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Autoantibodies to the 65-kDa isoform of glutamate decarboxylase GAD65 (GAD65Ab) are strong candidates for a pathological role in Stiff-Person syndrome (SPS). We have analyzed the binding specificity of the GAD65Ab in serum and cerebrospinal fluid (CSF) of 12 patients with SPS by competitive displacement studies with GAD65-specific rFab-derived from a number of human and mouse mAbs specific for different determinants on the Ag. We demonstrate considerable differences in the epitope specificity when comparing paired serum and CSF samples, suggesting local stimulation of B cells in the CSF compartment of these patients. Moreover, these autoantibodies strongly inhibit the enzymatic activity of GAD65, thus blocking the formation of the neurotransmitter gamma-aminobutyric acid. The capacity of the sera to inhibit the enzymatic activity of GAD65 correlated with their binding to a conformational C-terminal Ab epitope. Investigation of the inhibitory mechanism revealed that the inhibition could not be overcome by high concentrations of glutamate or the cofactor pyridoxal phosphate, suggesting a noncompetitive inhibitory mechanism. Finally, we identified a linear epitope on amino acids residues 4-22 of GAD65 that was recognized solely by autoantibodies from patients with SPS but not by serum from type 1 diabetes patients. A mAb (N-GAD65 mAb) recognizing this N-terminal epitope was successfully humanized to enhance its potential therapeutic value by reducing its overall immunogenicity.

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BACKGROUND: Stiff-person syndrome (SPS) is an immune-mediated central nervous system disorder characterized by fluctuating muscle stiffness, disabling spasms, and heightened sensitivity to external stimuli. Up to 80% of patients with SPS have anti-glutamic acid decarboxylase (GAD) antibodies in the serum or cerebral spinal fluid (CSF). Whether these antibodies are clinically relevant and correlate with disease severity is unknown. OBJECTIVE: To correlate anti-GAD antibody titers in the serum and CSF of patients with SPS with the degree of clinical severity. DESIGN: Patients studied the last 6 years. SETTING: The Clinical Center of the National Institutes of Health, Bethesda, MD. PATIENTS: Sixteen patients with typical SPS and elevated serum anti-GAD antibody titers. INTERVENTIONS: Antibody titers in serum and CSF were measured by radioimmunoassay, and the intrathecal anti-GAD-specific IgG production was calculated. MAIN OUTCOME MEASURES: Comparison of antibody titers with stiffness index and heightened sensitivity scores based on scales that reliably measure disease severity. RESULTS: The mean disease duration was 11 years (range, 5-30 years). The mean anti-GAD antibody titer in the serum was 51 500 U/mL (range, 24 000-200 000 U/mL); and in the CSF, 181 U/mL (range, 30-400 U/mL). A 10-fold increased intrathecal production of GAD-specific IgG antibodies was noted. No correlation was found between antibody titers in serum or CSF with disease severity. In 4 patients, the anti-GAD antibody titers measured serially during a 2-year period did not correlate with clinical fluctuations. CONCLUSIONS: In patients with SPS, the anti-GAD antibody titers in serum and CSF do not correlate with disease severity or duration. Anti-GAD antibodies are an excellent marker for SPS, but monitoring their titers during the course of the disease may not be of practical value.

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Interactions between inducible co-stimulatory molecule (ICOS) and ICOS-ligand (ICOS-L) are crucial for T-cell co-stimulation, effector cell differentiation and memory CD8+ T-cell activation. Because in the muscle of patients with sporadic inclusion body myositis (sIBM) clonally expanded CD8+ T cells invade major histocompatibility complex (MHC) class I-expressing muscle fibres, we investigated ICOS.ICOS-L interactions and correlated their expression with perforin, a marker for cytotoxic effector function by autoinvasive CD8+ T cells. The mRNA from 20 muscle biopsies of sIBM, 20 non-inflammatory or dystrophic controls, two dermatomyositis (DM) and two polymyositis (PM) patients was reverse transcribed and reamplified by semi-quantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR), using primers for ICOS, ICOS-L and perforin. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-normalized ratio of ICOS, ICOS-L and perforin expression was compared with the degree of endomysial inflammation. Protein expression of ICOS, ICOS-L and perforin was confirmed by immunohistochemistry. We demonstrate that ICOS-L mRNA was upregulated in sIBM (arbitrary units, median +/- SEM: 48.6 +/- 14.9) compared with controls (6.2 +/- 17.8, P < 0.05) and significantly correlated with the expression of ICOS (53.9 +/- 16.6 versus 6.7 +/- 8.9 in controls, P < 0.001). By triple labelling immunohistochemistry, the CD8+ T cells in sIBM and PM were found to invade ICOS-L- and MHC class I-co-expressing muscle fibres. Among the autoinvasive CD8+ T cells, however, only a subset of approximately 5-10% were ICOS positive, and thereby perceptive for ICOS.ICOS-L signalling at the immunological synapse. In contrast, in Duchenne muscular dystrophy and DM, although ICOS and ICOS-L mRNA expression was also increased, the majority of ICOS-L- and ICOS-positive cells were in the perimysial regions and connective tissue. The mRNA for perforin was increased in sIBM (28.1 +/- 8.7) compared with controls (4.3 +/- 11.2, P = 0.18), and significantly correlated with mRNA of ICOS, ICOS-L and the degree of endomysial inflammation as assessed in coded haematoxylin/eosin tissue sections. By triple immunohistochemical staining and cell counting, perforin granules were found in 71% of the autoinvasive CD8+ T cells that were also ICOS positive. Our data indicate that in sIBM there is upregulation of ICOS.ICOS-L co-stimulatory signalling in association with enhanced perforin expression by the autoinvasive CD8+ T cells. The findings support previous suggestions that in IBM, the muscle fibres have the capacity for antigen presentation, thereby activating a specific subset among the autoinvasive CD8+ T cells to exert a cytotoxic effect. The observations strengthen the immunopathogenesis of sIBM, and offer the basis for future therapeutic interventions targeting ICOS.ICOS-L co-stimulatory interactions.

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Because IFN-gamma-inducible chemokines, Mig (CXCL9), IP-10 (CXCL10), I-TAC (CXCL11) and their receptor, CXCR3, are critical molecules in T cell trafficking and generation of effector T cells, we examined their expression in the muscle biopsies of patients with sporadic inclusion body myositis (s-IBM) and disease controls. The functional role of these molecules was also studied by examining the effect and time kinetics of IFN-gamma in inducing Mig and IP-10 expression in human myotubes in vitro. We found significantly high levels of Mig and IP-10 mRNA expression in s-IBM muscles compared to controls. IFN-gamma upregulated the mRNA expression of Mig and IP-10 by human myotubes in a dose-dependent manner. By double-label immunohistochemistry, Mig was expressed on a subset of CD8(+) cells and the areas of the muscle fiber in contact or contiguous to the T cells; CXCR3 was expressed only on a subset of the autoinvasive CD8(+) T cells but not the myofibers. IP-10 and I-TAC were not detected by immunocytochemistry. The findings indicate that in s-IBM, IFN-gamma is involved in the upregulation and in situ production of proinflammatory chemokines, which, in turn, participate in the recruitment of activated T cells and contribute to the self-sustaining nature of endomysial inflammation.

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We utilized two methods to look for T cell clonal expansions in myasthenia gravis (MG). We analyzed TCRBV CDR3 length polymorphism (spectratyping) to look for evidence of clonal expansion of CD4 or CD8 T cells directly from peripheral blood of MG patients. No statistically significant differences were found between the diversity of TCR repertoires in MG patients compared to normal control individuals when analyzed as groups. Rare oligoclonal expansions were detected in some individual MG patients but the significance of these findings is unclear. Next, we analyzed a panel of T cell hybridomas from acetylcholine receptor (AChR) immunized, MG-susceptible HLA-DR3 transgenic mice. The epitope specificity, TCRBV gene usage and CDR3 sequences of these hybridomas were highly diverse. We conclude there is only limited evidence for restricted TCR repertoire usage in human MG and suggest this may be due to the inability of HLA-DR molecules to select for restricted TCR recognition of AChR epitopes.

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BACKGROUND: The interaction between host lymphocytes and endothelial cells on the transplanted organ is believed to play an important role in acute and chronic graft rejection. Trafficking and recruitment of lymphocytes to the site of inflammation is known to be controlled by several cytokines and chemokines. It is unclear whether endothelial cells themselves can be a source of inflammatory chemoattractant molecules on alloimmune induction. METHODS: Using a semiquantitative polymerase chain reaction method, the authors analyzed the expression of chemokine mRNA coding for interferon (IFN)-gamma-induced protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) in a pool of human aortic endothelial cells. Both of these chemokines are known to be induced by IFN-gamma. Endothelial cell-derived chemokine mRNA was assayed at rest, after IFN-gamma activation, and after co-culture with allogeneic peripheral blood mononuclear cells (PBMC) from normal blood donors with and without a monoclonal antibody to IFN-gamma. Finally, protein release into the media was assayed using an enzyme-linked immunosorbent assay to IP-10. RESULTS: Mig and IP-10 were expressed in human endothelial cells both after IFN-gamma treatment and after PBMC co-culture. Furthermore, the expression of both of these endothelial cell-derived chemokines was dependent on IFN-gamma because PBMC-induced expression was blocked with anti-IFN-gamma. IP-10 levels in the endothelial cell supernatant increased from a baseline of 13.4+/-10.8 pg/mL to 299.5+/-13.4 pg/mL (P<0.0001) with exposure to PBMC and was likewise inhibited by anti-IFN-gamma A-b (33.8+/-17.8 pg/mL). CONCLUSIONS: Vascular endothelial cells are capable of producing inflammatory chemokines when activated and potentially serve to amplify the allogeneic response.

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Experimental autoimmune myasthenia gravis can be induced in some strains of mice and rats by immunizing with acetylcholine receptor. Also, epidemiologic studies demonstrate an MHC linkage of myasthenia gravis in the man. In order to obtain direct experimental evidence for the influence of the genes of the MHC complex in the development of myasthenia gravis, we used mice transgenic to individual HLA molecules. We observed an increased susceptibility to the disease in HLA DQ8 transgenic mice compared to HLA DQ6 transgenic mice ( J. Immunol. 160:4169; 1998). These mice lacked endogenous mouse class II molecules. In the present study we mapped the cryptic and dominant sequences on the extra cellular region of human acetylcholine receptor. Although some epitopes (e.g., alpha11-30, alpha141-160, alpha171-190) were common between DQ8 and DQ6 transgenic mice, several others were disparately recognized. We also found a functional dichotomy in T cells from mice differing by one MHC molecule (HLA DQ8 or DQ6) when primed by sequences immunodominant in DQ8 and DQ6 tg mice. Differential disease manifestation in the two different HLA transgenic mice could be explained not only by differential recognition of peptides by these antigen presenting molecules, but also by the difference in the functional profile of T cells generated when primed by promiscuous sequence regions.

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