The intestinal epithelium is immature at birth and undergoes striking functional and structural changes in microvillus membrane (MVM) during suckling period. This is the stage that most incidents of toxigenic diarrhea take place. To study the role of intestinal maturation in modulating attachment/biological action of luminal pathogens, two well known bacterial enterotoxins, cholera toxin (CT) and E. coli heat-labile toxin (LT) will be used as probes to define toxin interactions with the developing intestine in the animal model. Developmental changes in the responsiveness of the target host (i.e. intestine) to CT and LT will be studied from the initial binding (to MVM or isolated enterocytes), followed by ADP-ribosylation, adenylate cyclase activation, cAMP accumulation, protein phosphorylation and finally fluid secretion. Membrane receptors (glycolipids and glycoproteins) for CT and LT will be identified, characterized and isolated from immature and mature intestine. Developmental regulation of intestinal glycosylation will be studied by glycosyltransferase assay, cell-surface carbohydrate analysis, labeled sugar incorporation and oligossacharide structural characterization. Results of these studies collectively will help to understand the role of intestinal glycosylation in receptor expression, a key factor in determination toxin attachment. Effects of hormones (cortisone and thyroxin) and nutrition (natural milk) on intestinal maturation and toxin interaction will be studied in order to explore additional means in the reduction of toxin attachment and toxin action on the immature intestine. Both in utero and postnatal effects of hormones will be evaluated, and the role of milk oligosaccharides on the inhibition of toxin binding will be studied. Information obtained from this project should help to understand the biochemical mechanism by which intestinal development may modulate the host response to toxin binding and action. This information may lead to a better prevention of toxin attachment during postnatal development, and a better treatment of toxin-induced hypersecretion in the neonate.
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