The long-term goals of these studies are to define the cellular mechanisms of host-pathogen interaction in infectious enteritis and identify rational approaches to nutritional and pharmacologic enhancement of epithelial defense and repair. Our preliminary studies have shown that inducible nitric oxide synthase (iNOS) is a key mediator of epithelial defense in C. parvum infection. Nitric oxide synthesis is increased in infected piglets and arises from induction of iNOS mRNA and protein expression by infected epithelial cells and lamina propria at the apical villi. Treatment of infected piglets in vivo with an iNOS-selective inhibitor (L-NIL), abolishes the increase in NO synthesis arising from infection and significantly worsens epithelial parasitism. Our Central Hypothesis is that the induced transport of extracellular L-arginine plays a key role in epithelial cell defense against C. parvum infection. Specifically, in response to C. parvum infection, the subepithelium mediates induction of epithelial iNOS and the L-arginine transporter, CAT-2B. CAT-2B-mediated uptake of L-arginine results in synthesis of NO by epithelial iNOS, which promotes defense against C. parvum infection. To address this hypothesis we will 1) examine CAT-2B expression and characterize ARG transport by freshly isolated epithelium and Ussing-chambered ileaf mucosa from C. parvum -infected piglets, 2)examine the specific roles of the epithelium, iNOS and CAT-2B in epithelial defense using porcine epithelial cell monolayers (IPEC-J2) and intact porcine ileal mucosa infected with C. parvum in-vitro and 3) examine whether entera! ARG promotes epithelial defense in an in-vivo model of C. parvum infection. Regulation of CAT expression and the availability of extracellular ARG may be key targets for augmentation in the pathway leading to NO biosynthesis and epithelial defense in C. parvum infection. The PI is presently in the 3rd year of a 5-year K08 from NIDDK. These studies are anticipated to generate key preliminary data for the PI's first R01 application
