`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 Í³·¬¸÷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
`(REPRINTED) ARCH OPHTHALMOL/VOL 118, NOV 2000
`øÎÛÐÎ×ÒÌÛÜ÷ ßÎÝØ ÑÐØÌØßÔÓÑÔ ñ ÊÑÔ ïïèô ÒÑÊ îððð
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`1489
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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
`øÎÛÐÎ×ÒÌÛÜ÷ ßÎÝØ ÑÐØÌØßÔÓÑÔ ñ ÊÑÔ ïïèô ÒÑÊ îððð
`1490
`ïìçð
`
`©2000 American Medical Association. All rights reserved.
`îððð 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®