INTERVENTIONAL STRATEGIES FOR HEMORRHAGIC COLITIS

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The broad aim of this proposal is to use a new animal model of enterohemorrhagic E.coli (EHEC) infection to understand the molecular pathogenesis of EHEC infection and develop practical interventional strategies to prevent and treat EHEC disease. It is now well recognized that some toxin-producing strains of E.coli, most commonly of serotype O157:H7, induce hemorrhagic colitis which may progress to fatal hemolytic-uremic syndrome. EHEC strains produce potent toxins which are referred to as Shiga-like toxins (SLTs) because of their relatedness to shiga toxin of shigella dysenteriae. Most EHEC share the ability to adhere intimately to intestinal epithelial cells by ~attaching and effacing~ (A/E) mechanisms. Although EHEC attachment mechanisms, with loss of microvilli, may contribute directly to diarrhea; the most severe intestinal and renal manifestations result from toxin-mediated damage to vascular endothelium with tissue edema, inflammatory infiltrates, cytokine production and vascular thrombi. A/E adherence may have profound influence on the delivery of toxin to the host, but this has not been adequately studied. At present, there is no effective prophylaxis or accepted treatment for EHEC disease. The specific aims of the proposal are to use an animal model of EHEC infection to: 1. Examine the influence of A/E adherence on SLT toxicity using mutants in the locus of enterocyte effacement (LEE). 2. Test the ability of passively administered immunoglobulin with anti- toxic activity to prevent EHEC disease. 3. Test the ability of intraluminal toxin-receptor analogs to prevent EHEC disease. 4. Determine whether antibiotic therapy has beneficial or harmful effects on the course of disease. 5. Examine the mediators of inflammation and determine whether anti- inflammatory strategies, in particular recombinant IL-1 receptor antagonist (IL-1ra), and recombinant TNF binding protein (TNF-BP) can alter the disease. 6. Examine strategies for active immunization against EHEC using the toxins, and products of the genes in the locus of enterocyte effacement (LEE). E.coli strain RDEC-H19A infection of rabbits will serve as the animal model of EHEC disease for these studies. RDEC-H19A, produced by the Transfer of the toxin-converting phage H19A of an O26:H11 EHEC to the rabbit entero-pathogenic E.coli RDEC-1, is an attaching and effacing rabbit pathogen. This strain produces high levels of Shiga-like toxin I (SLT-I), colonizes cecum and colon, and induces intestinal disease in rabbits with pathologic changes resembling human EHEC disease. Interventions will limit the interaction of SLTs with endothelium by neutralizing toxin within the vascular compartment; binging toxin in the gut lumen; eliminating the toxin-producing organisms; or inhibiting the action of pro-inflammatory cytokines.