The overall hypothesis for this application is that in patients with ulcerative colitis (UC) the epithelium expresses autoantigen (s) which may drive the immune destruction of the epithelial cells. Recently, we purified the Mr 40K colonic autoantigen (P-40), sequenced 2 proteolytic peptides and demonstrated that P-40 belongs to cytoskeletal tropomyosin (TM) family. Eighty to 90% of UC have circulating antibodies against P- 40/TM. We want to extend this observation at the cellular and molecular levels. Specifically, we plan to define the colon epithelial TM isoforms from normal and UC patients, and examine polymorphism, if any. We have shown both in UC mucosa as well as in selected colon cancer cells that UC associated autoantibodies bind at the same site as the 7E12H12 monoclonal antibody (developed by us earlier) does. We will purify and characterize the peptide reactive to the 7E12H12 moAb from colon epithelium/specific colon cancer cells and examine its relationship to TM-isoforms. The immunoreactivity of UC sera against the TM-isoforms and 7E12H12 reactive peptide will be examined. Disease specificity, type- specific IgG response, relation to disease state will be investigated. Cellular immune responses against the TM-isoform (s)/peptides will be studied using peripheral blood lymphocytes from patients with UC. We will isolate neutrophil-TM, compare with colonic TM and examine the cross-reactivity with various anti-TM antibodies and inflammatory bowel disease sera. We will test the hypothesis that anti-neutrophil cytoplasmic antibody (pANCA) associated with directed to TM-isoform(s). We will further isolate and characterize cDNA encoding the colon epithelial specific TM-isoform(s) from normal and UC. A human colon cDNA expression library from normal & UC colon mucosa and appropriate colon cancer cell line will be screened by UC serum, 7E12H12 moAb, anti-P40, anti-nonmuscle TM and synthetic oligonucleotides common to all known TM isoform e.g. REN-29. The specific cDNA inserts will be overexpressed so as to provide a large amount of recombinant protein to study specific cellular immune responses, provide uniform material for a serologic test and to explore therapeutic possibilities.
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