Strains PA149 and PA324 are clinical isolates obtained early in the course of infection from patients with CF

Strains PA149 and PA324 are clinical isolates obtained early in the course of infection from patients with CF. titers of antibody to antigens, including whole cells, purified lipopolysaccharide (LPS), and pili. Inhibition studies showed that adsorption Mutant IDH1-IN-4 of an antiserum to asialo-GM1 with cells could remove the reactivity of antibodies to asialo-GM1, and adsorption of this serum with asialo-GM1 removed antibody binding to LPS. Antibodies in sera raised to asialo-GM1 were observed to bind to cells by immunoelectron microscopy. Antibodies to asialo-GM1 inhibited formation of a biofilm by in the absence of mammalian cells, indicating a direct inhibition of bacterial cell-cell interactions. These findings demonstrate that asialo-GM1 is not a major cellular receptor for clinical isolates of and that commercially available antibodies raised to this antigen contain high titers of antibody to multiple antigens, which do not interfere with the binding of to mammalian cells but possibly interfere with the binding of cells to each other. Interactions of bacterial cells with host tissues initiates many processes, including the anchoring of microbes to host cells and extracellular matrices, the activation of innate host immune responses, and changes in gene expression in both the microbial and host cell (15, 25, 33, 48). A large array of adhesins for host mammalian receptors have been described for many bacterial species. Among the gram-negative bacteria, pili and flagella often play a prominent role in anchoring bacterial cells to host tissues (1, 45, 48). For present Mouse monoclonal to Neuropilin and tolloid-like protein 1 on murine and bovine corneal epithelial cells (16, 20, 47); others have disputed whether asialo-GM1 is expressed in the human cornea (52). Some of these studies confirmed that asialo-GM1 is a receptor for binding by using purified glycolipid to inhibit binding (24, 47) or commercially prepared antisera to this antigen (7, 9, 10, 20, 24). Finally, a role has been proposed for a possible neuraminidase in generating asialo-GM1 tetrasaccharide from the Mutant IDH1-IN-4 parental sialylated GM1 molecule (3, 9), although to date the only evidence for a gene that encodes a neuraminidase Mutant IDH1-IN-4 is the recent identification of a DNA sequence in PAO1 that has some homology to other bacterial neuraminidases (GenBank accession no. “type”:”entrez-protein”,”attrs”:”text”:”AAF60322″,”term_id”:”13027829″,”term_text”:”AAF60322″AAF60322). Although the reports noted above suggest a strong case for the involvement of asialo-GM1 as a receptor for on mammalian cells, careful scrutiny of these studies indicates that their general applicability to this host-pathogen interaction may be limited. Few of the studies used clinical isolates of (30); most used well-characterized laboratory strains such as PAO1, PAK, ATCC 19660, and PA103 (7, 9, 10, 21, 24). Only two studies presented evidence that purified asialo-GM1 ganglioside, or the purified tetrasaccharide, could inhibit the adherence of to cells (24, 47). Furthermore, Imundo et al. (24) found a very high concentration of the asialo-GM1 ganglioside (25 mM) or tetrasaccharide (250 M) was needed to inhibit binding to CF bronchial cells by only 57 to 75%, and Singh et al. (47) noted only a transient decrease in binding of to unwounded cornea after premixing the bacteria with asialo-GM1. In the Singh et al. study, monosialoganglioside (GM1), which is not considered a major receptor for binding to cells (24). Also, Davies et al. (9) could not inhibit binding of to CF epithelial cells with the asialo-GM1 tetrasaccharide. Additional concerns center on the use of commercially prepared polyclonal antibodies to asialo-GM1 in these studies. Although numerous investigators have found these antibodies to be effective at inhibiting the binding of to cells (2, 7, 9, 10, 22), essentially none of the studies confirmed the specificity of the antibodies by blocking the biologic activity of the antibodies with appropriate adsorbing or inhibiting Mutant IDH1-IN-4 reagents to demonstrate the specificity of the antibodies to asialo-GM1. Of even greater concern is Mutant IDH1-IN-4 that these polyclonal antibodies are raised in rabbits to essentially a self-antigen purified from bovine tissues emulsified in methylated bovine serum albumin (BSA) and complete Freund’s adjuvant. Such antisera would contain high levels of antibodies that would.