76 Poster Abstracts / Journal of Neuroimmunology 90 (1998) 13-105 425 Cerebrospinal Fluid (CSF) Indices of Inflammatory Markers Aernss the Clinical Spectrum of Multiple Sderosis (MS) S.A. McMillan. G.V. McDonnell, J.P. Douglas, S.A. Hawldns,RoyalGroupof Hospitals, Belfast, Ireland 428 Increased Spontaneous and Inducible Susceptibility toApoptosis of Peripheral Lymphocytes in Multiple Sclerosis M.P. Myeko. A. Walczak, MedicalAcademy of Lodz, Poland, L. Pokoca, Military MedicaIAcademy, Lodz, Poland, K. Selm aj, MedicalAeademy ofLodz, Poland Background: Accumulating evidence indicates significant heterogeneity in MS and soluble (s) adhesion molecules are postulated as markers of disease activity. We evaluated intrathecal production of these and other molecules across the clinical spectrum of MS. Methods: CgF indices of IgG, sE- selectin, sL-selectin, sVCAM-I, sICAM-I and sCD30 were calcnlated in the following patients: 17 prima~ progressives (PPMS), 15 secondary progressives (SPMS), 28 relapsing-remitting in relapse (RRMSR) and 14 RRMS in remission (RRMSNR). Controls were 66 patients with other neurological diseases (eNDs). Results: At 90% specificity, the most sensitive index was for IgG (92% sensitivity in RRIdSNR and 93% sensitivity in RRMSR). Corresponding sensitivity in PPMS and SPMS was 71% and 73% respectively. None of the other indices had sensitivity >50% apart from sVCAM-I (64% in RRMSR and 52% RRMSNR) and sCD30 (53% in PPMS). Conclusions: The strongest association in MS was with intrathecal lgG production. Similar results in PPMS and SPMS may reflect comparable progression rates. Otherwise only intrathecal sVCAM-I production is significantly associated with MS and only in RRMS. It ha~ been stlowh that MBP reactive mature T cell lines from multiple sclerosis (MS) patients are highly susceptible to apoptesis. To further investigate the importance of programmed cell death in pathogenesis of multiple sclerosis we analyzed the frequency of apoptosis in peripheral blood lymphocytes (PBL) in MS. We assessed the percentage of apoptotic PBL by using qosntitive pmpidium iodide cytofluorometfic method. Peripheral blood samples were obtained from 28 MS patients and 17 controls. We report here, for the first time, significantly increased, by more than 3 fold, frequency of apoptotic cells from MS patients comparing to controls; relapse phase: 12.2% v. 3.9%, p<0.001, remission phase: 8.7% v. 3.9%, p<0.03, in progressive MS: 7,6% v. 3.9%, p<0.04. The effect of dexamethason- induced apoptosis was present in both MS and control PBL cultures, with a similar stimulation index. For a phenotypic analysis of apoptotic cells we double stained them with FITC-eonjugatad anti-CD4, CD8, VLA4, Fas mAbs. Sunprisingly, we found that CD4+ and CD8+ T cells were significantly underreprcsentad in apoptmic population of PBL in MS patients comparing to controls (for CD4+: 17.3% v. 32.6% in controls, and for CD8+: 11.9% v. 29.3% in controls). Also, in contrast to previously reported higher frequency of Fas antigen on T cells during MS, we detected only 52% of Fas- positive apoptotic PBL versus 41.4% in control PBL. These results show a significant activation of programmed death cell in PBL during MS which is probably not associated with Fas/FasL induction and might result from other proapoptetic signals. 426 Assessment of a "Naive" vs"Memory" Origin of Human Autoreaetive CD4+ T Cell Clones Specific for Myelin Basic Protein (MBP) P.A. Muraro. Universityof Chieti, Italy, B. Bielekova, M. Perle, G. Afshar, D.E Pette, H.F. McFarland, R. Martin, NINDS, NIH, USA T lymphecytes reactive to myelin antigens are thought to be involved in the pathogenesis of demyelinafing diseases, including multiple sclerosis (MS). The human T call response to myelin basic protein (MBP) in MS has been extensively studied by limiting dilution analysis, in sn attempt to define its possible pathogenic role. Although most studies have shown that MBP-specific CD4+ T cells am present in healthy subjects with frequencies comparable to those found in patients, the specific contdbutinn of "naive" and =memory" calls to these responses has not been examined. Since the expression of the reciprocal CD45 isoforms CD45RA and CD45RO has proven useful for the differentiation of nffive and antigen-expadenced cells, we used highly purified CD45RO-(RA+) and CD45RA-(RO+ ) CD4+ T cell subsets isolated from MS patients and healthy donors to establish MBP-specific T cell clones (TCC), putatively originating from the naive and memory lymphocyte pools. Using this approach, we demonstrated that TCC derived from the CD45RO-(RA+) and CD45RA-(RO+) subsets remarkably differ in terms of epilope specificity, affinity for the antigen, cytokine secretion, call surface molecule expression and TCR usage, confirming and extending the notion that the differential expression of CD45 [soforms allows the identification and isolation of CD4+ T cefis emerging from distinct pathways of selection and maturation. Understanding these striking differences in the specificity and phenotype of naive and memory-originated TCC gives important hints on the role of innate versus antigen-driven T cell repertoires in physiological immune responses and autoimmunity. 429 ELISPOT Analysis of Proteolipid Protein (PLP) Cytokin¢ Responses in Multiple Sclerosis (MS) C.M. Pelfrey. B. Weinstock-Guttman, R.A. Rudick, Cleveland Clinic Foundation, USA, P.V. Lehmann, Case WesternRezerve University, USA Pm-inflmnmalo~ cytotdnes (rhl-like) am lhaught to be c~dal for the initiation and amptifi~fiun of inflanuna~y brain lcs/eas and din~t myetin dmasge in unfltiple sdero~s (MS). The role of Th2dike cytokines is unclear, as is the rote of CD4+ vem~ CDg+ T-ceI~ We ased the cytokine ELISRTr assay to study pcaeolipid protein &LP) psp~te ma~ivity infieshly LseimedPBMC. We tested responses flora 14 MS patients and l I controls asing 9-amim-ecid lmgth overlapping PLP pcptides to study fl~ fine specificity of lymphacytes as measured by their IFNy and IL-5 profile. PLP peplk~ giving IFNy-positive responses(mean s~ulation index > 3) v, ere sulxlivided into sin~.poa~ (p~ive c~ renxraes)ve~s ne~e-adj~=t-posinves ¢ma~ CD4 rc~soraas). The MS pafieres ~ 12 times as many nudges compared to controls. ~ the total mnnlm" of IFNy.p~tive spots (Le cells), spots in nadtilflo-adjacent..pmitiveswere 43 times as high in MS patiet~ as wt&u~s. The avenge numb~ of IL-5 ~ngle ix~fivcs was almost idmlical ~ MS t~ica-as and e0mds and an IL-5 anaeple-edjacent poatives were otswvcd, suggeaing that R..p- sl~itic IL-5rcspom~ do mt ditt~" significantly baw~a MS I~ientsann cor~o~. Our data suggea that MS patin~ts give a nm~ mong~ MHC Class ~ immune response to PLP peptides c ~ to ce~m~s, which is ~ by s~zetion of IFNy, an inflammato~ cytokine that has beea smmgly implicated in the disease precass. 427 Down-regulation of the Th-2 Associated GATA-3 Transcription Factor in Peripheral Blood Mononuclear Cells in Multiple Sclerosis M.I~ Mveko. M. Stasiolek, A. Walezak, M. Kwinkowski, K. Selmaj, Medical Academy of Lodz, Poland A central role in promoting inflammatory response in multiple sclerosis (MS) has been postulated to the presence of autoreactive Thl clones. Recently GATA-3, member of the GATA C4 zinc-finger transcription factors family, was identified as necessary and sufficient element for the Th2 cytokine gene expression including the IL-4 gene promoter activation. GATA-3 was found to be expressed selectively at high level in both naive CD4 T cells and Th2 cells, but at a minimal level in Thl cells. In our study we have assessed by electrophoretic mobility shift assay (EMSA) presence of the GATA DNA binding activity in the nuclear extracts from plasma blood mononuelear cells (PBMC) from 10 MS patients and 10 controls. We have found by EMSA supershift that GATA-3 is present in PBMC as one of two GATA DNA binding activities. We have shown here, for the first time, a significant decrease in the GATA DNA binding activity in PBMC during the relapse phase of MS, and also. albeit milder, during remission phase and in progressive patients comparing to controls. Simultaneously, we have not found any changes of DNA binding activities of other nuclear factors like ATF/CREB family transcription factors family. These data suggest that GATA-3 might play an important role in molecular mechanisms promoting Thl/Th2 shift in T cell subsets during MS. 430 The Frequency of Circulating T Cells Reactive to PLP184-209 Increases Prior to MS Relapse and Devdopment of New MR1 Lesions M.E Pendcr. P.A. Csurhes, J.M. Greer, The Universityof Queensland, Australia, ED. Mowat, R.D. Henderson, Royal Brisbane Hospital, Australia, K.D. Cameron, P.A. McCombe, The UniversityofQueensland, Australia, M.F. Good, QLDI~tinae of Medical Research, Australia We have recently reported that patients wnh relapalng-remitting or secondary progressive multiple sclerosis (MS) have increased blood T cell proliferative responses to the 184-209 region of myelin proteolipid protein (PLP) (Brain [1997] 120, 1447-1460). This region of PLP is immtmodominunt in humans and eneephalitogenie in mice. To determine whether this T cell reactivity correlates with disease activity, we have performed serial monthly limiting dilution assays on the blood and magnetic resonance imaging (MRI) of the brain in patients with relapsing-remihing MS. We have found that the frequency o f t cells reactive to PLPI84-199 or PLPI90-209 increases 1-3 weeks before the onset of relapses and decreases during relapses, whereas the frequencies of T cells reactive to the immunodominant 82-100 peptide of human myelin basic protein or the control antigen, tetanus toxoid, do not show this patte/n. An increase in the frequency of T cells reactive to PLPI84-209 also precedes or accompanies the development of new gadolinium-enhancing MRI brain lesions. Our findings indicate that T cells reactive to PLP184-209 expand in the periphery prior to the onset of MS relapse. The decrease in the frequency of these T cells in the blood during relapse may he due to their entr,/into the central nervous system. Thus these T cells may have a pathogenic role in MS.