Recently there has been increased understanding of the pathogenesis of bacterial enterocolitis and epithelial cell-bacterial interactions. Some enteric pathogens are invasive and disseminate systemically. However, there is no knowledge of subepithelial cellular bacteriocidal mechanisms of the human colon. Our objectives are to study these mechanisms to broaden our understanding of the pathogenesis of these serious infections. The project will: 1) enumerate and identify the phagocytic and bacteriocidal populations of subepithelial human colonic tissues, 2) evaluate chemotactic responses of subepithelial phagocytes in response to prototype pathogenic E. coli and Shigellae compared to avirulent strains, 3) assay phagocytosis and killing of the cellular bacteriocidal activity in subepithelial lymphocytes and phagocytes, 5) quantify Fc and C3b receptors of these populations and determine their relationship to bacteriocidal activity. Organisms to be studied include 1) avirulent E. coli, 2) non-invasive cytotoxic E. coli, 3) invasive E. coli, 4) avirulent Shigellae, 5) invasive pathogenic Shigellae. Subepithelial populations of normal colon to be studied include lymphocytes and phagocytes isolated by EDTA collagenase dissociation and elutriation centrifugation. These techniques provide 95% pure lymphocytes and up to 80% pure phagocytes. Normal tissue will be obtained from patients undergoing colonic resection. Normal control and autologous blood lymphocytes and phagocytes will be studied for comparison. Parallel studies will be conducted with blood and intestinal phagocytes of guinea pigs. Cells will be enumerated by hemocytometer, histochemical stains, and flow cytometry. Chemotactic assays will be conducted in Boyden chambers using bacteria as chemoattractants. Phagocytic assays will be conducted with 3H-and FITC labelled bacteria with scintillation counting and fluorescent flow cytometry respectively. Cellular bacteriocidal activity will be assayed by microbial culture and short term assays. Fc and C3b receptor assays will be conducted using fluorescent cytometric analysis. Bactericidal mechanisms of subepithelial colon populations will be identified. The increased understanding of these mechanisms will offer new opportunities for manipulating host defence against invasive enteric pathogens.
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| Meyer, D; Gump, D; Fabian, J et al. (1991) 140-MDa plasmids of Escherichia coli and Shigella flexneri 5 do not influence phagocytosis. Med Microbiol Immunol 180:143-7 |
