Phagocytic leukocytes play an important role in the body's defense against pathogenic microorganisms. Directed to a pathogen by chemoattractant factors, these cells engage invading organisms via surface receptors and initiate a phagocytic process mediated by a series of signals that allow the receptor-ligand interaction. Most bacteria are eventually destroyed by creating an environment in the phagosome that optimizes the effects of simultaneously released lytic and oxidative agents, but certain fungi and pathogenic mycobacteria subvert these in an as yet unknown way, so that destruction does not occur. Based on our earlier studied of neutrophils (PMN) and monocyte-derived macrophages (MC/MP), we have hypothesized that pathogens which evade PMN and/or MC/MP killing do so by, for their specific receptor(s), either (a) modifying the initiation of early signals that would eventually lead to lytic and oxidative attacks on normally processed stimuli, (b) mediating the conditions within the phagosome (e.g. pHp), as we have already shown occurs with Cryptococcus neoformans, so that they are unfavorable to lytic enzyme activity and/or oxidative entity (02, O2-, Ocl-, O-, OH.) formation (and, as we have shown for C. neoformans, which is not killed by ? MC/MP until pHp is increased with chloroquine), or (c) causing a disproportionation in the subpopulation responses we have previously demonstrated for at least one of the stimulation mediating classes of receptors, the FcR. We have also now shown that PMN possess a novel Ca2+/H+ channel which counteracts cytoplasmic and phagosomal acidification while MC/MP do not express the channel, perhaps accounting for the differences between PMN and MC/MP processing of some pathogens and the generally higher pHp observed but previously unexplained in PMN. We will study the PMN and MC/MP stimulation by normally killed vs defense-evading stimuli, using immune complexes (IC), """"""""benign"""""""" mycoorganisms such as M. avium and Staph. Epidermidis and then progressing to M. tuberculosis which can be handled in our BL-3 facility which also houses a recently arrived MoFIo flow cytometer. Using fluorescent probes of pH, oxidative burst and lytic enzymes and evaluating on a cell by cell basis, we will study the regulation of PMN and MC/MP destruction by normally attacked vs defense-evading phagocytized stimuli and, where destruction was circumvented, whether evasion can be reversed by altering the phagosomal conditions, thereby permitting design of a counterattack to this evasion of microbicidal and/or bactericidal functions. ? ? ?
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| Bernardo, John; Long, Heidi J; Simons, Elizabeth R (2010) Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis. Cytometry A 77:243-52 |
| Herrmann, Jens Martin; Bernardo, John; Long, Heidi J et al. (2007) Sequential chemotactic and phagocytic activation of human polymorphonuclear neutrophils. Infect Immun 75:3989-98 |
