The goal of the proposed research is to characterize mechanisms of innate immunity in the small intestine. Cryptdins are Paneth cell defensins, microbicidal peptides that are released continually into the small intestinal lumen. The specific objectives of this proposal are focused at defining the mechanisms of cryptdin action and the regulation of their synthesis. Six mouse cryptdins have been characterized, and they are potent antimicrobial agents with selective activities against varied target microbes. In addition to the six known peptides, fourteen novel cryptdin isoforms have been predicted by genetic evidence. Cryptdins are coded by separate genes, and certain genes are expressed differentially during postnatal crypt ontogeny and along the longitudinal axis of the adult small bowel. Abundant data support the hypothesis that Paneth cell defensins are important components of mucosal immunity, and that concept will be tested by analyzing biological interactions between murine cryptdins and by characterizing the corresponding genes. To test hypotheses regarding mechanisms of Paneth cell defensin action, potential synergistic interactions between cryptdins will be investigated by quantitating the regional cryptdin isoform content of mouse small intestine, evaluating the microbicidal action of modified cryptdins isolated from the intestinal lumen, and by assaying cryptdins, both individually and in combinations that reflect their relative levels, for antimicrobial potency against a range of pathogens. Preparations of individual and combined Paneth cell defensins will be tested for potential cytotoxic effects against mammalian cell lines, and cytotoxicities will be compared with that of myeloid defensins. To investigate the role of pore-formation in the cryptdin killing mechanism, measurements will be made of the kinetics and stoichiometry of ion- permeable channel formation in lipid bilayers by cryptdins. To test hypotheses relevant to regulation of cryptdin synthesis, the primary structures of cryptdins isolated from newborn mouse small bowel will be determined, and the developmental appearance of cryptdin isoforms will be established. The relative placement and orientation of mouse cryptdin isoform genes will be established along the physical map. Finally, potential cis-acting determinants of cryptdin positional specificity will be studied by characterizing transgenic mice constructed with variable flanking and intronic regions of the cryptdin-4 gene. These studies will clarify mechanisms of Paneth cell defensin function in innate immunity by elucidating their mode of action, potential interactions, and the regulation of their synthesis in the adult and developing mouse small intestine.
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