Oct. 3OlConcurrent Session 12 - Bullous Diseases zyxwvutsr 38 163 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 166 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA THE PRECURSOR FREQUENCY OF PERlPHERAL BLOOD LYMPHOCYTES (PBLI DIFFERENTIAL REGULATION OF 180KO AND 230KD BULLOUS PEMPHIGOIO ANTIGENS IN THE FORMATION AND DISINTEGRATION OF HEMIOESMOSOMES. Yasu" Klta.ima K&sushi Owarlbe'. Yoshlaki Hirak"'. M.K"jl Owada' an 0Ii&i+& Dept of Uermatol. Jtchl Med School, "chlgl. Japan. 'Uept of M I B 1 School of Sci. Nagoya Univ. Nagoya. Japan. 'Inst of Mol & Cel?ul i?"i for Pharmaceut Sci. Kyoto Pharmaceut Univ. Kvoto. Ja"an To clarify the mechanisms of hemidesmosome form&ion. we studied the rearrangement of the lBOk0 and 230kO bullous pemphigold antigensIBPAs1 by low(O.O7mMj-high11.87mMl Ca*' shift and 12-tetradecanoylphorbol-13~ acetate(TPAi treatment. and their TPA-induced phosphorylation I" cultsed keratlnocytes(OJM-II by immunolag~cal methods using m"noclonal antibodies to these BPAs. The IliOkD BPA was distributed on the whole cell surface in paraformaldehyde-fined cells. Pretreatment vlth 0.5% Trlton X-IOOlTrXI erased the antigen from the non-ventral membrane. but retal"ed that an the ventral membrane. The 230kO BPA was distributed 1" the cytoplasm and on the ventral surface without TrX treatment. and only on the ventral membrane after TrX treatment. Since TrX does not solublize the proteins bound to keratln Intermediate filaments(KIFsJ. but does those free from KIFs. it is suggested that the 180kO BPA on the non-ventral surface IS drifting in the cell membrane. the 230k0 BPA is pooled in the cytoplasm. and both BPAs on the ventral membrane are bound to KIFs. In low Ca" cells. both BPAs on the ventral cell membrane were distributed evenlr as fine dots. whereas they formed a concentric ring "I- arch pattern in high Ca*+cells as studied by inmunofluorescence. TPA (16nYl. which activated protein hinase C. disrupted translently(l-21 the ring pattern. TPA treatmentll5mln) caused a profound increase I" phosphorylatian Iserlnel and an lncreaze Ilk01 in molecular weight of the 18OkO BPA on Immunoprecipitation and SOS-PAGE. Phospharylati"" of the 230kO BPA was not detected. These data swgest that these two BPAs are differentially controlled t" form hemidesmosomes on the ventral membrane. 1 164 FROM PATIENTS WlTH B”LLt,“S PEMPHlGOlD (BP) IS INCREASED AGAINST AMPHIPATHMIC SYNTHETIC PEPf1DE.S FROM BPAGl MJ Ric”. RD Slrsilei”. RP m. Duke Univsnity and the Durham VA Medical Ccntlr from Patients with BP contains autoantibudw rpcfific for mult,plc epitopcs on two diSfi”C, b&wl”ent membrane zone proteins. and elevated IL-2 recOpt”rE. nese ,,alatasuggest that the autoantihody response in tbrsc patirntr is antngen drive”, and suppon, a potential role for T cells in the initiation and regulation of autoantihody production. The purpose of Ihe present EtUdy was to determine If PBL from patients with BP and c”ntrOls display increared precursor fryuencier against amphrphUhic md hydmphilrc synthetic peptides encoded by BPAG,, PBL were ,ro,ated from patients and wntmls and CL>cultured for 7 days in the presence of 18.22 amino acnd long synthetic hydmpbdic (6 peptides) or ampbipathic (8) peptides. 3H-thymidine “puke was dctcrmind and the precvrrar frequency for each peptide dewmined by li”l”i”g. dilution analysis bared 0” the Poisson disrributio”. 7/1O patient* with BP and 3/tCl controls demonstrated a” increased precursor frequency peptides. lncreascd precursor frequwlc,es in the BP patients were noted against synthetic p+iJCS wdxn or adjacent 1” the 5’ region of the coiled~coil regmn: 6/lO BP patie”& and ?/lo co”troI~ had increaed precursor frequencies to pq”l&D at rhe 5’ and of the CLXIcd coil region. a*,acenr to a region in whxh we have previously mapped autoanlihody binding actwity. This rcgw” also includes a region with syucncc homology t” the human hat shock prmci”, hsp60. These data demonstrate a” incrcasilsedPrecursor fryucncy in the PBL of pahc”,, with BP a”* in some ““rm~l ~““trols agamst amphipathic psptidcs frum LtPAGl and supports the role ofT crllb in the pathogen& of BP. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE Durham. NC. sera c>1:3co,oa), against atkartone amphlpathic peptKt< Minimal reactivity was noted inpatients orcontmls agamt hydrophilic 167 DEVELOPMENT OF AN ELISA THE SEP.A OF BULLOUS LA. Diaz. Medical K. Wilske. TO DETECT PEMPHIGOID A. Tavlor. ANTI-BP180 AND HERPES AUTOANTIBODIES GESTATIONIS i D.J. Em- IN PATIENTS. COMPARATIVE SERA REACTIVITY PEMPHIGOID (BP) FROM JAPAN FUSION PROTEINS SYNTHESIZED Dept. of Dermatology, College of Wisconsin and VAMC. Milwaukee, WI, USA One of the primary antigenic targets of autoantibodiesproduced by bullous BPAGI. Medicine OF PATIENTS WITH BULLOUS AND THE UNITED STATES FROM THE COILED-COIL RP Hall. T Hashimoto. K Watanabe. MI Rico.T (Dermatology) and Dermatology, Duke University (“S) AGAINST REGION OF Nlshikawa, Departments of Medical Center, Durham. pemphigoid (BP) and herpes gestationis (HG) patients is a 180 kD hemidesmosomal protein, BP180. Our labaatoty has recently cloned the BP180 antigen and has identified B NC, and Keio University School of Medicine, Tokyo, Japan. Recent collaborations have demonstrated that sera from Japanese, British dominant epitope recognized by BP and HG autoantibodies. patients with BP share similar antigen profiles for reactiwty with the 230 kD BPAGI. Sera from US and Japanese patients identify a major epitope on the carboxyl temunal region of BPAG I. The purpose of the present study was to determine if epitope In this investigation we have developed an ELISA system IO detect autoantibody reactivity against this cloned epitope. A 42 amino acid segment of the BP180 NCl6A domain has been expressed in E. coli as a glutathione system, S-transferax fusion pmtein (GST-BPIEOSAI) An affinity-purified preparation microtiter wells and incubated with a I: la, endemic pemphigus foliaceus (EPF)(n: normal human sera (NHS)(n:22). MicrMiter Overall, in this assay. patients was predominantly of the IgG class (no IgM. LE or NHS) Specific (99.7%) identical activity sera (64%) None of sera recognize a Based on its high sensitivity (56 to 64%) for detecting BP and HG autoantibody activity, here may prove to be a valuable of diagnoadc the ELISA assay PEMPHIGOID RECOGNIZE A AND COMMON HERPES GESTATIONIS NON-COLLAGENOUS, AUTOANTIBODIES SITE ECTODOMAIN. W. Giudice. D.J. Emew. B.D. Zelickso L.A. Diar. Depts of Deromtology, Med. Coil. of Wiseat.& Univ. of Minnesota, Baltmmre, MD, Bullour characterized (HD) Minneapolis. USA pemphigoid (BP) by suhepidermal antigens, encompassing MN, and the VAMC, BP230 and blisters and BPIEO. sera domain, tested. sera. antiserum agaimt rawd autoantibody-reactive to the HD. lamiwa component. NCl6A. contains blister form is localized These findings antigen. and may (HG) against BP180 Z. Liu. a d WI, US;; Sch. are of Med., diseases skin two hemidesmosomal fusion proteins (FPs) major function a” BP180 segment site recognized directed against affinity-purified basal that lamiru type rabbit this BP/HG immediately with domain projecti a” extracellular of the BP180 by autoantibodies of HG as a” 11 mansmembrane collagenous as a site of interaction in BP and HG. acids of the BP180 showed the predicted C-terminal non-collagenous Autoantibodies using of agaiost a panel of BP, 14 amino of BP sera and by 63% this region of BP180 to the epidermal confirm The long, a” tmmunodominant formation localization a recombinant region The of BP and HG sera. epidermal Univ. investigation site, comprising Ultrastructural of the BP180 into the basal matrix gestationis and immunoadsorption One antigenic site. onentation herpes and autoantibodies was shown to be recognized by 60% lmmunoadsorption analysis identified mmwnodomioant adjacent Hopkins WI THE various segmenti of the BP180 antigen were expressed in a prokxyot~c and control NC16A Johns lo the present system and assayed by immunoblotting HG USA; Mdwaukee, ON G.J. Aohalt. Milwaukee, ectodomain, from a majority this site may be relevant in sub- with BPAGI) and 30 US patients to 3 fusion proteins generated from (AA683-853. 18 kD) adjaw.“, to the 5‘md of the coiled-coil reactivity was as follows (“umber positive/number tested): srr;r &!!?.I !w Japanese U”“ed state, Cuntrul *Statistically compared 168 BULLOUS for reactivity BPAGI cDNA: FP3 (AAIOO3-I 193. 21 kD)from the amino end of dte coiled-coil reeion: FP7 (AA1623-1812, 21 kD) from the carboiyl end of the coil&coil region; and FPi signdicant to controls; not universally 165 Sera from 34 Japanese patients (cxculating titer t 1:8O, 16134 reactive on immunoblot region. lmmunoblot Fp9 II20 2120 3131 8134’ 10130’ 10/30* o/19 ,122 Z/IO rrrctwty of US patients to FP3 and FP7 w&t see” when Japanese patients reacted to FP7 but not to FP3 suggest that epitops reactwity toot. Japanese and US BP patients to fusion proteins domain. antiBMZ The.% results confirmed that BP and HG terminal with BP were assayed by immunoblot in both sets of IgA or IgE was detected). were positive in this assay. patterns are shared betwee” sod product was and 18 of 28 HG reactivity outside of the carboxyl HG amounts Ig. The chromogenic autoantibody from our laboratory common amigenic site on the BP180 ectodomain. and specificity described &E)(n:IS) on Bound autoantibodies were labeled with goat-anti-human 35 of 62 BP sera (56%) and extended z previous finding wra: BP (n:62), wells coated with showed specific reactivq the control sera (EPF, expression WBS immobilized 17). lupus crythematosus recombinant GST were used as a negative control. measured at 492 not. (1.7pg) dilution of the following (x28). z horseradish peroxidasesoojugated using the pGEX of GST-BP180SAI and US to fusion proteins “ear the wile&cod shared between Japanese and US patients with BP. These findings region of BPAGl is