The overall hypothesis of this proposal is that necrotizing enterocolitis (NEC) is a disease of immature intestinal host defenses triggered by the introduction of artificial feedings and propagated by inappropriate microbial colonization. To test this hypothesis we will use newly established in vitro models for study of human intestinal host defense including a recently established human fetal small intestinal cell line, organ cultures of small intestine from NEC patients and age matched controls, Ussing chamber techniques to quantitate antigen uptake and bacterial translocation, and a transplanted fetal human intestinal loop model for simulated intraluminal conditions in the immature intestine. Using these methods, we will study aspects of developmental regulation of bacterial colonization, antibacterial properties of developing Paneth cells, the functional significance of a newly discovered intestinal Fc receptor for human IgG and selective aspects of intestinal inflammatory responses and immune function. If, as expected, we note developmental differences in these studies, we plan to assess developmental regulation of these host defenses by growth factors (IGF-l, EGF, TGF-alpha and beta, cortisone) and cytokines (INF-gamma, TNF-alpha, PAF, etc). To study bacterial colonization and toxin interactions, we will extend previous animal studies to examine glycosyltransferases and microvillus membrane glycoconjugates (the putative bacterial gut receptor) developmentally and fimbrial adhesion molecules from two strains of enterotoxigenic E.Coli in intestine from NEC patients and at various ages (fetal-childhood). Because Paneth cells have been shown to produce and secrete antibacterial substances including TNF-alpha, lysozyme, alpha-l-antitrypsin and cryptdins, an intestinal form of defensin, we will study the ontogeny of Paneth cell protective function and specifically its anti bacterial properties in NEC intestine. We will define the significance of the newly described Fc receptor by first quantitating IgG and their subclass levels in amniotic fluids and preterm colostrum. We will then use the same concentrations of IgG to test for preferential binding to the fetal enterocyte and transport across the epithelium into the circulation. We will also test IgG-IgA at concentrations reported to protect premature infants against necrotizing enterocolitis to see if these concentrations actually bind to this receptor. Finally, we will measure the response to stimulus of inflammatory cytokines (IL-1, IL-6, IL-8, PAF) and class II antigen expression in various ages of intestinal enterocytes and intestine from NEC patients for developmental differences and determine if growth factors affect their expression at the mRNA and post transcriptional levels. These studies in human immature small intestine should help our understanding of the pathogenesis of NEC and may lead to new strategies for prevention and treatment.
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