The overall objectives of the proposed research are: 1) the study of the surface membrane changes occurring during differentiation, embyonic and postnatal development in the intestinal epithelium; 2) the identification of the factors which trigger and modulate the expression of differentiated functions in cultured intestinal cells; 3) the study of the growth characteristics of cultured intestinal crypt cells; and 4) to compare tumor intestinal cell lines with normal cells with respect to their surface membrane antigens and growth characteristics. During the next year of support, our major efforts will be directed towards the purification of an apparently specific intestinal growth inhibitor, previously shown to be present in villus cell homogenates and in the spent culture medium of fetal intestinal organoid cultures, and the preparation and characterization of monoclonal antibodies to intestinal surface membrane and cytoskeletal components. Purification of the inhibitory factor from villus cell homogenates has progressed slowly until recently, due to its apparently heterogeneous behaviour under a variety of experimental conditions, but the recent finding that a similar or identical inhibitor is secreted by fetal intestinal organoid cultures into the culture medium suggests to us that this may represent a much better source of inhibitor, from which its purification may be attempted and carried out in the absence of luminal proteases and mucus components. Many of the studies which are planned as part of this proposal are centered on the use of monoclonal antibodies as specific reagents for both in vivo and in vitro studies. A large number of well characterized antibodies are already available in our laboratory. During the next year of support we are planning to prepare new antibodies using different antigens for immunization: surface membrane fractions purified from intestinal crypt cells, brush border membranes from fetal and newborn (5-9 days old) rats, cytoskeletal components purified from adult villus cell brush borders, and cultured human tumor cells (CaCo-2 cells). Mice will be immunized with these different antigens, and tested: the two mice from each group having the highest serum titer will be selected, and their spleen cells will be fused with NS1 mouse myeloma cells. Hybridoma cultures, selected with HAT medium, will be tested for the production of specific antibodies by ELISA and radioimmunobinding assays. Cultures of interest will be cloned and used for the production of large amounts of monoclonal antibodies. Antibodies will be purified and used for the identification and characterization of the antigens recognized.
