Browsing by Subject "Phagocytosis"
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Item Activation of macrophages by peroxidases(Texas Tech University, 1986-05) Lefkowitz, Doris LynneThe role of peroxidase in biological systems is not well understood. The specific aims of the present study were to determine (a) if peroxidases are able to stimulate the production of superoxide by macrophages; (b) if peroxidases are able to activate macrophages to the tumoricidal state; (c) if so, is the cytotoxic triad of peroxidase, HgOg, and a halide ion responsible for promoting such activation; and (d) if simple heme-type compounds can replace peroxidases in promoting such activation. The results obtained demonstrated that various peroxidases (horseradish peroxidase, lactoperoxidase, microperoxidase) can stimulate both the respiratory burst and promote the inhibition of tumor cell growth by macrophages in a dose-dependent manner. Similar results were obtained using an immobilized peroxidase. The addition of iodide did not markedly affect either process, but the addition of a peroxidase substrate caused a significant increase in the tumoricidal activity of the macrophages. Furthermore, the tumoricidal activity of peroxidase-activated macrophages was inhibited in the presence of cytochrome c, indicating a requirement for superoxide. Hemin and hematoheme were unable to stimulate either process in macrophages, indicating that the presence of peroxidative activity is a requirement to obtain a stimulation of either process. These results suggest that enzymatically active peroxidases are able to stimulate the respiratory burst as well as induce tumoricidal activity in thioglycollate-induced peritoneal macrophages. The fact that certain compounds, such as interferon, were able to elicit macrophage-mediated tumor cell inhibition without affecting the respiratory burst, suggests that activation of the respiratory burst may not be a prerequisite for tumor cell inhibition. Furthermore, the activation of macrophages is not promoted by the cytotoxic triad, but rather may be promoted by a product of the peroxidative activity of the peroxidases, possibly a free radical species.Item An electron microscopic, cytochemical study of the in vitro phagocytosis of latex particles by peritoneal phagocytes(Texas Tech University, 1971-08) Burk, Kenneth HNot availableItem Exogenous myeloperoxidase enhances bacterial phagocytosis and intracellular killing by macrophages(Texas Tech University, 1995-12) Lincoln, John AnilIt is well documented that myeloperoxidase (MyPo) contributes to the bactericidal activities of neutrophils and monocytes. Since mature macrophages (M0) are devoid of this enzyme, its participation in M0-mediated phagocytosis and bacterial killing has not been completely defined. The present study demonstrates that exogenously added MyPo, at physiological levels, enhances both phagocytosis and killing of Escherichia coli (E. coli). Murine peritoneal M0 were exposed to various concentrations of MyPo for different time intervals. Viable opsonized E. coli were added either prior to or after addition of MyPo. Thioglycollate-induced (TG) but not resident M0 exhibited an increase in phagocytosis. Both resident and TG-induced M0 demonstrated increased bactericidal activity. Physiological levels of soluble MyPo also induced a significant increase in both TGinduced and resident M0 chemiluminescence (CL). Since luminol-dependent CL measures reactive oxygen intermediates (ROI) production, studies were done to determine whether superoxide anion or H2O2 were involved in MyPo-induced M0 killing. Both superoxide dismutase and catalase significantly reduced MyPo-induced bactericidal activity. The above data suggest that soluble MyPo, released from neutrophils at a site of infection or inflammation, can enhance both phagocytosis and killing of microorganisms.Item Functions of Phosphatidylinositol 4-Phosphate 5 Kinases in Actin Cytoskeletal Regulation during Phagocytosis(2009-06-18) Mao, Yuntao; Yin, Helen L.Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a crucial signaling phosphoinositide at the plasma membrane (PM) which mediates a variety of biochemical activities and cellular functions. It is primarily synthesized by type I phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) through the phosphorylation on the D-5 position of the inositol ring of phatidylinositol 4-phosphate [PI(4)P]. Mammals have three PIP5K isoforms named a, b, and g (human isoform designation) which have a highly conserved central kinase homology domain and divergent amino and carboxyl terminal extensions. There is now extensive evidence suggesting that PIP5Ks have unique functions and regulations in many cellular processes which provide the key to understand how functionally, and possibly physically, segregated PIP2 pools are generated. The actin cytoskeleton is dynamically remodeled during Fcg receptor (FcgR)-mediated phagocytosis in a PIP2-dependent manner. I investigated the role of PIP5Kg and a isoforms, which synthesize PIP2, during phagocytosis. PIP5Kg-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgR clustering. Delivery of exogenous PIP2 rescued these defects. PIP5Kg knockout BMM also have more RhoA and less Rac1 activation and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5Kg by RNA interference (RNAi) inhibits particle attachment. In contrast, PIP5Ka knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein (WASP) activation and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5Kg but not a is transiently activated by spleen tyrosine kinase (Syk)-mediated phosphorylation. I propose that PIP5Kg acts upstream of Rac/Rho and that the differential regulation of PIP5Kg and a allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.Item The LspA Proteins Of Haemophilus Ducreyi: Extracellular Virulence Factors(2007-05-22) Mock, Jason Robert; Hansen, Eric J.Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. This inhibitory activity was previously shown to be abrogated by inactivation of the lspA1 and lspA2 genes of this pathogen. Avoidance of phagocytosis has been suggested to be a primary method by which H. ducreyi persists in humans, and subsequently allows the ulcerative disease process to occur. Like other genital ulcerative diseases, chancroid is associated with an increased risk for acquisition and transmission of the human immunodeficiency virus (HIV), and is an important cofactor in HIV spread in areas where chancroid is prevalent. My studies have focused on the regulation and mechanism of action of the LspA proteins. The regulation studies have demonstrated that transcriptional regulation of these large extracellular virulence factors does occur, and also suggest that other outer membrane proteins may be regulated by a similar method. Work elucidating the mechanism of action of the LspA proteins has identified a decrease in catalytic activity of Src family protein tyrosine kinases in macrophages exposed to wild-type H. ducreyi, leading to a halt in phagocytic cup formation and a subsequent decrease in uptake of opsonized targets. Additional experiments suggest a possible membrane component in immune cells may be responsible for relaying the LspA proteins' inhibitory signal. This is the first example of a bacterial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in host cells.Item Peroxidase-mediated oxygenation and microbicidal activity(Texas Tech University, 1999-05) Vigerust, David JohnIt is well documented that a peroxidase, H2O2, and a halide form a "cytotoxic triad." As a result of the interactions of the components of the triad, reactive oxygen intermediates (ROI) are formed and these ROI help to destroy various invading pathogens including Candida. The present study was undertaken to determine if equivalent units of peroxidase activity also induced equivalent macrophage-mediated killing of Candida. Peritoneal macrophages were obtained from age matched C57BL/6J mice and exposed to various concentrations of eosinophil peroxidase (EPO), myeloperoxidase (MPO), and horseradish peroxidase (HRP). Equivalent units of peroxidase as determined by oxidation of guaiacol, did not induce equivalent production of ROI. Luminol-dependent chemiluminescence studies indicated that 10 units of EPO induced more ROI than either HRP or MPO. Candidicidal activity and phagocytosis of M(t) was measured using a fluorescence acridine orange phagocytosis assay. The following pattern EPO>MPO>HRP emerged for both assays. Therefore, enzymatic activity does not directly correlate with candidicidal activity. These data indicate a distinct order of peroxidases relative to their ability to stimulate chemiluminescence and macrophage-mediated killing.Item Polymannan enhancement of macrophage function(Texas Tech University, 1999-12) Gnade, Bryan ThomasPrevious studies have shown that mannosylated bovine semm albumin (mBSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans and Escherichia coli by resident murine peritoneal macrophages (M0). Upregulation of the above M0 functions was associated with binding of mBSA to the macrophage mannose receptor. The present study was done to detennine if certain polymannans, and other glyconutrients could stimulate M0 functions in a similar manner. Resident peritoneal murine M0 collected from C57BL/6 mice were exposed to the glyconutrients for 10 and 60 minutes. The RB was measured using chemiluminescence. Both phagocytosis and killing were measured after incubation with one of the following microorganisms: Candida albicans, Escherichia coli and S. aureus. The percent phagocytosis and killing were determined using fluorescence microscopy. Results indicated little or no effect of four of these compounds on phagocytosis and killing Compound 3, which was identified as Ambrotose, caused a dose and time dependent effect on M0 induced killing of all 3 microorganisms.Item The elaboration of extracellular capsular polysaccharide by Klebsiella pneumoniae and its relationship to virulence(Texas Tech University, 1983-12) Domenico, PhilipNot available