This was designed to improve the understanding of the pathogenesis of alpha-hemolysin (hly)-producing E. coli infections commonly seen in surgical and critically ill patients. The applicant hypothesizes that the in vivo pathogenicity of E. coli hly is predominantly neutrophil-mediated, and that the hemolytic activity is necessary to confer hly-induced alterations in neutrophil function by a mechanism independent of the LPS receptor, CD14, despite the physiologic binding of LPS to hly. Preliminary data demonstrates a hly-induced early mortality (6-8h) in mice with concomitant findings of altered neutrophil activity.
The specific aims of the proposed project are: 1) to determine the role of the neutrophil in the mediation of the early mortality induced by hly-producing strains of E. coli on neutrophil respiratory burst activity, degranulation, phagocytosis, and chemotaxis and to determine if such effects are dependent upon CD14, and 3) to determine the role of the hemolytic activity of hly-induced alterations in neutrophil adhesion and expression of adhesion molecules and to determine if such effects are dependent upon CD14. Neutrophil-depleted monocyte-inhibited, and immunologically intact mice will be inoculated with these isogeneic strains of E. coli producing active hly, no hly, or intact, but hemolytically inactive hly, and examined for mortality, tissue-specific histpathogenesis and tissue myeloperoxidase formation. In vitro, neutrophils will be exposed to these three isogenic strains of E. coli and examined for chemiluminescence, myeloperoxidase formation, superoxide formation, phagocytosis, chemotaxis, adhesion to endothelial cells, and expression of adhesion molecules. Separate groups of neutrophils we will also be pre-exposed to anti-CD14 MAb. Efforts to delineate the pathogenicity of this clinically relevant, virulent bacteria are designed to improve the understanding of the complex host-bacterial interaction in order to develop better therapies in the management of bacterial sepsis and infections.
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| Crabtree, T D; Jin, L; Raymond, D P et al. (2001) Preexposure of murine macrophages to CpG oligonucleotide results in a biphasic tumor necrosis factor alpha response to subsequent lipopolysaccharide challenge. Infect Immun 69:2123-9 |
| Jin, L; Raymond, D P; Crabtree, T D et al. (2000) Enhanced murine macrophage TNF receptor shedding by cytosine-guanine sequences in oligodeoxynucleotides. J Immunol 165:5153-60 |
