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Project Title:
Pathogenesis of Experimental Necrotizing Enterocolitis
Funding Agency:
National Institutes of Health
Total Project Period:
Dec 01, 2001 - Jan 31, 2005
Principal Investigator:
Mitchell Fink, MD
Co-Investigator(s):
Henri R. Ford, MD (Project Director); Rosemary A. Hoffman, Ph.D.; Ronald Jaffe
MB, BCh; Ruben Zamora, PhD; Timothy Billiar, MD; Simon C. Watkins, PhD; Seth J. Corey, MD, MPH; Ian J Reynolds, PhD; Anthony J Bauer, PhD
Project Summary:
Necrotizing enterocolitis (NEC) is the most frequent and the most lethal disease that affects the gastrointestinal tract of the premature infant. The exact etiology of the disease is undefined. The only consistent identifiable epidemiologic precursors for NEC are prematurity and enteral alimentation. We have previously shown up-regulation of inducible nitric oxide (NO) synthase (NOS-2) mRNA and protein in intestinal segments from infants with acute NEC. NOS-2 co-localized with enterocyte apoptosis and nitrotyrosine immunoreactivity. NOS-2 was down regulated at the time of intestinal stoma closure when the acute inflammation had subsided. We have developed a reproducible model of gut inflammation in neonatal rats that mimics many of the morphological changes characteristic of human NEC. We simply formula-feed hypoxic neonatal rats thereby simulating the conditions associated with human NEC. These pups show
NOS-2 mRNA up-regulation, nitrosative stress, increased enterocyte apoptosis and decreased enterocyte proliferation in the crypts. Intraepithehal lymphocytes (IEL) from hypoxic formula-fed rats secrete more TNF-D and IFN-D compared to breast-fed rats. In vitro studies suggest that co-culture of IEL with the rat intestinal epithelial cell line IEC-18 in the presence of IL-lp induces IFN-y, TNF-a and NOS-2 production that can be abrogated with antibody to IFN-y, furthermore, peroxynitrite (ONOO) induces apoptosis and inhibits proliferation ofIEC-6 cells. The data suggests that intestinal necrosis in NEC may be the result of NO-induced imbalance between tissue injury and repair mechanisms. Mucosal injury resulting from perinatal insults leads to bacterial-epithelial interactions, local release of cytokines such as [FN-y and TNF-a by IEL and lamina propria (LP) lymphocytes. These mediators induce NOS-2 up-regulation with production of NO and ONOO by enterocytes or LP macrophages. NO or ONOO in turn promotes further tissue injury enterocyte apoptosis) and concurrent inhibition of tissue repair mechanisms (enterocyte proliferation) leading to gut earner failure and NEC.
To test our hypothesis, we propose the following Aims: I) To define the mechanism of NOS-2upregulation in human and experimental rodent NEC. II) To define the mechanisms by which NOS-2 up-regulation promotes intestinal epithelial injury and alters tissue repair mechanisms in rodent NEC. Ill) To determine the effects of scavengers of NO or inhibitors of NO production on the development of experimental rodent NEC.
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