Critical to understanding the pathogenesis of disseminated candi- diasis is elucidation of the events of escape of Candida from the intravascular compartment and its penetration through vessel walls during invasion of target organs. This process occurs in a milieu rich in circulating humoral and cellular elements and across the boundary of the vascular endothelial cells. During the last grant period, utilizing a model of the vascular compartment-endothelial cell interface consisting of human vascular endothelial cells in tissue culture, we found evidence for a ligand-dependent interaction in adherence of Candida to endothelium, made a polyclonal antiserum that blocks adherence, and showed, for the first time, a protective role of leukocytes for endothelium during Candida tissue penetration. Additionally, we developed an ELISA for quantifying endothelial cell adherence, an elutriator system for adding additional dimensions to monitoring neutrophil and endothelial cell secretory physiological responses, and methods to focus on facets of Candida physiology such as cell wall composition, cell morphology, and secretory products likely to be important in adherence and penetration of endothelium. Using this background knowledge and the newer methods we developed during the previous funding period, we will characterize the nature of the adhesin ligand using immunoprecipitation of cell wall adhesins with antiserum that blocks adherence and with biochemically extracted cell wall constituents, elucidate mechanisms of Candida penetration of endothelium with selected cerulenin resistant, germ tube negative, and protease negative mutants, determine mechanisms by which neutrophils protect endothelium from Candida damage, characterize chemotactic factors secreted by endothelium, and determine whether endothelial cells enhance circulating neutrophil-Candida phagocytosis and killing through secretion of cytokines and/or contiguous contact with neutrophils.
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