application) Primary sclerosing cholangitis (PSC) includes features of autoimmunity. The production of autoantibodies in the majority of patients is highlighted by the prevalence of atypical pANCAs and specific anti-catalase immunoglobulins. In addition to self-reactive antibodies, autoreactive T lymphocytes likely participate in PSC as CD4- and CD8-positive T lymphocytes infiltrate affected bile ducts. In the proposed experiments, we will investigate the role of a conserved autoantigen, catalase. Catalase is highly conserved in bacteria and mammals and may induce autoimmune responses by molecular mimicry. Previous studies indicate that catalase is highly expressed in both hepatocytes and biliary epithelium. Affinity-purified autoantibodies from PSC patients will be used to map epitopes of cloned human catalase cDNA as well as selected enterohepatic Helicobacter catalases. Immunodominant epitope-containing peptides isolated by filamentous phage display will be used to examine immunopositivity in PSC patients. To investigate T cell epitopes within this candidate antigen, human and bacterial catalase peptides capable of binding MHC class I antigens have been identified by a computer-assisted HLA-A2.1 peptide binding algorithm. Candidate peptides will be evaluated for in vitro binding with IHLA-A2.1 on T2 hybridoma cells. Peptides which bind HLA-A2.1 will be used to isolate cytotoxic T lymphocyte (CTL) clones from healthy blood donors and the common bile ducts of PSC patients. Molecular mimicry will be examined in vivo by inoculation of wild type and IL-10-deficient mice with H. hepaticus catalase and subsequent analysis of anti-murine catalase immune responses. Splenocyte transfer into syngeneic animals will be performed to address contributions of anti-catalase cell mediated immune responses to the development of sclerosing cholangitis. Finally, we propose to establish a mouse model linking IBD and sclerosing cholangitis in IL-10-deficient mice. AJ/Cr (IL-10 -/-) mice pruned by immunization with immunodominant H hepaticus catalase peptides will be infected with Helicobacter hepaticus. These mice will likely develop chronic colitis, cholangitis, anti-neutrophil and anti-catalase autoantibodies, an unopposed Thl immune response (consistent with PSC), and an inherent predisposition to fibrosis. Hepatobiliary pathology will be correlated with anti-catalase immune responses. The proposed studies will enhance our understanding of the role of molecular mimicry and autoreactive T cells in inflammatory diseases of the hepatobiliary tract. The proposed animal model will facilitate the evaluation of diagnostic and therapeutic strategies for PSC in vivo.
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