We wish to define the pathogenesis of Crohn's disease (CD). Using patients' tissues and athymic (nu/nu) mice, we plan to identify isolate and characterize putative antigen(s) related to CD. In our initial studies, lymph node and intestinal filtrates of CD tissue produced lymphoma or hyperplastic lymph nodes in 16% of nu/nu mice. Both lymphomas and hyperplastic lymph nodes immunologically react with 50-80% of sera from patients with symptomatic CD and not with control sera. We further demonstrated that immunoreactive B cells occur in normal nu/nu lymph nodes. Using hybridoma technology, we will """"""""immortalize"""""""" antigen(s) present in these cells. We will examine the effects of chemical and physical treatments of the tissue filtrate on appearance of antigen in lymph nodes. Since the """"""""antigen"""""""" detected by CD sera in nu/nu B cells may be an """"""""anti-idiotypic antibody"""""""", we will purify IgG from CD tissue filtrates and inject it into nu/nu. Secretory proteins from nu/nu Beta cells-myeloma hybrids and nu/nu serum IgG will be examined for anti-idiotypic antibodies. Using a fluorescence-activated cell sorter, we will separate immunoreactive cells. Using immunohistochemical techniques, EM and immunoelectron microscopy, we will localize the antigen and search for viral particles in these cells. If viral particles are seen, detailed virologic studies will be performed. Using gel filtration techniques, CD and control intestinal tissue homogenates, we identified several CD tissue-specific proteins of 160 kd, 120 kd and 110 kd mol. wts. Specific recognition of the proteins by CD serum IgG was demonstrated by transblot. Using HPLC, we purified the 160 kd protein which reacted with CD sera and not with control sera by dot blot assay. We plan to purify the protein(s), determine their molecular structure and raise monoclonal antibodies. These will enable us to examine their origin (from host tissue or an extrinisic agent(s)), determine whether they are derived from a single source, and whether they are associated with lymphoid hyperplasia and the appearance of immunoreactive cells in nu/nu, and perform molecular studies for cloning their genes. These proteins may provide important probes for studies of the etiology of CD and lead to a better undertanding of its pathogenesis.
