Improvements in surgery, transfusion therapy, emergency and intensive care have generated increasing numbers of patients who initially survive resuscitation from circulatory (hypovolemic and cardiogenic) shock, only to succumb later from the multiple organ failure synidrome (MOF). Having previously defined the hemodynamic mechanism, and a major toxic pathway by which splanchnic organs (intestine, colon, stomach, and liver) are targets for ischemia and reperfusion injury during shock, this project now seeks to better understand the mechanisms by which these injured splanchnic organs influence injury in distant (non-splanchnic) organs in a porcine model of MOF. Specific hypotheses to be evaluated include: a) The intestine and/or pancreas are sources of important circulating toxins, some of which have proteolytic activity. b) The microvascular endothelial cell is an important initial target of these mediators; c) Toxic oxygen metabolites generated by xanthine oxidase (XO), activated by these circulating toxins from xanthine dehydrogenase (XD) on the endothelial cell surface, are the initial trigger of this microvascular injury. This hypothesis is based upon the exciting new finding that enzymatically active and immunoreactive XO is present in high concentration on the outside surface of the EC plasma membrane. d) Neutrophil accumulation in the microvasculature, with its own important toxic consequences, is secondary to this primary endothelial cell injury. In porcine in situ, porcine cross circulation, and ex situ perfused organ (porcine lung and rat liver), and mouse reticuloendothelial function preparations, and in cultured endothelial monolayers, these hypotheses will be tested by probing with proteases (chymotrypsin) antiproteases (soybean trypsin inhibitor), specific antioxidants (superoxide dismutuse and catalase) and xanthine oxidase inhibition both with allopurinol and with a new monoclonal antibody that blocks XO activity. Endothelial XO will be quantitatively distinguished from XD in situ by a new histochemical method, and by MoAb's that distinguish XO from XD. These methods should allow us to evaluate the central hypothesis, that endothelial cell plasma membrane surface xanthine oxidoreductase, via post-translational XD to XO conversion, transduces circulating toxic and inflammatory mediators into the end organ injury that constitutes multiple organ failure.
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