Candidate and Environment: The candidate, Dr. Bloom completed his G.I. fellowship and is now a junior faculty member of the G.I. Division at the University of Maryland. With the support of an NRSA, he studied attenuated Shigella vaccine constructs as vectors for foreign antigen delivery in an animal model. This work provided basic experience in molecular genetics. The candidate now seeks to become expert in these techniques under the supervision of Dr. James B. Kaper of the Center for Vaccine Development (CVD), the co-sponsor of this proposal and an authority in microbial molecular genetics. Because of his interest in hemolytic uremic syndrome (HUS) stemming from his documentation of an outbreak of Shigella dysenteriae in Souther Africa, Dr. Bloom will focus his attention on an animal model of hemorrhagic colitis developed by the co- sponsor, Dr. Edgar C. Boedeker of the G.I. Division and CVD. UNDER Drs. Boedeker and Kaper, the candidate hopes to acquire the molecular tools to investigate the pathogenesis of hemorrhagic colitis due to Shiga toxin producing E. coli (STEC) and to further develop his career as an independent investigator in the outstanding research environment of the CVD. Research: The broad aim of this proposal is to use a new animal model of STEC infection to understand the molecular pathogenesis of this disease. This approach should aid in the development of therapeutic regimens to prevent and treat STEC disease. Over the past decade STEC have emerged as important pathogens, causing life threatening food-borne illness with numerous reports of hemorrhagic colitis, often complicated by the HUS, occurring in sporadic and epidemic outbreaks throughout the world. STEC produce potent protein toxins named Shiga toxins (Stx). In addition to Stx production, STEC share the ability to adhere intimately to intestinal epithelial cells by """"""""attaching and effacing"""""""" (A/E) mechanisms. The most severe intestinal and renal manifestations of STEC infection result from toxin-mediated damage to microvascular endothelium, with tissue edema, inflammatory infiltrates, cytokine production, and vascular thrombi. Endotoxin and pro-inflammatory cytokines up-regulate Stx mediated tissue injury in vitro, but these effects have not been studied in vivo. Furthermore, A/E adherence of bacteria to intestinal epithelial cells, which is encoded for in the genetic locus of enterocyte effacement (LEE) may have a profound influence on the effectively delivery of toxin to the host.
Specific aims of the proposal are to use an animal model of STEC infection to: 1. Examine the influence of A/E adherence on Stx toxicity by: a. producing a deletion mutation in Tir (the translocated intimin receptor) a critical virulence gene of the LEE, in the toxin-producing strain RDEC-H19A; b. using the products of the genes of the LEE, intimin and TIR, to actively immunize against STEC using a vaccine vector system developed for use in the rabbit model by the candidate. 2. Examine the sequential steps in the initiation and maintenance of inflammation, and the induction of vascular injury, by ut8ilizing a selected group of antagonists in the in vivo model including: a. the cytokine IL-11 which has effects on the maintenance of intestinal epithelial barrier function as well as inhibitory effects on macrophage activation; b. intraluminal and systemic endotoxin antagonists: i. Neutrophil BPI (bactericidal/permeability increasing); and ii. Limulus ENP (endotoxin neutralizing protein); c. anti IL- 8 antibody to study the acute inflammatory effects of this chemokine; d. platelet activator factor (PAF) antagonists which affect platelet aggregation and acute inflammation.
