Treatment of log phase cultures of Escherichia coli with cell wall active antibiotics results in increased exposure of immunologically reactive lipid A epitopes of lipopolysaccharide (LPS) and release of soluble LPS into culture supernatants. Comparison of the efficacy of two cell wall active antibiotics, ceftazidime, a penicillin-binding protein 3 selective antibiotic, and imipenem, a penicillin-binding protein 2 selective antibiotic, for their relative efficacy in mediating LPS release indicated quantitative but not qualitative differences, with the former antibiotic manifesting a significantly broader range of concentrations at which LPS release could be demonstrated. Comparison of the relative efficacy of these two antibiotics in a mouse bacteraemia model in which animals were made hypersensitive to the lethal effects of endotoxin by treatment with D-galactosamine indicated that the latter antibiotic may provide a greater level of protection. These studies suggest that the release of endotoxin mediated by antibiotic treatment may contribute to the pathogenesis of disease in infectious due to gram-negative organisms.
Lipopolysaccharide (LPS), purified from gram-negative bacteria, is well known to induce proinflammatory responses in monocytes and macrophages, and release of LPS from the microbial surface has been suggested to be an important initiating event in the sepsis syndrome. However, numerous studies have documented that a variety of constituents present in the outer cell membrane of gram-negative bacteria have the capacity to activate cells of the immune system. Given that the majority of immunotherapeutic approaches designed to intervene in gram-negative sepsis have to date targeted the LPS molecule, it would be of value to assess the relative proinflammatory properties of LPS and other gram-negative structures. Experiments were therefore undertaken to assess stimulation of human monocytes by components released from Escherichia coil following bacteriolysis by the cell wall-active antibiotic ceftazidime. As assessed by both induction of procoagulant activity and release of tumor necrosis factor, bacterial culture supernatants contain significant proinflammatory activity. When culture supernatants are fractionated via either velocity sedimentation in sucrose gradients or isopycnic density gradient ultracentrifugation in cesium chloride, the predominant monocyte-stimulating activity is identified in LPS-containing fractions. Further, such activity can be readily abrogated by the addition of polymyxin B. These results provide support for the hypothesis that LPS may be responsible for the
Specific binding of two monoclonal IgM antibodies previously investigated as therapeutic agents for treating gram-negative septic shock, HA-1A and E5, was assessed with respect to lipid A and lipopolysaccharide (LPS). Both antibodies bound to lipid A; however, binding of HA-1A was significantly greater than that of E5 to LPS derived from rough strains of bacteria. Reciprocal competitive inhibition experiments supported the concept that HA-1A and E5 bind to distinct epitopes on lipid A. Further, competitive inhibition studies using a monoclonal anti-idiotype antibody with specificity for the variable region of HA-1A suggested that HA-1A and E5 do not share a common idiotype. Finally, studies using double-stranded DNA as antigen indicated that E5 but not HA-1A will bind to DNA. Collectively, these data indicate that HA-1A and E5 are different lipid A-specific antibodies that bind to distinct epitopes on lipid A.
The human immunoglobulin M monoclonal antibody HA-1A was first described as an antibody which bound specifically to the lipid A region of lipopolysaccharide (LPS) (N. N. H. Teng, H. S. Kaplan, J. M. Herbert, C. Moore, H. Douglas, A. Wunderlich, and A. Braude, Proc. Natl. Acad. Sci. USA 82:1790-1794, 1985) and provided significant protection when administered to patients with gram-negative bacteremia and shock (E. J. Ziegler, C. J. Fisher, Jr., C. L. Sprung, R. C. Straube, J. C. Sadoff, G. E. Foulke, C. H. Wortel, M. P. Fink, R. P. Dellinger, N. N. H. Teng, I. E. Allen, H. J. Berger, G. L. Knatterud, A. F. LoBuglio, C. R. Smith, and the HA-1A Sepsis Study Group, New Engl. J. Med. 324:429-436, 1992). Since that original report, questions have arisen in the scientific literature concerning the specificity of this antibody in LPS and/or lipid A binding. Experiments have, therefore, been carried out with a variety of assay formats to determine the capacity of this HA-1A antibody to bind to lipid A and LPS. Direct binding experiments with a sensitive enzyme-linked immunosorbent assay (ELISA) system have established that HA-1A will bind to purified lipid A from both Escherichia coli and Salmonella spp. These results have been confirmed by using a fluid-phase antigen-antibody competitive inhibition assay with purified lipid A and an antibody-antibody competitive inhibition assay with a monoclonal antibody with known specificity for lipid A. The HA-1A monoclonal antibody has also been shown to bind to a panel of R-chemotype LPS by ELISA and, unlike many other previously reported anti-lipid A antibodies, binding of HA-1A to R-chemotype LPS and lipid A is comparable. Although binding of HA-1A to S-LPS (smooth, wild-type LPS) could not be detected by direct ELISA, competitive inhibition experiments with some preparations of S-LPS have been able to show specific HA-1A binding. Collectively, these data confirm the binding specificity of HA-1A for the lipid A component of LPS and provide evidence that this monoclonal antibody manifests a relatively uncommon profile in its capacity to bind lipid A and R-chemotype LPS as well as some preparations of S-LPS.
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