virus (HCV), and the sequence homology between HCV and Peripheral blood mononuclear cells (PBMC) of pa-LKM suggests cross-reactivity or HCV-related autoimmutients with autoimmune hepatitis (AIH) and controls nity. [4][5][6][7] Immunohistological studies in AIH patients showed were studied for their proliferative response to six overa predominance of CD4 / T cells within dense lymphocytic lapping synthetic peptides covering the 33-amino acid infiltrations. [8][9][10] In vitro, T-cell lines and clones derived from immunodominant region of cytochrome P450IID6, the the blood and liver tissue of AIH patients showed a preferenmain target antigen of LKM-1 antibody-positive type II tial activation of CD4 / CD8 0 cells compared with the accumu-AIH. PBMC from 8 of 8 type II AIH patients (100%), 6 of lation of CD4 0 CD8 / T cells in the liver of patients with 12 LKM-1 antibody-negative type I AIH patients (50%), chronic active hepatitis B or C. 11,12 The clonal analysis but only 4 of 31 patients with chronic hepatitis C (12.9%)showed that in AIH patients the T cells predominantly reacted with a 23-amino acid LKM peptide and mainly showed the Th1-like cytokine release, but the frequency of with a shorter 18-amino acid LKM peptide. Follow-up interleukin-4 (IL-4)-producing Th2 helper T cells was also showed that LKM-specific T-cell responses decreased increased. 12 after immunosuppression had started. Fine specificity, It has been demonstrated recently that liver-infiltrating HLA restriction, and cytokine release of LKM-specific T T-cell clones from patients with type II AIH recognized a cells were analyzed with 16 CD4 / peptide-specific T-cell recombinant LKM-1 antigen carrying the immunodominant lines and 21 CD4 / T-cell clones isolated and expanded 33-amino acid region of cytochrome P450IID6, the main tarfrom blood and liver tissue of six AIH patients. Activated get of LKM-1 antibodies. 4,13 Thus, the LKM-specific T-cell LKM-specific T cells released interferon gamma (IFN-g)response in AIH may contribute to the immunopathogenesis, but no or little interleukin-4. In three AIH patients, although further functional characterization is needed to PBMC showed specific recognition of autologous LKMshow pathogenetic significance. specific T cells, suggesting the presence of a regulatoryThis study focused on (1) the fine specificity of the cellular T-cell network. These T cells also showed the CD4 / pheimmune response to main T-cell epitopes within the immunonotype and secreted large amounts of IFN-g. Furtherdominant LKM-1 antigen, (2) analysis of the cytokine release more, it was assessed that the regulatory T-cell response of T cells in response to LKM peptides, and (3) demonstration is clonotypic. To conclude, we describe a major T-cell of anticlonotypic T cells that may control autoreactivity. ies, hypergammaglobulinemia, and response to immunosuppressive therapy, were studied. Eight of the 20 (40%) had LKM-1 antibodypositive type II AIH, and 12 of the 20 (60%) had LKM-1 antibodyAutoantibodies against intracellular structures s...
Analysis of the variable chains (V alpha/V beta) of the specific T cell receptor (TCR) of organ-infiltrating T cells may provide further insights into the pathogenesis of many infectious diseases, malignancies, and autoimmune disorders. To determine the TCR V beta repertoire of these small T cell populations antigen-independent in vitro expansion is necessary but may select for certain T cell subpopulations. In this study various antigen independent T cell activation protocols were used to stimulate peripheral blood mononuclear cells (PBMC) of six healthy blood donors, and TCR V beta molecules were analyzed by flow cytometry and semiquantitative reverse-transcriptase polymerase chain reaction. In addition, the analysis of in vitro expanded liver-infiltrating T cells and autologous peripheral blood T cells derived from five patients with autoimmune hepatitis but none of six controls revealed a selective overexpression of single TCR V beta molecules in the liver tissue. In contrast to freshly isolated PBMC, no preferential expansion of single TCR V beta families was observed using phytohemagglutinin, anti-CD3 antibodies, or oxidative stress for antigen-independent T cell activation. In conclusion, antigen-independent T cell activation offers the chance to analyze small populations of organ-infiltrating T cells without skewing the TCR V beta repertoire.
Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and co-digested with HaeIII to reduce fragments to 50-750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P < or = 1 x 10(-6) to 1 x 10(-24)). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P < or = 1 x 10(-7) to 1 x 10 (-12). The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P < or = 1 x 10(-7). The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18-20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.
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