°¿®¬³»²¬ ±º Ñ°¸¬¸¿´³±´±¹§ô
`progressive lymphocytic infiltration,pri-
`ers such as HLA-DR (MHC classII) and
`°®±¹®»­­·ª» ´§³°¸±½§¬·½ ·²º·´¬®¿¬·±²ô °®·ó
`»®­ ­«½¸ ¿­ ØÔßóÜÎ øÓØÝ ½´¿­­ ××÷ ¿²¼
`Harvard Medical School,
`Ø¿®ª¿®¼ Ó»¼·½¿´ ͽ¸±±´ô
`marily consisting of CD4+ T andBcells.*”
`ICAM-1 (intercellular adhesion mol-
`³¿®·´§ ½±²­·­¬·²¹ ±º ÝÜìõ Ì ¿²¼ Þ ½»´´­òìôë
`×ÝßÓóï ø·²¬»®½»´´«´¿® ¿¼¸»­·±² ³±´ó
`Boston, Mass (Drs Kunert and
`Þ±­¬±²ô Ó¿­­ øÜ®­ Õ«²»®¬ ¿²¼
`ecule-1) in the conjunctivaofpatients with
`This infiltration is believed to be respon-
`̸·­ ·²º·´¬®¿¬·±² ·­ ¾»´·»ª»¼ ¬± ¾» ®»­°±²ó
`»½«´»óï÷ ·² ¬¸» ½±²¶«²½¬·ª¿ ±º °¿¬·»²¬­ ©·¬¸
`Gipson, Ms Tisdale); Allergan,
`Ù·°­±²ô Ó­ Ì·­¼¿´»÷å ß´´»®¹¿²ô
`sible for the destruction of normal secre-
`Sjogren syndrome.’””? To date, there is
`Ͷ±X ¹®»² ­§²¼®±³»òïîôïí ̱ ¼¿¬»ô ¬¸»®» ·­
`­·¾´» º±® ¬¸» ¼»­¬®«½¬·±² ±º ²±®³¿´ ­»½®»ó
`Inc, Irvine, Calif (Dr Stern);
`ײ½ô ×®ª·²»ô Ý¿´·º øÜ® ͬ»®²÷å
`little information on the effect of modu-
`tory function.* Lymphocytic infiltration of
`¬±®§ º«²½¬·±²òê Ô§³°¸±½§¬·½ ·²º·´¬®¿¬·±² ±º
`´·¬¬´» ·²º±®³¿¬·±² ±² ¬¸» »ºº»½¬ ±º ³±¼«ó
`and the National Eye Institute,
`¿²¼ ¬¸» Ò¿¬·±²¿´ Û§» ײ­¬·¬«¬»ô
`the lacrimal gland has also been de-
`lating these molecules in the conjunctiva
`¬¸» ´¿½®·³¿´ ¹´¿²¼ ¸¿­ ¿´­± ¾»»² ¼»ó
`´¿¬·²¹ ¬¸»­» ³±´»½«´»­ ·² ¬¸» ½±²¶«²½¬·ª¿
`Bethesda, Md (Ms Smith).
`Þ»¬¸»­¼¿ô Ó¼ øÓ­ ͳ·¬¸÷ò
`of patients with Sjogren and non-
`scribed in patients with non-Sjégren
`­½®·¾»¼ ·² °¿¬·»²¬­ ©·¬¸ ²±²óͶ±X ¹®»²
`±º °¿¬·»²¬­ ©·¬¸ Ͷ±X ¹®»² ¿²¼ ²±²o
`DrStern is an employee of
`Ü® ͬ»®² ·­ ¿² »³°´±§»» ±º
`KCS.’* Although the immunopathologic
`Sjogren syndrome.
`ÕÝÍòéôè ß´¬¸±«¹¸ ¬¸» ·³³«²±°¿¬¸±´±¹·½
`Ͷ±X ¹®»² ­§²¼®±³»ò
`Allergan Inc.
`ß´´»®¹¿² ײ½ò
`
`
`WWW. ARCHOPHTHALMOL.COM
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`øÎÛÐÎ×ÒÌÛÜ÷ ßÎÝØ ÑÐØÌØßÔÓÑÔ ñ ÊÑÔ ïïèô ÒÑÊ îððð
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`TEVA - EXHIBIT 1012
`
`©2000 American Medical Association. All rights reserved.
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`TEVA - EXHIBIT 1012
`
`

`

`
`
`SUBJECTS AND METHODS
`ÍËÞÖÛÝÌÍ ßÒÜ ÓÛÌØÑÜÍ
`
`SUBJECTS
`ÍËÞÖÛÝÌÍ
`
`Conjunctival biopsy specimens from 32 patients were ex-
`ݱ²¶«²½¬·ª¿´ ¾·±°­§ ­°»½·³»²­ º®±³ íî °¿¬·»²¬­ ©»®» »¨ó
`amined; 13 patients were treated with 0.05% CsA, 6 with
`¿³·²»¼å ïí °¿¬·»²¬­ ©»®» ¬®»¿¬»¼ ©·¬¸ ðòðëû Ý­ßô ê ©·¬¸
`0.1% CsA, and 13 with vehicle alone. This subject group
`ðòïû Ý­ßô ¿²¼ ïí ©·¬¸ ª»¸·½´» ¿´±²»ò ̸·­ ­«¾¶»½¬ ¹®±«°
`was randomly chosen from a double-masked, vehicle-
`©¿­ ®¿²¼±³´§ ½¸±­»² º®±³ ¿ ¼±«¾´»ó³¿­µ»¼ô ª»¸·½´»ó
`controlled clinical study designed by Allergan,Inc, Irvine,
`½±²¬®±´´»¼ ½´·²·½¿´ ­¬«¼§ ¼»­·¹²»¼ ¾§ ß´´»®¹¿²ô ײ½ô ×®ª·²»ô
`Calif, to investigate the efficacy and safety of topical CsA
`Ý¿´·ºô ¬± ·²ª»­¬·¹¿¬» ¬¸» »ºº·½¿½§ ¿²¼ ­¿º»¬§ ±º ¬±°·½¿´ Ý­ß
`in the treatment of moderate to severe KCS.”' The study
`·² ¬¸» ¬®»¿¬³»²¬ ±º ³±¼»®¿¬» ¬± ­»ª»®» ÕÝÍòîï ̸» ­¬«¼§
`was conducted in compliance with Good Clinical Prac-
`©¿­ ½±²¼«½¬»¼ ·² ½±³°´·¿²½» ©·¬¸ Ù±±¼ Ý´·²·½¿´ Ю¿½ó
`tices, investigational site institutional review board regu-
`¬·½»­ô ·²ª»­¬·¹¿¬·±²¿´ ­·¬» ·²­¬·¬«¬·±²¿´ ®»ª·»© ¾±¿®¼ ®»¹«ó
`lations, sponsor and investigator obligations, informed con-
`´¿¬·±²­ô ­°±²­±® ¿²¼ ·²ª»­¬·¹¿¬±® ±¾´·¹¿¬·±²­ô ·²º±®³»¼ ½±²ó
`sent regulations, and the Declaration of Helsinki. Potential
`­»²¬ ®»¹«´¿¬·±²­ô ¿²¼ ¬¸» Ü»½´¿®¿¬·±² ±º Ø»´­·²µ·ò ᬻ²¬·¿´
`patients signed a prescreening informed consent form and
`°¿¬·»²¬­ ­·¹²»¼ ¿ °®»­½®»»²·²¹ ·²º±®³»¼ ½±²­»²¬ º±®³ ¿²¼
`a second written informed consent form prior to actual en-
`¿ ­»½±²¼ ©®·¬¬»² ·²º±®³»¼ ½±²­»²¬ º±®³ °®·±® ¬± ¿½¬«¿´ »²ó
`rollment.”' The protocol for this study is described briefly
`®±´´³»²¬òîï ̸» °®±¬±½±´ º±® ¬¸·­ ­¬«¼§ ·­ ¼»­½®·¾»¼ ¾®·»º´§
`here. Adult patients of either sex wereeligible for partici-
`¸»®»ò ß¼«´¬ °¿¬·»²¬­ ±º »·¬¸»® ­»¨ ©»®» »´·¹·¾´» º±® °¿®¬·½·ó
`pation if they had a diagnosis of moderate to severe KCS
`°¿¬·±² ·º ¬¸»§ ¸¿¼ ¿ ¼·¿¹²±­·­ ±º ³±¼»®¿¬» ¬± ­»ª»®» ÕÝÍ
`at initial examination as defined by the following criteria:
`¿¬ ·²·¬·¿´ »¨¿³·²¿¬·±² ¿­ ¼»º·²»¼ ¾§ ¬¸» º±´´±©·²¹ ½®·¬»®·¿æ
`(1) Schirmertest results (without anesthesia) less than or
`øï÷ ͽ¸·®³»® ¬»­¬ ®»­«´¬­ ø©·¬¸±«¬ ¿²»­¬¸»­·¿÷ ´»­­ ¬¸¿² ±®
`equal to 5 mm/5 min in at least 1 eye (if Schirmertest re-
`»¯«¿´ ¬± ë ³³ñë ³·² ·² ¿¬ ´»¿­¬ ï »§» ø·º ͽ¸·®³»® ¬»­¬ ®»ó
`sults without anesthesia equaled 0 mm/5 min, then Schirmer
`­«´¬­ ©·¬¸±«¬ ¿²»­¬¸»­·¿ »¯«¿´»¼ ð ³³ñë ³·²ô ¬¸»² ͽ¸·®³»®
`test results with nasal stimulation had to be >3 mm/5 min
`¬»­¬ ®»­«´¬­ ©·¬¸ ²¿­¿´ ­¬·³«´¿¬·±² ¸¿¼ ¬± ¾» òí ³³ñë ³·²
`in the same eye); (2) sum of corneal and interpalpebral con-
`·² ¬¸» ­¿³» »§»÷å øî÷ ­«³ ±º ½±®²»¿´ ¿²¼ ·²¬»®°¿´°»¾®¿´ ½±²ó
`junctival staining greater than or equal to +5 in the same
`¶«²½¬·ª¿´ ­¬¿·²·²¹ ¹®»¿¬»® ¬¸¿² ±® »¯«¿´ ¬± õë ·² ¬¸» ­¿³»
`eye where cornealstaining was greater than or equal to +2;
`»§» ©¸»®» ½±®²»¿´ ­¬¿·²·²¹ ©¿­ ¹®»¿¬»® ¬¸¿² ±® »¯«¿´ ¬± õîå
`(3) a baseline Ocular Surface Disease Index” score of 0.1
`øí÷ ¿ ¾¿­»´·²» ѽ«´¿® Í«®º¿½» Ü·­»¿­» ײ¼»¨îî ­½±®» ±º ðòï
`with no morethan 3 responses of “not applicable”; and (4)
`©·¬¸ ²± ³±®» ¬¸¿² í ®»­°±²­»­ ±º v²±¬ ¿°°´·½¿¾´»få ¿²¼ øì÷
`a score greater than or equal to 3 on the Subjective Facial
`¿ ­½±®» ¹®»¿¬»® ¬¸¿² ±® »¯«¿´ ¬± í ±² ¬¸» Í«¾¶»½¬·ª» Ú¿½·¿´
`Expression Scale.”’ Signs and symptoms must have been
`Û¨°®»­­·±² ͽ¿´»òîï Í·¹²­ ¿²¼ ­§³°¬±³­ ³«­¬ ¸¿ª» ¾»»²
`present despite conventional management.
`°®»­»²¬ ¼»­°·¬» ½±²ª»²¬·±²¿´ ³¿²¿¹»³»²¬ò
`Patients were excluded from thestudy if they had par-
`п¬·»²¬­ ©»®» »¨½´«¼»¼ º®±³ ¬¸» ­¬«¼§ ·º ¬¸»§ ¸¿¼ °¿®ó
`ticipated in an earlier clinical trial with CsA ophthalmic
`¬·½·°¿¬»¼ ·² ¿² »¿®´·»® ½´·²·½¿´ ¬®·¿´ ©·¬¸ Ý­ß ±°¸¬¸¿´³·½
`emulsion or had used systemic or topical ophthalmic CsA
`»³«´­·±² ±® ¸¿¼ «­»¼ ­§­¬»³·½ ±® ¬±°·½¿´ ±°¸¬¸¿´³·½ Ý­ß
`within 90 days prior to the study. Other exclusioncriteria
`©·¬¸·² ç𠼿§­ °®·±® ¬± ¬¸» ­¬«¼§ò Ѭ¸»® »¨½´«­·±² ½®·¬»®·¿
`were the presenceor history of any systemic or ocular dis-
`©»®» ¬¸» °®»­»²½» ±® ¸·­¬±®§ ±º ¿²§ ­§­¬»³·½ ±® ±½«´¿® ¼·­ó
`orderor condition (including ocular surgery, trauma, and
`±®¼»® ±® ½±²¼·¬·±² ø·²½´«¼·²¹ ±½«´¿® ­«®¹»®§ô ¬®¿«³¿ô ¿²¼
`disease); currentor recentuse of topical ophthalmic or sys-
`¼·­»¿­»÷å ½«®®»²¬ ±® ®»½»²¬ «­» ±º ¬±°·½¿´ ±°¸¬¸¿´³·½ ±® ­§­ó
`temic medications that could affect a dry eye condition;
`¬»³·½ ³»¼·½¿¬·±²­ ¬¸¿¬ ½±«´¼ ¿ºº»½¬ ¿ ¼®§ »§» ½±²¼·¬·±²å
`knownhypersensitivity to any componentof the drug or
`µ²±©² ¸§°»®­»²­·¬·ª·¬§ ¬± ¿²§ ½±³°±²»²¬ ±º ¬¸» ¼®«¹ ±®
`procedural medications such as stains or anesthetics;
`°®±½»¼«®¿´ ³»¼·½¿¬·±²­ ­«½¸ ¿­ ­¬¿·²­ ±® ¿²»­¬¸»¬·½­å
`
`required contact lens wear during the study;recent (within
`®»¯«·®»¼ ½±²¬¿½¬ ´»²­ ©»¿® ¼«®·²¹ ¬¸» ­¬«¼§å ®»½»²¬ ø©·¬¸·²
`1 month) oranticipated use of temporary punctalplugs dur-
`ï ³±²¬¸÷ ±® ¿²¬·½·°¿¬»¼ «­» ±º ¬»³°±®¿®§ °«²½¬¿´ °´«¹­ ¼«®ó
`ing the study; permanentocclusionoflacrimal puncta within
`·²¹ ¬¸» ­¬«¼§å °»®³¿²»²¬ ±½½´«­·±² ±º ´¿½®·³¿´ °«²½¬¿ ©·¬¸·²
`3 monthsof the study;or if the patients were pregnant, lac-
`í ³±²¬¸­ ±º ¬¸» ­¬«¼§å ±® ·º ¬¸» °¿¬·»²¬­ ©»®» °®»¹²¿²¬ô ´¿½ó
`tating, or planning a pregnancy. Patients were also ex-
`¬¿¬·²¹ô ±® °´¿²²·²¹ ¿ °®»¹²¿²½§ò п¬·»²¬­ ©»®» ¿´­± »¨ó
`cludedif they appeared to have end-stage lacrimal gland
`½´«¼»¼ ·º ¬¸»§ ¿°°»¿®»¼ ¬± ¸¿ª» »²¼ó­¬¿¹» ´¿½®·³¿´ ¹´¿²¼
`disease (Schirmerreading with nasal stimulation <3 mm/5
`¼·­»¿­» øͽ¸·®³»® ®»¿¼·²¹ ©·¬¸ ²¿­¿´ ­¬·³«´¿¬·±² ôí ³³ñë
`min) or if their KCS was secondary to the destruction of
`³·²÷ ±® ·º ¬¸»·® ÕÝÍ ©¿­ ­»½±²¼¿®§ ¬± ¬¸» ¼»­¬®«½¬·±² ±º
`conjunctival goblet cells or scarring.
`½±²¶«²½¬·ª¿´ ¹±¾´»¬ ½»´´­ ±® ­½¿®®·²¹ò
`A retrospective diagnosis ofSjogren syndrome was used.
`ß ®»¬®±­°»½¬·ª» ¼·¿¹²±­·­ ±º Ͷ±X ¹®»² ­§²¼®±³» ©¿­ «­»¼
`with modified criteria reported by Vitali et al” to ensure
`©·¬¸ ³±¼·º·»¼ ½®·¬»®·¿ ®»°±®¬»¼ ¾§ Ê·¬¿´· »¬ ¿´îí ¬± »²­«®»
`that a consistent definition of Sjagren syndrome was as-
`¬¸¿¬ ¿ ½±²­·­¬»²¬ ¼»º·²·¬·±² ±º Ͷ±X ¹®»² ­§²¼®±³» ©¿­ ¿­ó
`signed to the patients enrolled. Diagnosis included pres-
`­·¹²»¼ ¬± ¬¸» °¿¬·»²¬­ »²®±´´»¼ò Ü·¿¹²±­·­ ·²½´«¼»¼ °®»­ó
`enceof at least one of the following autoantibodies in sera:
`»²½» ±º ¿¬ ´»¿­¬ ±²» ±º ¬¸» º±´´±©·²¹ ¿«¬±¿²¬·¾±¼·»­ ·² ­»®¿æ
`antinuclear antibody (ANA), rheumatoid factor (RF), and
`¿²¬·²«½´»¿® ¿²¬·¾±¼§ øßÒß÷ô ®¸»«³¿¬±·¼ º¿½¬±® øÎÚ÷ô ¿²¼
`Sjégren syndrome autoantibodies class SS-A (Ro) and class
`Ͷ±X ¹®»² ­§²¼®±³» ¿«¬±¿²¬·¾±¼·»­ ½´¿­­ ÍÍóß øα÷ ¿²¼ ½´¿­­
`SS-B (La). In addition, oral and ocular symptoms were used
`ÍÍóÞ øÔ¿÷ò ײ ¿¼¼·¬·±²ô ±®¿´ ¿²¼ ±½«´¿® ­§³°¬±³­ ©»®» «­»¼
`to classify patients with Sjégren syndrome.
`¬± ½´¿­­·º§ °¿¬·»²¬­ ©·¬¸ Ͷ±X ¹®»² ­§²¼®±³»ò
`Patientsinstilled 1 drop of 0.05% or 0.1% CsA oph-
`п¬·»²¬­ ·²­¬·´´»¼ ï ¼®±° ±º ðòðëû ±® ðòïû Ý­ß ±°¸ó
`thalmic emulsionsor vehicle of CsA ophthalmic emulsion
`¬¸¿´³·½ »³«´­·±²­ ±® ª»¸·½´» ±º Ý­ß ±°¸¬¸¿´³·½ »³«´­·±²
`twice daily in each eye for 6 months; once on waking in
`¬©·½» ¼¿·´§ ·² »¿½¸ »§» º±® ê ³±²¬¸­å ±²½» ±² ©¿µ·²¹ ·²
`the morning and onceat bedtime. Patients were allowed
`¬¸» ³±®²·²¹ ¿²¼ ±²½» ¿¬ ¾»¼¬·³»ò п¬·»²¬­ ©»®» ¿´´±©»¼
`to use assigned artificial tears (REFRESH Lubricant Eye
`¬± «­» ¿­­·¹²»¼ ¿®¬·º·½·¿´ ¬»¿®­ øÎÛÚÎÛÍØ Ô«¾®·½¿²¬ Û§»
`Drops; Allergan Inc) as needed up to month 4.
`Ü®±°­å ß´´»®¹¿² ײ½÷ ¿­ ²»»¼»¼ «° ¬± ³±²¬¸ ìò
`Full-thickness conjunctival biopsy specimens ofa stan-
`Ú«´´ó¬¸·½µ²»­­ ½±²¶«²½¬·ª¿´ ¾·±°­§ ­°»½·³»²­ ±º ¿ ­¬¿²ó
`dard size (2-3 mm) were removed from the “worse” eye
`¼¿®¼ ­·¦» øîóí ³³÷ ©»®» ®»³±ª»¼ º®±³ ¬¸» v©±®­»f »§»
`by surgeonsfollowing standard procedure. The worse eye
`¾§ ­«®¹»±²­ º±´´±©·²¹ ­¬¿²¼¿®¼ °®±½»¼«®»ò ̸» ©±®­» »§»
`was defined as the eye with the worse Schirmertear test
`©¿­ ¼»º·²»¼ ¿­ ¬¸» »§» ©·¬¸ ¬¸» ©±®­» ͽ¸·®³»® ¬»¿® ¬»­¬
`value (without anesthesia) and the worse sum of corneal
`ª¿´«» ø©·¬¸±«¬ ¿²»­¬¸»­·¿÷ ¿²¼ ¬¸» ©±®­» ­«³ ±º ½±®²»¿´
`and interpalpebral conjunctival staining. If both eyes were
`¿²¼ ·²¬»®°¿´°»¾®¿´ ½±²¶«²½¬·ª¿´ ­¬¿·²·²¹ò ׺ ¾±¬¸ »§»­ ©»®»
`comparable, then the right eye was used. At the baseline
`½±³°¿®¿¾´»ô ¬¸»² ¬¸» ®·¹¸¬ »§» ©¿­ «­»¼ò ߬ ¬¸» ¾¿­»´·²»
`visit, the conjunctival biopsy specimen was obtained from
`ª·­·¬ô ¬¸» ½±²¶«²½¬·ª¿´ ¾·±°­§ ­°»½·³»² ©¿­ ±¾¬¿·²»¼ º®±³
`the inferonasal quadrant close to midline. At the 6-month
`¬¸» ·²º»®±²¿­¿´ ¯«¿¼®¿²¬ ½´±­» ¬± ³·¼´·²»ò ߬ ¬¸» êó³±²¬¸
`visit, the sample was removed from the sameeye but from
`ª·­·¬ô ¬¸» ­¿³°´» ©¿­ ®»³±ª»¼ º®±³ ¬¸» ­¿³» »§» ¾«¬ º®±³
`the inferotemporal quadrant, also close to midline.
`¬¸» ·²º»®±¬»³°±®¿´ ¯«¿¼®¿²¬ô ¿´­± ½´±­» ¬± ³·¼´·²»ò
`
`TISSUE PROCESSING FOR
`Ì×ÍÍËÛ ÐÎÑÝÛÍÍ×ÒÙ ÚÑÎ
`IMMUNOHISTOCHEMICAL ANALYSIS
`×ÓÓËÒÑØ×ÍÌÑÝØÛÓ×ÝßÔ ßÒßÔÇÍ×Í
`
`After removal, the baseline biopsy specimens were imme-
`ߺ¬»® ®»³±ª¿´ô ¬¸» ¾¿­»´·²» ¾·±°­§ ­°»½·³»²­ ©»®» ·³³»ó
`diately frozen in OCT embedding compound(Tissue-Tek;
`¼·¿¬»´§ º®±¦»² ·² ÑÝÌ »³¾»¼¼·²¹ ½±³°±«²¼ øÌ·­­«»óÌ»µå
`Miles Laboratories, Elkhart, Ind) in a cryomold (Miles
`Ó·´»­ Ô¿¾±®¿¬±®·»­ô Û´µ¸¿®¬ô ײ¼÷ ·² ¿ ½®§±³±´¼ øÓ·´»­
`Laboratories) and stored at -80°C until patient-matched
`Ô¿¾±®¿¬±®·»­÷ ¿²¼ ­¬±®»¼ ¿¬ "èðWÝ «²¬·´ °¿¬·»²¬ó³¿¬½¸»¼
`
`Currently, administration ofartificial tears is the most
`Ý«®®»²¬´§ô ¿¼³·²·­¬®¿¬·±² ±º ¿®¬·º·½·¿´ ¬»¿®­ ·­ ¬¸» ³±­¬
`commontherapyavailable for lubricating a dry ocular sur-
`½±³³±² ¬¸»®¿°§ ¿ª¿·´¿¾´» º±® ´«¾®·½¿¬·²¹ ¿ ¼®§ ±½«´¿® ­«®ó
`face. This palliative treatment gives only temporary and in-
`º¿½»ò ̸·­ °¿´´·¿¬·ª» ¬®»¿¬³»²¬ ¹·ª»­ ±²´§ ¬»³°±®¿®§ ¿²¼ ·²ó
`complete symptomaticreliefand does not address the cause
`½±³°´»¬» ­§³°¬±³¿¬·½ ®»´·»º ¿²¼ ¼±»­ ²±¬ ¿¼¼®»­­ ¬¸» ½¿«­»
`of the symptoms, which may include immune-mediated
`±º ¬¸» ­§³°¬±³­ô ©¸·½¸ ³¿§ ·²½´«¼» ·³³«²»ó³»¼·¿¬»¼
`inflammationofthe ocularsurface. Evidence ofinflamma-
`·²º´¿³³¿¬·±² ±º ¬¸» ±½«´¿® ­«®º¿½»ò Ûª·¼»²½» ±º ·²º´¿³³¿ó
`tory processes in the pathogenesis of KCSled to the de-
`¬±®§ °®±½»­­»­ ·² ¬¸» °¿¬¸±¹»²»­·­ ±º ÕÝÍ ´»¼ ¬± ¬¸» ¼»ó
`velopmentofcyclosporine (CsA)asa first attemptto treat
`ª»´±°³»²¬ ±º ½§½´±­°±®·²» øÝ­ß÷ ¿­ ¿ º·®­¬ ¿¬¬»³°¬ ¬± ¬®»¿¬
`this condition therapeutically. Cyclosporine is an immu-
`¬¸·­ ½±²¼·¬·±² ¬¸»®¿°»«¬·½¿´´§ò ݧ½´±­°±®·²» ·­ ¿² ·³³«ó
`nosuppressive agent commonly used systemically to treat
`²±­«°°®»­­·ª» ¿¹»²¬ ½±³³±²´§ «­»¼ ­§­¬»³·½¿´´§ ¬± ¬®»¿¬
`inflammatory diseases such aspsoriasis or rheumatoid ar-
`·²º´¿³³¿¬±®§ ¼·­»¿­»­ ­«½¸ ¿­ °­±®·¿­·­ ±® ®¸»«³¿¬±·¼ ¿®ó
`thritis or to prevent organ transplant rejection.'* Topical
`¬¸®·¬·­ ±® ¬± °®»ª»²¬ ±®¹¿² ¬®¿²­°´¿²¬ ®»¶»½¬·±²òïì ̱°·½¿´
`CsA has been usedas treatmentofocular conditions such
`Ý­ß ¸¿­ ¾»»² «­»¼ ¿­ ¬®»¿¬³»²¬ ±º ±½«´¿® ½±²¼·¬·±²­ ­«½¸
`as vernal keratoconjunctivitis, ° corneal transplants,"* cor-
`¿­ ª»®²¿´ µ»®¿¬±½±²¶«²½¬·ª·¬·­ôïë ½±®²»¿´ ¬®¿²­°´¿²¬­ôïê ½±®ó
`neal ulcers,"’ and herpetic stromal keratitis.'* The effect of
`²»¿´ «´½»®­ôïé ¿²¼ ¸»®°»¬·½ ­¬®±³¿´ µ»®¿¬·¬·­òïè ̸» »ºº»½¬ ±º
`this drug on inflammatory diseasesis dueto its ability to
`¬¸·­ ¼®«¹ ±² ·²º´¿³³¿¬±®§ ¼·­»¿­»­ ·­ ¼«» ¬± ·¬­ ¿¾·´·¬§ ¬±
`
`inhibit T-cell-mediated inflammation by preventing the ac-
`·²¸·¾·¬ Ìó½»´´o³»¼·¿¬»¼ ·²º´¿³³¿¬·±² ¾§ °®»ª»²¬·²¹ ¬¸» ¿½ó
`tivation of T cells (by antigen-presenting cells or
`¬·ª¿¬·±² ±º Ì ½»´´­ ø¾§ ¿²¬·¹»²ó°®»­»²¬·²¹ ½»´´­ ±®
`cytokines).!*”° Activated T cells are responsible for the pro-
`½§¬±µ·²»­÷òïçôîð ß½¬·ª¿¬»¼ Ì ½»´´­ ¿®» ®»­°±²­·¾´» º±® ¬¸» °®±ó
`duction of inflammatory substances such as cytokines,
`¼«½¬·±² ±º ·²º´¿³³¿¬±®§ ­«¾­¬¿²½»­ ­«½¸ ¿­ ½§¬±µ·²»­ô
`whichleadto furthertissue damageand,in turn,to the ac-
`©¸·½¸ ´»¿¼ ¬± º«®¬¸»® ¬·­­«» ¼¿³¿¹» ¿²¼ô ·² ¬«®²ô ¬± ¬¸» ¿½ó
`tivation of more T cells and the production of even more
`¬·ª¿¬·±² ±º ³±®» Ì ½»´´­ ¿²¼ ¬¸» °®±¼«½¬·±² ±º »ª»² ³±®»
`inflammatory substances.
`·²º´¿³³¿¬±®§ ­«¾­¬¿²½»­ò
`Clinical trials with this drug have shown improve-
`Ý´·²·½¿´ ¬®·¿´­ ©·¬¸ ¬¸·­ ¼®«¹ ¸¿ª» ­¸±©² ·³°®±ª»ó
`mentin various objective measures of KCS suchas cor-
`³»²¬ ·² ª¿®·±«­ ±¾¶»½¬·ª» ³»¿­«®»­ ±º ÕÝÍ ­«½¸ ¿­ ½±®ó
`neal staining and Schirmertest values.”’ To attempt to
`²»¿´ ­¬¿·²·²¹ ¿²¼ ͽ¸·®³»® ¬»­¬ ª¿´«»­òîï ̱ ¿¬¬»³°¬ ¬±
`find tissue correlates in these patients, conjunctival bi-
`º·²¼ ¬·­­«» ½±®®»´¿¬»­ ·² ¬¸»­» °¿¬·»²¬­ô ½±²¶«²½¬·ª¿´ ¾·ó
`opsy specimens from patients with Sjogren and non-
`±°­§ ­°»½·³»²­ º®±³ °¿¬·»²¬­ ©·¬¸ Ͷ±X ¹®»² ¿²¼ ²±²ó
`Sjogren KCStreated with CsA or vehicle were evaluated
`Ͷ±X ¹®»² ÕÝÍ ¬®»¿¬»¼ ©·¬¸ Ý­ß ±® ª»¸·½´» ©»®» »ª¿´«¿¬»¼
`immunohistochemically for the presence of activated T
`·³³«²±¸·­¬±½¸»³·½¿´´§ º±® ¬¸» °®»­»²½» ±º ¿½¬·ª¿¬»¼ Ì
`cells (CD3+ [Pan-T cell], CD4+ [T helpercell], and CD8+
`½»´´­ øÝÜíõ Åп²óÌ ½»´´Ãô ÝÜìõ ÅÌ ¸»´°»® ½»´´Ãô ¿²¼ ÝÜèõ
`[cytotoxic T cell]) and lymphocyte-activation markers
`Ž§¬±¬±¨·½ Ì ½»´´Ã÷ ¿²¼ ´§³°¸±½§¬»ó¿½¬·ª¿¬·±² ³¿®µ»®­
`
`
`WWW. ARCHOPHTHALMOL.COM
`(REPRINTED) ARCH OPHTHALMOL/VOL 118, NOV 2000
`ÉÉÉòßÎÝØÑÐØÌØßÔÓÑÔòÝÑÓ
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`1490
`ïìçð
`
`©2000 American Medical Association. All rights reserved.
`ƒîððð ß³»®·½¿² Ó»¼·½¿´ ß­­±½·¿¬·±²ò ß´´ ®·¹¸¬­ ®»­»®ª»¼ò
`Downloaded From: http://archopht.jamanetwork.com/ by a University of Michigan User on 01/25/2016
`ܱ©²´±¿¼»¼ Ú®±³æ ¸¬¬°æññ¿®½¸±°¸¬ò¶¿³¿²»¬©±®µò½±³ñ ¾§ ¿ ˲·ª»®­·¬§ ±º Ó·½¸·¹¿² Ë­»® ±² ðïñîëñîðïê
`
`

`

`andrightof that area. In this mannertheentire biopsy area
`¿²¼ ®·¹¸¬ ±º ¬¸¿¬ ¿®»¿ò ײ ¬¸·­ ³¿²²»® ¬¸» »²¬·®» ¾·±°­§ ¿®»¿
`was usually captured.
`©¿­ «­«¿´´§ ½¿°¬«®»¼ò
`
`COUNTING PROCEDURE
`ÝÑËÒÌ×ÒÙ ÐÎÑÝÛÜËÎÛ
`
`Measurementof 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 (AdobeSystems 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 recordedforcells 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 orcells 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 Photoshopandthefact 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 recordedascells per millimeter squared
`­½±°»ò Ü¿¬¿ ©»®» ®»½±®¼»¼ ¿­ ½»´´­ °»® ³·´´·³»¬»® ­¯«¿®»¼
`for all markers, andstatistical analysis was based on these
`º±® ¿´´ ³¿®µ»®­ô ¿²¼ ­¬¿¬·­¬·½¿´ ¿²¿´§­·­ ©¿­ ¾¿­»¼ ±² ¬¸»­»
`measurements.
`³»¿­«®»³»²¬­ò
`
`Sjogren subset.
`
`6-month biopsy specimenswere obtained and similarly fro-
`êó³±²¬¸ ¾·±°­§ ­°»½·³»²­ ©»®» ±¾¬¿·²»¼ ¿²¼ ­·³·´¿®´§ º®±ó
`zen. Six-micrometersections 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 aspairs
`·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
`
`Immunohistochemicalstaining 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 werefixed
`¿²¼ ØÔßóÜÎ øи¿®Ó·²¹»²÷ò Ý®§±­¬¿¬ ­»½¬·±²­ ©»®» º·¨»¼
`in cold acetone (—20°C)for 3 minutes andair 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
`pg/mL; CD4, 5.0 pg/mL; CD8, 2.5 pg/mL; CD11a, 10.0
`¡¹ñ³Ôå ÝÜìô ëòð ¡¹ñ³Ôå ÝÜèô îòë ¡¹ñ³Ôå ÝÜïï¿ô ïðòð
`pg/mL; and HLA-DR,1.0 pg/mL.Sections were rinsed in
`¡¹ñ³Ôå ¿²¼ ØÔßóÜÎô ïòð ¡¹ñ³Ôò Í»½¬·±²­ ©»®» ®·²­»¼ ·²
`PBSalone, 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 temperaturein 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 Jackson 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 undera 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 numberof positive cells, followed by imagestotheleft
`»­¬ ²«³¾»® ±º °±­·¬·ª» ½»´´­ô º±´´±©»¼ ¾§ ·³¿¹»­ ¬± ¬¸» ´»º¬
`
`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 continuousvariables 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 numberofcells 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󩿧
`ANOVAwith main effect for treatment was used totest
`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 groupsbyvisit. 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-Sjégren 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 | patient in the
`¬®»¿¬³»²¬ ¹®±«° ·² ©¸·½¸ ¬¸»®» ©¿­ ±²´§ ï °¿¬·»²¬ ·² ¬¸»
`Ͷ±X ¹®»² ­«¾­»¬ò
`
`(CD11a and HLA-DR)to further understand the under-
`øÝÜïï¿ ¿²¼ ØÔßóÜÎ÷ ¬± º«®¬¸»® «²¼»®­¬¿²¼ ¬¸» «²¼»®ó
`lying mechanism of CsA treatment.
`´§·²¹ ³»½¸¿²·­³ ±º Ý­ß ¬®»¿¬³»²¬ò
`
`Eady
`ÎÛÍËÔÌÍ
`PATIENT POPULATION
`ÐßÌ×ÛÒÌ ÐÑÐËÔßÌ×ÑÒ
`
`The mean+SDage of our subjects was 59.0+13.5 years
`̸» ³»¿² € ÍÜ ¿¹» ±º ±«® ­«¾¶»½¬­ ©¿­ ëçòð € ïíòë §»¿®­
`(range, 28.8-84.2 years), including 27 women and 5 men.
`ø®¿²¹»ô îèòèóèìòî §»¿®­÷ô ·²½´«¼·²¹ îé ©±³»² ¿²¼ ë ³»²ò
`Within this group, there were 12 Sjogren and 20 non-
`É·¬¸·² ¬¸·­ ¹®±«°ô ¬¸»®» ©»®» ïî Ͷ±X ¹®»² ¿²¼ îð ²±²ó
`Sjogren patients.
`Ͷ±X ¹®»² °¿¬·»²¬­ò
`
`LYMPHOCYTIC MARKERS
`ÔÇÓÐØÑÝÇÌ×Ý ÓßÎÕÛÎÍ
`
`In general, there was a decrease from baseline in the num-
`ײ ¹»²»®¿´ô ¬¸»®» ©¿­ ¿ ¼»½®»¿­» º®±³ ¾¿­»´·²» ·² ¬¸» ²«³ó
`ber ofcells positive for CD3, CD4, and CD8 following
`¾»® ±º ½»´´­ °±­·¬·ª» º±® ÝÜíô ÝÜìô ¿²¼ ÝÜè º±´´±©·²¹
`
`treatmentwith either concentration of CsA. The only ex-
`¬®»¿¬³»²¬ ©·¬¸ »·¬¸»® ½±²½»²¬®¿¬·±² ±º Ý­ßò ̸» ±²´§ »¨ó
`ception was that there was a mean increase from base-
`½»°¬·±² ©¿­ ¬¸¿¬ ¬¸»®» ©¿­ ¿ ³»¿² ·²½®»¿­» º®±³ ¾¿­»ó
`line in the CD4-positive T helper cell population follow-
`´·²» ·² ¬¸» ÝÜìó°±­·¬·ª» Ì ¸»´°»® ½»´´ °±°«´¿¬·±² º±´´±©ó
`ing 0.05% CsA treatment. In comparison,all cells positive
`·²¹ ðòðëû Ý­ß ¬®»¿¬³»²¬ò ײ ½±³°¿®·­±²ô ¿´´ ½»´´­ °±­·¬·ª»
`for the lymphocytic markers increased from baselinefol-
`º±® ¬¸» ´§³°¸±½§¬·½ ³¿®µ»®­ ·²½®»¿­»¼ º®±³ ¾¿­»´·²» º±´ó
`lowing vehicle treatment.
`´±©·²¹ ª»¸·½´» ¬®»¿¬³»²¬ò
`Figure 1 showsthe percent change from baseline
`Ú·¹«®» ï ­¸±©­ ¬¸» °»®½»²¬ ½¸¿²¹» º®±³ ¾¿­»´·²»
`for cells expressing the lymphocytic markers (CD3, CD4,
`º±® ½»´´­ »¨°®»­­·²¹ ¬¸» ´§³°¸±½§¬·½ ³¿®µ»®­ øÝÜíô ÝÜìô
`and CD8)after 6 months of treatmentfor the overall pa-
`¿²¼ ÝÜè÷ ¿º¬»® ê ³±²¬¸­ ±º ¬®»¿¬³»²¬ º±® ¬¸» ±ª»®¿´´ °¿ó
`tient population. Note that there was a reduction from
`¬·»²¬ °±°«´¿¬·±²ò Ò±¬» ¬¸¿¬ ¬¸»®» ©¿­ ¿ ®»¼«½¬·±² º®±³
`baseline in the number of CD3-positive cells in the CsA-
`¾¿­»´·²» ·² ¬¸» ²«³¾»® ±º ÝÜíó°±­·¬·ª» ½»´´­ ·² ¬¸» Ý­ßó
`treated groups, while there was an increase from base-
`¬®»¿¬»¼ ¹®±«°­ô ©¸·´» ¬¸»®» ©¿­ ¿² ·²½®»¿­» º®±³ ¾¿­»ó
`line in the vehicle-treated group. There wasalso an in-
`´·²» ·² ¬¸» ª»¸·½´»ó¬®»¿¬»¼ ¹®±«°ò ̸»®» ©¿­ ¿´­± ¿² ·²ó
`crease from baseline in the numbers of CD4-positivecells
`½®»¿­» º®±³ ¾¿­»´·²» ·² ¬¸» ²«³¾»®­ ±º ÝÜìó°±­·¬·ª» ½»´´­
`in the vehicle group, with a smallerincrease in the 0.05%
`·² ¬¸» ª»¸·½´» ¹®±«°ô ©·¬¸ ¿ ­³¿´´»® ·²½®»¿­» ·² ¬¸» ðòðëû
`CsA group and a slight decrease in the 0.1% CsA group.
`Ý­ß ¹®±«° ¿²¼ ¿ ­´·¹¸¬ ¼»½®»¿­» ·² ¬¸» ðòïû Ý­ß ¹®