Anti-Gal is a natural human IgG antibody that is constantly produced to high titer throughout life in response to immune stimulation by human gut flora. We have found that this abundant and ubiquitous antibody binds significantly more often to Gram-negative organisms that cause sepsis than to Enterobacteriacae recovered from normal stool cultures. In addition, anti-Gal blocks the alternative complement pathway (ACP) lysis of a representative Serratia marcescens #21 human blood isolate by binding to its lipopolysaccharide (LPS) and is responsible for its resistance to lysis by normal human serum. The same anti-Gal antibody however has a modest, but significant, promoting effect on the ACP- mediated lysis of another S. marcescens human blood isolate (Serratia #7) when it binds to this organism's capsular polysaccharide (K antigen). Anti-Gal does not alter the number of molecules of C3b deposited on either Serratia. In addition, it does not change the site of binding of C3b to Serratia #21 LPS nor does it modify the molecular form of the C3 fragments bound (mostly iC3b, with small amounts of C3b and C3d) or change C9 deposition onto this organism. Yet Serratia #21 is lysed by sera depleted of anti-Gal. It follows that the generation of an effective MAC depends on the exact nature of the carbohydrate to which C3b has bound and that the many OH residues that are available on a polysaccharide are not equivalent in their ability to foster a stable MAC. This research seeks to study the chemical nature of the C3b binding sites on Serratia #21 LPS in the presence and absence of anti-Gal, in an effort to define the chemical structures of promoters and demoters of complement activation on bacterial outer membranes. This will be done by chemical and enzymatic modification of the polysaccharide, by compositional and by linkage analysis, and lastly by purifying the C3 fragment- oligosaccharide complexes and identifying the carbohydrates to which the C3 fragment is bound in the presence and absence of anti-Gal. We will also investigate whether the effects of anti-Gal binding are functions of the antibody or of its binding sites and find out how anti-Gal affects the stability of C9 and the MAC. In addition, we will study whether anti-Gal affects PMN uptake or killing of target enteric bacteria when it binds to LPS or K antigens, and examine how naturally-occurring anti- Gal differs from anti-Gal synthesized in response to immunization or infection with regards to its ability to activate complement and promote opsonophagocytosis. These goals should provide important information on the role of this abundant antibody in host defense against Gram-negative bacterial infections. Since many sepsis-associated enterics bind anti- Gal, active and passive immunization to prevent sepsis may well be directed in part at anti-Gal epitopes. As we begin using passive immunization clinically, the need to answer these questions becomes more urgent.
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