The main goal of this proposal is to purify the Mr 40K colonic epithelial protein to homogeneity, characterize it biochemically, investigate its polymorphism in different tissue and examine its biological role by establishment of an experimental autoimmune model for ulcerative colitis (UC) in mice. Recent data strongly suggest that this protein is a target self-antigen that triggers an autoimmune reaction which may be important in the pathogenesis of UC. The protein is also present in experimental animals, immunogenic in mice and preliminary studies demonstrated gross and histological changes in colonic mucosa upon injection in the appropriate adjuvant. We will characterize the T cell response to the purified Mr 40K protein and will analyze the T cell proliferative responses to different peptides derived from the Mr 40K protein by chemical or enzymatic cleavage followed by purification on HPLC. We will then determine which of the immunogenic peptide(s) induces an immune response leading to inflammation in the colon. The MHC restriction and T cell receptor usage by these T cell clones will be analyzed by two general approaches: a) The Mr 40K protein/peptide(s) will be injected with various adjuvants into susceptible mouse strains (high responder strains) using various protocols developed for the induction of other autoimmune diseases. b) T cell lines/clones will be adoptively transferred into normal and into immune-compromised mice including SCID mice. Disease induction will be assessed by gross examination and histology. Two main questions concerning the immune mechanisms responsible for the onset of the experimental disease will be addressed: a) What is the phenotype of the antigen presenting cell (APC) that presents the Mr 40K protein to the antigen-specific T cell that mediates tissue damage? Irradiation bone marrow chimeras will be constructed such that the hematopoietic compartment is of a different haplotype than the rest of the host's tissues. T cell clones histocompatible with either the hematopoietic or the host tissue will be adoptively transferred into these chimeras to determine whether the APC is a classical bone marrow-derived cell or a colon epithelial cell. b) How is tissue damage inflicted upon the colon epithelium? Three possible mechanisms will be tested: antibody-dependent cell-mediated cytotoxicity.
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