CL LNfCAL rMMlJNOLOC Y AND LMMUNOPATIlOLOCY

Vo1 87 No 3 J une rIP 276 - 2HI 1998 Aruclo No 11984536

Eye Muscle Antibodies in Patients with Ocular Myasthenia Gravis Possible Mechanism for Eye Muscle Inflammation in Acetylcholine-Receptor Antibody-Negative Patients

K Gunji C_ Skolnick T Bednarczuk S Benest B A C Ackrell B Cochran t J S Kennerdellmiddot and J R Wall middot1

Thyroid Elf Dj~ R~reoh LaboroWry 3290 Wm Piu Way U- PARC Bldg 84 Room J25 PilUburgh Pt fl~haflUl 15238 t Olpltmtnl ( Ophthalmology Ohio Stale Ufli ll~n ity Colum bu$ Ohio and tVltmn A(OiN Mrdictll CeM OM

Dtpotme flt of 8 iochtmlStry afld BiophY$its U il)frity oCoUfom i4 SO fl Fro I IlCO Colian Ia

bodles reactive with pwificd calsequestrin (63 kDa) Mynstbenia ~avis is an organ-specific autoimmune were deteeted in 2 1 of patieocs with OMG but in no

disorder ienerally tbougbt to be calUed by an anti shy patient with GMG Antibodies re-cogniring purifted body-mediated attack against tbe skeletal muscle Qicoshy succinate dehydrogenase (67 kOal were found in 42 tinic acetylcboline (Acb) receptor (AchR) at tbe neuroshy o(patients with OMG in 100 (5 o(5 ) ofparients with muscular junction ExtraocuJar muscle weakness aud GMG and in 48 ofalJ patients with myasthenia gravis double vision are pre8ent in about OOClt of patients with n ot associated with Graves hyperthYroidism There myasthenia gravis and are the predominant comshy was D O close correla t ion between any e~e muscle-reacshyplaints in about 20 of patients when the condition is tive antibody and 8ntibodie9 agaiost tbe AchR in eishycal led ocular myasthenia gravis (OMG) While serum ther group or myasthenic patients The findiogs supshyantibodie9 again8t the AchR are detected in mOit pashy port the notion that immunoreactivity agoiost skeletal tients with generaJiozed myasthenia gravis (GMG) they Qusele proteins other than the AcbR may playa role are not round in about one-third of patients with the in tbe development of the mWlcle wtakness in AchR oeular variety and epidemiolotjfical clinical and seremiddot antibody-negative patienls witb OIG and G~IG alshylogcaJ studies s uggest that OMG and GMG are two tbough it is uoJikely that any oftbe antibodies demonshyseparate diseases Both fo rms efmyasthenia gravis are strated in this study are directly impUcattd Similarly sometimes associated with tbyroid autoimmunity or while the demonstration of antibodits reacrive with thyroid-associated ophthalmopathy (TAO) We have eye muscle antigen9 associated with TAO in patient9 therefore tested tbe sera of patientlt with GMG and with OMG raise the possibility that tbt link between OMG by Western blotting for antibodies agawt pormiddot the oculax lesions of myasthenia p-a-is and Graves cine eye m uscle membrane proteins in general acd by disease may be autoimmuoity Ggairut a common antimiddot enzyme-liuked immunOliorbent assays (ELISA) lipeshy en(s) it is more like ly that both disorders are medimiddot cifically fOJ reaction with two skeletal muscle antia-eus ated by cytotoxic T cells recognizing anotber cell memshywbich are prominent marker antigens for TAO brane untiien s uc h as tbe novel th)TOid and eye mU9shynamely the calcium-binding protein calsequestrin cle shared protein G2s and that iCnun antibodies and be so-called 64middotkDe protein The 64-kDa protein reactive witb s u ccinate dehydrogenase fp subunit and has recently been identified as the fievoprotein submiddot calseqveatrin are markers of 81l immunemediated eye unit of mitochondrial succinate debydrogenase_ P ashy musele reaction 0 __ ACa ~ ~

tients with ophthalmopathy and myasthenia were exshy Key Words myasthenia gravU ocuJar myastbenia cluded Nine or tbe patients bad associated Graves by gravis eye muscle s ke letal muscle immunoblottinii perthyroidiam without evide nt ophthalmopatby and thyroid-ossoc iated ophthalmopathy S-I kDa protein one had H ashirootos thyroiditis_ Antibodjes against suceinte dehydrogenase calsequesrrio porcine eye muscle membrane antigens of M Hi-110 kDa were detec t ed in patients with GJG or OMG one or more antibodies be ing detected in looao of patient9 with GMG and in 88 of those with OMG The most

INTROD UCTlO-frequently found antibodies were those target ing e ye muscle membrane proteins of 15 67 and 110 kDa Anti middot

Myasthenia gravis is an orgsn peciric aUhLmmu ne

disorde r ge ne ra lly th ought t o be ca u3ed b~ Rn 1n1ibody shymedia ted a ttack agai nst t he skeleal muscle nicot inic T whom ~ rrt po nd middotllcmiddot all ri -Cp-II 1lmiddotJt~ ~houd be Idshy

d middot1 E-mai wuIl9pl h bull lUhstJ acetylcholine (Ach ) receptor (AchR middot at the nlU fo mUSCU-

I_~ ~ _ I ~ bullbull ~ 1 m WJI h Gdmiddot ~ middotromiddot _ u 11 10 bull 10 bull bulllt1

277

e

OCULAR MYASTHENIA GRAVIS

lar junction (1-3 ) Loss of functi onal receptors wi th lmpainnent of the neuromuscular signal transmission Iesults in weakness and fatigability in selected skeleta l muscles (n There is also evidence of skeleta l muscle inRammation (4) and production of serum antibodies reactive against a variety of muscle antigens (5 6) Extraocular muscle weakness and double vision are present in a bout 90 of patients with myasthenia grashyis aod are the initial compiai n13 in about 20 ofcases This latter condition is called ocuJar myasthenia gravis ~OMG) Epidemiologic clinica] and serologic studies have supported the notion that OMG and generalized myasthenia gravis (GMG) are different diseases (7 - 9) That serum antibodies agai nst the AchR are detected in 90 ofpatients with GMG but in only 65 with the oc ular variety (10 11) suggests that othe r antibodies may playa role in the development of eye muscle weakshyness in OMG

Myasthenia gravis is li nked with autoimmune thyshyroid disease in that approximately 5 of the patients also have Graves hyperthyroidism or H ashimotos thyshyroiditis (12 13) Thyroid-associated ophthalmopathy (TAO) 1S an au toimmune disorder of the extraocular (eye) muscle and orbital connective ti ssue closely assoshyclated with Graves hyperthyroidism (reviewed in 14) OMG and TAO share some clinical features and may occur in the same patient rn a recent study Manno et al ( l S) s howed tha t when myasthenia gravis was associated with thyroid autoimmunity it tended to have a milder clinical expression with preferential ocushylar involvement and lower frequencies of thymic disshyease and AchR antibodies This association was particshyu la rly strong in patients with ophthalmopa thy a nd thyshyroid autoimmunity (15) We ha-e thus studied patients with GMG aod OMG for serum autoantibodies reactive with eye muscle antigens including two recently idenshytified as being associated with TAO namely the Fp s ubunit of mitochondrial s uccinate dehydrogenase (the 64 kDa protein) (16) and calsequestrin a 63 -kDa calshycium-binding protein localized in the sarcoplasmic reshyt iculum of the skeletal muscle fiber (17) We demonshystrated antibodies agains t one or more eye muscle proshyteins of M IS- 110 kDa in 100laquo of patients vith OMG and 88 of patients with GMG and against succinate dehydrogenase Fp in 42 of patients with OMG and 100 with GMG

CUNICAL SUBJECTS ~lJ METHODS

Clinical Subjects

The studies concerned patients with the following

(Il Generalized m_vasthcl1io grOlIS (GMG J Five males and two females of whom twO had associ a ted Craves hyperLhyro idism were rudied The diagnosis was made from the typi ca l hlsOlY and cl inical examishy

nation including oph thalmologic assessment and canmiddot firmed by t he tensilon test Only one patient had detectmiddot able serum antibodies against the AchR

(ii) Ocular myasthenia gravis (OMG) Sixteen m ales a nd 17 females of whom 7 had ass ociated Graves hyperth)Toidism and 1 had Hashimotos thyshyroiditis we re a lso studied The diagnosis was made from the typicaJ history and clinical examinalion inshycluding ophth almologic assessment and confirmed by the tensilon test Eighteen of the patients had detectshyable serum antibodies against the AchR

Patients with ophtha lmopathy and myasthenia were excluded from the study

(iiij Normal subjects Tweoty-one mal es and 33 feshymales aged-matched with myasthenic patients with no personal or family history of myasthenia gravis thyshyroid disease ophthalmopa th~ or other autoimmune disease were r ecruited from ancillary hospital and labshyoratory s taff

The study was IRE approved and informed written consent was obtained from aU patients and normal subshyjects stud ied

SDS - PAGE and Western Blornng

Antibodies reactive with porcine eye muscle memshybrane proteins and purified beef beart succinate dehyshydrogenase Fp subunit were dececced following standard Laemmli SDS-PAGE (18) using an 85 separating gel and a 4 stacking gel in a minigel apparatus as reported previously (19 20 Primary antibodies were patie nts sera dilu ted 110 and a rabbit antimiddotflavoprotein subunit antiserum diluted L2000 and secondary antishybody was an alkaline phosphatase-conjugated anti-hushyman IgG (y chain specific antisennn diluted 112000 for patients sera or anti-rabbit rgG (whole molecule) antiserum diluted mooo for the anti-Fp antiserum Tests were read by two obseTers and results were exshypressed as band density A band density of + or greater was taken as a positive test

Isolation of Purified Beef Heart Huscle Succinate Dehydrogenase

Succinate dehydrogenase conraini ng the Fp and Ip (iron - su lfur protein) subunits was solubili zed by pershychlorate treatment (2 1) of suc(inatecoenzyme Q modoshyreducta se (complex H of the respiratory chain) which had been isolated from beef hegtan mitochondria by the method ofBaginsky and Hal eR 22 J The enzyme prepashyratlon was gt90Ck pure baed on gel analysis and conshytent of cova le ntl y bound fta n adenine din ucleotide Pure enzyme was excised from SDS-polyacry lamide gel s according to th e meth od of I lerii e( 0 (23 ) and used DS a ntigen in We5ttrn blotting a nd EUSA

278 GllNJ] ET AL

Isolation of Purified Porcine Eye Muscle Calsequestnn

Pig eye muscle was homoge nized in a prechilled blender and cent rifuged at 13OOOg for 30 min Ammoshynium sulfate was added to the supernatant to a final concentration of 92 and the pH was adj usted to 47 -with phosphoric acid The precipitate from this step was collected by centrifugation at 13 OOOg fo r 30 min dissolved in Tris-phosphate buffer and dialyzed against the same buffer The sample was then applied to a DEAE-SephaceJ COIUTTUl and preequilibrated with buffer A (01 M potassium phosphate pH 71 1 mM EGTA 50 mM NaCD Buffer B CO1 M potassium phosshyphate pH 71 1 roM EGTA 700 mM NaCI) was then applied and 3-ml fractions were collected moni toring absorbance at 280 nM Two-hundred-microliter alishyquots were taken from every third fraction and subshyjected to Western blot analysis to check reactivity with an anti-calseQuestrin monoclonal antibody in order to identify those tubes containing calsequestrin Purified calsequestrin was used as antigen in EUSA

Enzyme Lmked Immunosorbent Assay

The method has been described n pre ous publicashytions from this laboratory (24 25) The oprimal antigen concentration was found to be 1 ugml for both calsequestrin and succinate dehydrogeoase and the opshytimal serum dilution was 1125 The setood antibody is an alkaline phosphatase-labeled goat anti-human IgG (U1500 dilution) Results are expressed as optical denshysity (00) at 410 nm and positi ve reactivi ty against test antigen is taken as mean + 2 SD for DOrmal subjects tested concurrently

S tatistica l Analysts

Differences in mean (=SE) values between patient groups and normals in EUSA was assessed statistishycally using the Student t test Differences in prevashylences of serum autoantibodies reactive with eye musshycle membrane antigens in imruunoblottiog and with Fp and calsequestrin in ELISA be tween patient and control groups were assessed using X2 tests

RESULTS

We tested sera from 7 patients with GMG 33 pashytients with OMG and 54 normal subjects as controls for antibodies against porcine eye muscle membrane antigens utilizing 80S - PAGE and Western blotting and agai nst succinate dehydrogenase fp and caJseshyquestri n in ELISA Patients with OMG Wfre furt her classified as a nti-AchR antibody positi middote 18 pa tients grou p n or anti-AchR ontibody negatiye 15 pa tie nts grou p 10 Seven patien ts with OMG had asocia ted

Graves hyperthyroid ism but no ophthalmopathy Pashyti ents with GM G of whom only one Wa3 anti-AchR a nhbody pos itive and two had assooJUd Graves hy_ perthyroidism were studied as a sirgle group (7 pashytients group III ) The resu lts are su-nmanzed in Fig 1 and Table 1 One or more serum an tibodi es against porcine eye muscle membrane proteins of M 15-110 illa were detected in 100 of palient3 with G1G and in 88 with OMG (Table 1) Next w~ tested for serum anti bodies against purified Fp and calsequestrin in ELISAs Anti-Fp a ntibody levels in the hree groups of patients and the normals are depicteC in Fig 1 Only 29 of the sera we re available for Fp antibody testing Results are expressed as 00 at 4 10 om vIeao (=SE) val ues were 0327 (=0068) fo r patients of group l which was significantly greater than ihar for normals (0 146 0016 t test P lt 005) 0250 1= 0062) for group II (P NS) and 0584 (0129 for pa tients of c=

group III (P lt ooon Taking an 00 of 0285 (mean + 2 SD for normals) as the upper limit of normal tests were positive in 6 of 12 (50) patient r fgroup I tested 4 of 12 (33) of gTOup II and 5 of 5 lOOCpound ) patients of group Ill but in none of 18 normal subject Taking an 00 of 049 (mean + 2 SO for nOrIDaG a5 the upper Umit of normal antibodies against caequestrin were de tected in 6 of 18 patients of group I 33t(1 1 of 15 patients of group II (7) in no pac-cJ[ of group TIl (0) and in 2 of 54 (4) no rmal submiddotea results not shown ) Mean (SE) va lues fo r parmiddotlt~t group were significantly different compared to nc-Jai only fo r pashytients ofgroup I (P lt 005) OveraU axtbodies against calsequestrin we re de monstrated in 2ltf and against succinate dehydrogenase Fp in 42lk of ill patient3 with OMG and a ntibodies against succinak dehydrogenase Fp subuni t were demonstra ted in ~~~ of all patients wi th OMG or GMG not associa ted ( Graesmiddot hypershythyroidism There was no significan correlation beshytween any eye muscle antibody and a a51 or preseut Graves hyperthyroidism or (b) antiboWes against the AchR in either group of myasthenic patieots

DISCUSSION

We studied patients with GMG anaO~IG for serum au toantibodies reactive with porcine -~ muscle memshybrane antigens including two antigel closely associ ated with TAO namely the succinau d~hydrogeoase Fp subunit which is incorrectly referred to 111 the litershyature as the 64-kDa protein and cals-fUeHnn a 63 shykDa calcium-binding proteio To sumoanze the main results antibodies reactive with na t ca~equestrin were detected in 21 of patients Ili t -_OgtIG bur in nO patient with GMG whil e antibodie - ~Jl zmg succishynate dehydrogenase Fp the 64-kDa p =- ~ were demshyons trated in 42 of patients with 0gt10 and lOOGf of patients with GM G Because pa ti ent middot- h rhYToid aushy

278 GllNJ] ET AL

Isolation of Purified Porcine Eye Muscle Calsequestnn

Pig eye muscle was homoge nized in a prechilled blender and cent rifuged at 13OOOg for 30 min Ammoshynium sulfate was added to the supernatant to a final concentration of 92 and the pH was adj usted to 47 -with phosphoric acid The precipitate from this step was collected by centrifugation at 13 OOOg fo r 30 min dissolved in Tris-phosphate buffer and dialyzed against the same buffer The sample was then applied to a DEAE-SephaceJ COIUTTUl and preequilibrated with buffer A (01 M potassium phosphate pH 71 1 mM EGTA 50 mM NaCD Buffer B CO1 M potassium phosshyphate pH 71 1 roM EGTA 700 mM NaCI) was then applied and 3-ml fractions were collected moni toring absorbance at 280 nM Two-hundred-microliter alishyquots were taken from every third fraction and subshyjected to Western blot analysis to check reactivity with an anti-calseQuestrin monoclonal antibody in order to identify those tubes containing calsequestrin Purified calsequestrin was used as antigen in EUSA

Enzyme Lmked Immunosorbent Assay

The method has been described n pre ous publicashytions from this laboratory (24 25) The oprimal antigen concentration was found to be 1 ugml for both calsequestrin and succinate dehydrogeoase and the opshytimal serum dilution was 1125 The setood antibody is an alkaline phosphatase-labeled goat anti-human IgG (U1500 dilution) Results are expressed as optical denshysity (00) at 410 nm and positi ve reactivi ty against test antigen is taken as mean + 2 SD for DOrmal subjects tested concurrently

S tatistica l Analysts

Differences in mean (=SE) values between patient groups and normals in EUSA was assessed statistishycally using the Student t test Differences in prevashylences of serum autoantibodies reactive with eye musshycle membrane antigens in imruunoblottiog and with Fp and calsequestrin in ELISA be tween patient and control groups were assessed using X2 tests

RESULTS

We tested sera from 7 patients with GMG 33 pashytients with OMG and 54 normal subjects as controls for antibodies against porcine eye muscle membrane antigens utilizing 80S - PAGE and Western blotting and agai nst succinate dehydrogenase fp and caJseshyquestri n in ELISA Patients with OMG Wfre furt her classified as a nti-AchR antibody positi middote 18 pa tients grou p n or anti-AchR ontibody negatiye 15 pa tie nts grou p 10 Seven patien ts with OMG had asocia ted

Graves hyperthyroid ism but no ophthalmopathy Pashyti ents with GM G of whom only one Wa3 anti-AchR a nhbody pos itive and two had assooJUd Graves hy_ perthyroidism were studied as a sirgle group (7 pashytients group III ) The resu lts are su-nmanzed in Fig 1 and Table 1 One or more serum an tibodi es against porcine eye muscle membrane proteins of M 15-110 illa were detected in 100 of palient3 with G1G and in 88 with OMG (Table 1) Next w~ tested for serum anti bodies against purified Fp and calsequestrin in ELISAs Anti-Fp a ntibody levels in the hree groups of patients and the normals are depicteC in Fig 1 Only 29 of the sera we re available for Fp antibody testing Results are expressed as 00 at 4 10 om vIeao (=SE) val ues were 0327 (=0068) fo r patients of group l which was significantly greater than ihar for normals (0 146 0016 t test P lt 005) 0250 1= 0062) for group II (P NS) and 0584 (0129 for pa tients of c=

group III (P lt ooon Taking an 00 of 0285 (mean + 2 SD for normals) as the upper limit of normal tests were positive in 6 of 12 (50) patient r fgroup I tested 4 of 12 (33) of gTOup II and 5 of 5 lOOCpound ) patients of group Ill but in none of 18 normal subject Taking an 00 of 049 (mean + 2 SO for nOrIDaG a5 the upper Umit of normal antibodies against caequestrin were de tected in 6 of 18 patients of group I 33t(1 1 of 15 patients of group II (7) in no pac-cJ[ of group TIl (0) and in 2 of 54 (4) no rmal submiddotea results not shown ) Mean (SE) va lues fo r parmiddotlt~t group were significantly different compared to nc-Jai only fo r pashytients ofgroup I (P lt 005) OveraU axtbodies against calsequestrin we re de monstrated in 2ltf and against succinate dehydrogenase Fp in 42lk of ill patient3 with OMG and a ntibodies against succinak dehydrogenase Fp subuni t were demonstra ted in ~~~ of all patients wi th OMG or GMG not associa ted ( Graesmiddot hypershythyroidism There was no significan correlation beshytween any eye muscle antibody and a a51 or preseut Graves hyperthyroidism or (b) antiboWes against the AchR in either group of myasthenic patieots

DISCUSSION

We studied patients with GMG anaO~IG for serum au toantibodies reactive with porcine -~ muscle memshybrane antigens including two antigel closely associ ated with TAO namely the succinau d~hydrogeoase Fp subunit which is incorrectly referred to 111 the litershyature as the 64-kDa protein and cals-fUeHnn a 63 shykDa calcium-binding proteio To sumoanze the main results antibodies reactive with na t ca~equestrin were detected in 21 of patients Ili t -_OgtIG bur in nO patient with GMG whil e antibodie - ~Jl zmg succishynate dehydrogenase Fp the 64-kDa p =- ~ were demshyons trated in 42 of patients with 0gt10 and lOOGf of patients with GM G Because pa ti ent middot- h rhYToid aushy

bull bull bull bull bull bull

279

0900 I

1 0 800

0700

I 06

lt C)s oo bullbull 0 0_00

0

0300

0200

0100

0000

J-middot _ bullbull

bull ~ bull

Normals GROUP

fiG 1 Serwr clbodlea agail)st pwil1ld flayoprolein lIubWliI of $uccina~ deh~droenllae in palienU with rnYlUlthenia rrai and oonnallubjen ~ ci~=ld usmg an emymeJ~d llnOlwwsorbenl aAS) (E LlStu Rtauts a re expressed as opLl tlli dn~ty tOOl at ltIll om The broken bO-1olta ) hilI at 0285 run is t he upper limit of nannal defined a mean - 2 SO for nonnaJ 6ubj-eu Mall (SE) Yaluei art mdicaltG for ea lfl)Up Group I patients with ocular myasthenia gnVlO fOIG and detelaquoable serum 8lItibociu ~ampI in ~t the aCfwlclu lioe receplor ~~bR poup It patienLa wHh OMG negative for atlli middotAchR aDlibodit group lu pauents with general ~-d myaethenia irvi5

toimmuniy and Oert ophthalmopathy were excluded though antibodies reactive with the Ach R play an imshyfrom the s lUdy 1I ieems highly likely that the antlbodshy portant role (1 While myasthenia gravilgt is usually a ies were associlled with myastherua r ather than TAO generalized fuese the skeletal mude autoimmun e

The pathogenesis of myasthenia gravis is unclear a1- reaction and resulting weakness and fatiga bili ty m ay

TABLE 1

PreaeoCE-s of Antibodies agampinst Porcine Eye Muscle Membrant Antigens in Patients with Ceneralized or Ocular Myasthenia Gravi s by lmlDunoblotting

Antigen

GIttIlP 30 kDa 45 kDa 67 kDlI 11 0 ill15 illmiddot AcbR_Abi pos O~IG group II In 18) 9 I5Oir 1 IS 8 (44 A) 713941 5 (28l

P lt 006 NS NS P lt OCJf NS AcoR-Ab ata OMG group If) ( Il 15) 7 (47) 9 (6(Yb 4 ( 27~ 717 8 fS3~1

P lt 005 P lt 005 NS P lt O(J~ NS GMGliroup III J j 3 (431 23~ 2 (29lraquo 6 (801 (71 middot

NS NS NS P lt 0 0) 1 P 005 Nonnala In 38 4 (lOij )0 C26i 12132) 4 ( (IA II 129

bull of of W~fI IDlilen ~ AcnRb Icelyk toJiIle recepOr antibodies OMG ocular ooyUt hem a gravia ltI n ll) rlR(1jve t r the an ligen in 5DS- PACE of porcine eje muscle membampIllt=t and Weter n blot ting with patintE rum SLauitlcal analrrE-l refer to dilTerence eampared vith nDrfflP1 determined ULci t i1~ te~ t NS not signincliOl I GMG generaJ~ cI_~ampJtheni8 gravis

280

be limited 10 hI txtmiddotmiddotmiddot_ 6r muscles Illdeed t hen i (vidence th H GIG - u IG are separate dIsorders with diffelln t path0fi---( mecha ni sms 112 13 1 Sev eral auLo8nllbodle~ ep-ii non-AchR skel etal muscle componen5 hayeuro hEe- ~cri bed in myasthenia gTavis mcluding tho~e reactmiddot Ith the ryan idine receptor Ithe calcium Ca ~- 1 c-Llel j (3) and various muscle proteins identified by i]unoblotting (4) hemaggluti middot nation (5) ELISA 4 -c immunoRuorescence (4 5) Since AchR amibode~ cf not detected in a ll patients with myasthenia gras there may be a subgroup esmiddot pecially among those wi-l the ocular variety in whom another antibod)11e iUch as those detected in the present study or more hkely antibodies not yet identimiddot fied may be implicated in rhe development of the mus shyde weakness

Both myasthenic sndromes occur in association with auroimmune tb~TOid disease and TAO Indeed OMG and TAO share some clinical features and may occur in the same patient Wbile T lymphocyte reactivmiddot ity against eye muscle and orhital connective tissue antigens is likely to be roe primary event in the develmiddot opment of ophtbalmopath~ the rol e of circulating a utomiddot antibodies reacnve with orbital antigens has been more extensively swdied Ire~E-ed in 26) Of these a protein with a reponed MT of amp illa is recognized as an immiddot portant auwantigen in T-O the corresponding serum autoantibodies being good markers of the eye disorder (19 20 27 281 and peurodicrors of its development in patients with thToid auroimmuruty especially Graves hyperthyroidism (2930middot The 64-kDa protein has reo cently been partially sequenced and identified as the Fp suburut of mitochoncal succinate dehydrogenase (16) The recalculated Jf of the 64-kDa protein is 67 kDa In a recent study antibodies against purified suemiddot cinate dehydrogenase ere detected in 67 of patients with active TAO in 30r vrith stable TAO and in 30 of patients with Grae~ hyperthyroidism without ophshythalmopathy but in only ik of age- and sexmatched nonnal subjects by immunoblotting ( 16) Another proshytein associated with TAO is calsequestrin a 63-kDa calcium-binding protein which is localized in the sarcoshyplasmic reticulum of the muscle fiber (17) Calsequesmiddot trin is known to share an epiwpe ti th heat shock promiddot tein 60 (3U Antibodie against calsequestrin were found in 401k ofpatieD Oth active TAO but in only 4 of those with stable burnt out eye disease and in 5 of nonnal subj~ by immunoblotting (G unji et al submitted ) In conciuoion while antibodies agai nst eye muscle antigens associated with TAO are found in the majori ty or patieot with myasthenia gravis it is like ly that the musel damage of myasthenia is medi aled by CDS- (cytOtoxic T lymphocytes targeti ng an other cell membrane antigen s uch as the recently doned thyroid and eye muscle shared protein G2s (32) and that the antibodies measured in this study are

SlCOlldlr ngtIlkr of immUlIc-Ol diatcd 1 ll1 u~dt damage in both di~ord(rs

AC~OWLEOG1I1poundNTS

This wMk wa~ ~uppon(d b~ a gran from Aleghen~- Smgtr Rmiddotmiddot ~c(trch 1lmiddot~IIl( amplC - ~lMI H~I~U1C~ of Hcnlth GrlIll HL1625J IB - C I Wt thank AGH Centrsl Ammal fac tlit) for pomiddot yiding porClnt t~middot t muscle

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19S 74-75 1977

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rem concepts regarding pathogenesis and manaCemenl Edocr ReI) 14 747- 7931993

15 Manno M Ricciardi Pinchera A Barbes mo G Manettl L Brayernan L Rossi B MuralOrio A and Manotlt S Mild clInical upression of m~middotllSIhes iTSVamp associated with lIuloim shymllne t hyroid dJSeases J Ct Elldocrinol dob_ in p~

16 Kubota S Gun K Acknll B A C Cochran B Swlarlt- C Wengf()cz S tiJ rOlTlaUU Y Kenne rdell J S fi nd Wlltl J R The 64 kDa eye muscle protein i8 the Ravoproteln subunit of mltOChondrial 6Uerinate dehydrocemse TIll corrMpondmj IOr uCl a ntibodiet U( good markers of a n immunemedIIgtd

I

(JeULtH MYASTHENI A GH I ~8 1

camaj~ 10 th t ~ - ~ de in ~IH nts wih Gnle~ hplrl hyrltlid l~m J (ln E - lfNnb 83 44 3_447 If1S

1 ~ Sill- (111 K [kluche r A Slol ar~k l C Kuhouta S a nd all J ~L ltCular cJnnlllg lind Cha raclCrlZallOn of I 64 kDa e l t nlU~cl ~lIl lCn II I~ociafpd wIth thvNlld-assOCl1l tfd oph th llinlopnh ~ltW e ed i nlls S Ixty-Ninth Annunl Mel lll l oflh Amtricar r~7d 5soclation San Diego 1996 IAbstract I

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25 Kapuna M Sal ~ ~L InJler H Gardmi E Bernard N and

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2 KilJmski J --t~ Ilnd WIIJ R T ht Kula r mu~d l- Ihe lall(( of lh~ immuntgt $Is em III en dOCrine IJphth fllmulgt~th middot pro ira Arc) Ali [m lllullul 106 204 -21 2 1995

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