We have developed a model of ischemic bowel necrosis, or necrotizing enterocolitis (NEC), in rats and mice by injecting PAF or tumor necrosis factor (TNF) with endotoxin (LPS). We found that synergism between TNF, LPS and PAF is important in causing shock and bowel necrosis. These agents induce their own, and each other's production. The present proposal aims to expand the original pathophysiological studies of experimental NEC to new aspects of biochemistry and molecular biology. We plan to test the following hypotheses: (1) Regulation of PAF forming enzymes (phospholipase A2 and acetyltransferase) and PAF degrading enzyme in the bowel is controlled by LPS, TNF or PAF, whose effects may be additive. (We have shown that these 3 agents induce PAF production. Preliminary results showed that LPS decreases acetylhydrolase activity). PLA2 gene expression will also be examined. (2) LPS, TNF or PAF may induce intestinal PAF receptor expression. (3) PAF synergizes with LPS to induce TNF transcription and production in the bowel. (Preliminary results showed that LPS induces TNF mRNA in gut tissue). (4) PAF induces its own formation via activation of PMNs, complement, and release of other mediators, and possibly, EDRF. (5) LPS is required for development of severe NEC lesions. We will assay tissue PAF, PAF regulating enzymes and PAF receptors, plasma PAF, TNF and LPS, and measure various pathophysiological parameters. The biochemical results will be supplemented by morphological studies: localization of transcription of TNF, PLA2, and PAF receptor by immunohistochemistry and in situ hybridization. We are particularly interested in intestinal macrophages in our model, since these cells are likely to be the source of PAF, TNF, an LTC4, and may be the """"""""missing link"""""""" in PAF-induced complement activation. We plan to deplete macrophages in vivo and examine the effects of LPS, TNF and PAF. In addition, we will isolate gut macrophages and examine their ability to produce these mediators upon stimulation. Lastly, we showed that human patients with NEC have high PAF and TNF levels and low acetylhydrolase activity, underscoring the relevance of these mediators in NEC. We plan to investigate the role of PAF, TNF and LPS in human NEC patients by examination of fecal PAF and LPS, serum LPS, and blood TNF(+) monocytes in early (stage I) NEC. If the results are consistent, these tests could be used for diagnosing early (stage I) NEC. The mortality and morbidity of this disease may be considerably reduced with the possibility of early diagnosis, heretofore unavailable.
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