Research is proposed to: 1) use specifically designed probes to identify the toxins generated by stimulated phagocytes and to monitor their intracellular reaction. 2) characterize their oxidation-reduction chemistry in sufficient detail that their potential role in cellular reactions can be properly evaluated, and 3) investigate the microbicidal mechanisms of putative phagocyte-generated toxins on representative test organisms. The oxidants include HOCl, H2O2, ONO2- and secondary oxidants derived from them. Experiments will use stopped-flow kinetics, esr spin trapping and Raman spectroscopy to identify the reaction products and to characterize their behavior toward biological materials. Recent studies have demonstrated that lethal reactions of very reactive oxidants cannot be accurately assessed by in vitro microbicidal assay systems. Fluorescein-conjugated polyacrylamide beads which are capable of discriminating between chlorine-based oxidants, ONO2- ion, and carbon-based oxidants will be used to probe events in the phagocytic vacuole. The bactericidal mechanisms of these oxidants will be examined by the protocols used for HOCl-inflicted cell damage. The studies will focus on metabolic functions associated with the bacterial plasm membrane. Cellular studies will use control neutrophils, a macrophage cell line, and MPO-deficient and CGD neutrophils.
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