Necrotizing enterocolitis (NEC) is an abrupt and devastating inflammatory necrosis of the bowel affecting 10% of premature infants born weighing less than 1500 grams. Although the etiology of this complex disorder is poorly understood, the evidence suggests that intestinal ischemia, formula feeding, and bacterial challenge activates platelet activating factor (PAF) and other inflammatory mediators leading to intestinal injury. Results from our previous NIH funded project have shown that PAF receptor activation by PAF leads to a series of physiological and/or pathological changes in the intestinal epithelium, including the stimulation of transepithelial electrolyte transport, cytoplasmic acidification, caspase activation and DNA fragmentation. In addition, endogenous bacteria stimulate the inflammatory cascade and are a prerequisite for neonatal NEC. Specific bacterial cell wall products stimulate toll receptors (TLR's) on multiple cell types that result in significant downstream events. In a healthy intestinal environment, the mucosal barrier is poorly responsive to bacterial cell wall products, partly because of low or absent TLR expression. Nonetheless, in the neonate during early development and bacterial colonization, intestinal expression of TLR and regulation of this process is incompletely understood. We hypothesize that during the developmental acquisition of neonatal bacterial colonization with the addition of asphyxia stress, there is abnormal intestinal gene expression of TLR-4, and that PAF contributes to this abnormal upregulation. Following TLR activation, downstream signaling initiates a series of events that culminate in the final common pathway of intestinal necrosis and NEC. Utilizing a novel neonatal mouse model of NEC that will allow testing of genetically-altered animals and cell culture studies that will provide additional mechanistic detail, the research plan is designed to 1) characterize the developmental regulation of intestinal TLR expression and study the importance of TLR in human NEC and the neonatal mouse model, 2) delineate the signaling mechanisms responsible for PAF-induced TLR expression in intestinal epithelial cells from humans and rodents, and 3) investigate the role of PAF on intestinal TLR gene expression and protein synthesis in vivo. The proposed studies will elucidate the interactions between PAF and TLR signaling, and clarify whether these pathways play an underlying role in the initiation of neonatal NEC.
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