The general goal of this project is to gain insight into the pathophysiologic basis of clinical Cryptosporidium infection. The fundamental basis of diarrhea in this illness is unknown; the host epithelial response to infection is poorly characterized. We believe that rational interventional therapy will develop from understanding the biological and functional effects of epithelial infection with Cryptosporidium. For our model in vivo/in vitro system, we have chosen the neonatal piglet because infected piglets develop highly symptomatic disease with diarrhea, unlike other laboratory animals, e.g. mice. In a novel approach we have developed a model of Cryptosporidium infection in a well- characterized cultured intestinal epithelial cell line in which both structural and functional correlates can be monitored and manipulated during infection. Thus our specific aims are: 1) To detail the temporal course of alterations in epithelial cell transport function during infection in the neonatal piglet using in vitro approaches and to make appropriate structure-function correlations. We will extend these studies in epithelial permeability in the second specific aim: 2) To assess alterations in macromolecular permeability and determine the sites at which these leaks occur in the infected epithelium in vitro. The functional abnormalities described in Specific Aims 1 and 2 are a consequence of parasite attachment and entry in host enterocytes. Thus our third aim is: 3) To characterize the remodelling of the host enterocyte cytoskeleton during Cryptosporidium infection and to study the alterations in epithelial cell transport events that may accompany cytoskeletal remodelling during infection. To accomplish these goals we will use infected neonatal piglet small intestine from defined sites in in vitro electrophysiologic Using chamber techniques, bi-directional transepithelial ion flux, and extracellular tracer molecule studies. Analysis by fluorescence and electron microscopy will be performed for structure-function correlations. Finally, we will apply similar structure-function approaches in an in vitro cell culture model of infection to assess the role of the enterocyte cytoskeleton in mediating parasite attachment/entry into host enterocytes and in regulating host cell transport function.
