The objective of this research program is to investigate the ability of antibody and other immunomodulatory agents to support and augment the activity of the host immune system in order to afford protection during experimental gram-negative bacillary sepsis. A major portion of this program concerns characterization of the ability of antibody directed against gram-negative bacterial lipopolysaccharide (LPS, endotoxin). The core LPS-lipid A portion of endotoxin represents an antigenic determinant common to many common gram-negative microorganisms. This region of LPS is expressed extensively on the cell surface of rough mutants of Escherichia coli and Salmonella minnesota. These organisms or their derived outer membrane LPS thus represent suitable immunogens for cross-reactive antibody production. Although murine monoclonal antibodies directed against the core LPS-lipid A region have been demonstrated to protect in vivo, the exact binding site specificity and antibody class which will maximize protection has not been established. Thus IgG and IgM monoclonal antibodies of similar binding specificity, and monoclonal and polyclonal preparations have not been directly compared. The major thrust of this work will therefore be to develop and test both monoclonal and polyclonal antibody preparations which bind to different portions of LPS. Different classes of monoclonal antibodies with similar binding specificity will be developed. These antibody preparations will be tested in vitro for: (1) antibody titer (enzyme-linked immunosorbent assay), (2) binding specificity (Dot blot and Western blot analysis, immunofluorescent bacterial binding), and opsonophagocytic character (acridine orange slide phagocytosis assay) Initial in vivo testing will establish protective capacity by exact LD50 calculations in three murine sepsis models: (1) iv bacteremia, (2) ip hemoglobin peritonitis, and (3) iv endotoxemia. Further in vivo testing will center around maximizing the protective effect of suitable antibody preparations. The ability of antibody to promote survival as a therapeutic agent administered after the bacterial challenge, either alone or in conjunction with antimicrobial agents, steroids, and the immunoenhancing agent muramyl dipeptide will be examined in the above models and a chronic peritonitis-sepsis model.
Showing the most recent 10 out of 17 publications
