Biomedical subjects
Publications and source records attributed to G B Pier.
We studied the effects of in vitro and in vivo coating of catheters with human blood proteins on binding of coagulase-negative staphylococci. Coating resulted in no enhancement of binding. Catheters coated in vitro bound fewer organisms than uncoated catheters. Host proteins do not enhance adherence of coagulase-negative staphylococci to biomaterials.
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BACKGROUND: Staphylococcus epidermidis is the principal pathogen in prosthetic valve endocarditis. The capsular polysaccharide adhesin (PS/A) has been shown to mediate attachment of bacteria to medical devices. In this study, we investigated the efficacy of active and passive immunization against PS/A in preventing S. epidermidis endocarditis in a rabbit model. METHODS AND RESULTS: Aortic valve vegetations were produced by inserting a Teflon catheter into the left ventricle through the right carotid artery. Bacteremia and endocarditis were then established by implanting in the left jugular vein a catheter that was attached to an osmotic pump and contaminated with S. epidermidis strain RP62A. During a 3-week study period, of 64 blood cultures taken every second or third day from six nonimmune rabbits, 54 (84%) yielded strain RP62A. In rabbits actively immunized with PS/A, eight of 60 blood cultures (13%) were positive (odds ratio 5.0, 95% CI, 2.0-12.3, p = 0.005). At death, all six nonimmune rabbits had infected vegetations that yielded 10(6)-10(11) colony-forming units (cfu)/g of vegetation, whereas only one PS/A-immunized rabbit had an infected vegetation. Immunization protocols designed to elicit antibody to teichoic acid but not to PS/A afforded no protection against bacteremia or endocarditis. Infusion of monoclonal antibody to PS/A through a catheter in the right jugular vein provided a level of protection against both bacteremia and endocarditis comparable to that produced by active immunization. In vitro, antibody against PS/A was opsonic for S. epidermidis. CONCLUSIONS: Immunoprophylaxis targeted at staphylococcal PS/A is a promising new approach to the prevention of prosthetic valve endocarditis.
Chronic endobronchial infection with mucoid Pseudomonas aeruginosa accounts for much of the morbidity and mortality in patients with cystic fibrosis (CF). Reduced morbidity is observed when infection is absent. Clinical investigations have implicated opsonizing antibody specific for the mucoid exopolysaccharide (MEP) surrounding these bacteria as a potential immunologic protective mechanism, whereas nonopsonizing antibody to MEP is not protective. Mice and rats immunized with doses of MEP that elicited opsonizing antibody had reduced levels of infection compared with nonimmune controls after intratracheal challenge with mucoid P. aeruginosa enmeshed in agar beads. Doses of MEP that elicited nonopsonizing antibody were not protective. Parallel experiments in which passive transfer of polyclonal and monoclonal opsonizing and nonopsonizing antibody were used yielded similar results. These data indicate that MEP-specific opsonizing antibody can protect against chronic P. aeruginosa infection in this model of disease.
Antibody directed to the O-specific polysaccharide (Ps) side chain of Pseudomonas aeruginosa LPS provides immunotype-specific protection against infection by virtue of enhancing opsonophagocytosis. We have developed a syngeneic anti-idiotypic antibody (mAb2) directed to a functionally active monoclonal immunotype 1 Ps-antibody (mAb1). The mAb2 performed as a molecular mimic of Ps as evidenced by 1) blocking of mAb1/mAb2 interaction by Ps, 2) blocking of mAb1/Ps binding by mAb2, 3) cross-species binding of mAb2 to human Ps antibodies from individuals immunized with the same immunotype 1 Ps, and 4) induction of anti-LPS antibody by immunization with mAb2 in syngeneic mice. Our studies thus show that an anti-idiotypic antibody may functionally mimic the O-polysaccharide of P. aeruginosa LPS, and bind to cross-reactive Id present in human Ps antibodies. We have further shown that this anti-idiotypic antibody induces anti-LPS antibody when used as an Ag in syngeneic mice, suggesting that this approach may eventually be used to successfully immunize humans.
A rabbit model of catheter-related bacteremia was developed to study immunity to the capsular polysaccharide/adhesin (PS/A) of coagulase-negative staphylococci. Catheters colonized by coagulase-negative staphylococci were inserted into the right jugular vein and attached to a subcutaneous osmotic pump, and blood cultures were obtained over 14 days. Nonimmune rabbits were bacteremic for 6-8 days after infection, hypoglycemic, and hyperlipidemic and had strong immune responses to teichoic acid but not to PS/A. PS/A immunization, but not teichoic acid immunization, reduced the number of bacteremic days by approximately 60%, diminished the hypoglycemia and hyperlipidemia, and ablated the immune responses to teichoic acid. Passive infusion of PS/A-specific polyclonal and monoclonal antibodies using a separate, noninfected catheter-pump combination implanted in the left jugular protected against both bacteremia and hematogenous colonization of this contralateral catheter.
Morbidity and mortality in cystic fibrosis (CF) is predominantly due to destruction of pulmonary tissue. The host immune response may, in part, play a pathogenic role in pulmonary destruction in these patients. To further understand host immune response in CF, we examined the state of activation of peripheral blood monocytes in CF. Baseline elastase activity was 2.2-fold greater in the CF monocytes than in controls. Pseudomonas aeruginosa mucoid exopolysaccharide (MEP) and high molecular weight polysaccharide (HMP) increased elastase activity in both control and CF monocytes, with a greater absolute increase in the CF monocytes. There was no difference in baseline or MEP-stimulated secretion of interleukin-1 (IL-1) or interleukin-6 (IL-6) between CF and control monocytes. Ibuprofen enhanced both MEP and HMP-stimulated elastase activity, whereas dexamethasone suppressed both baseline and stimulated elastase activity greater than 20% in both CF and control monocytes. These results suggest that circulating monocytes in CF are stimulated in vivo, resulting in a remarkably elevated elastase activity in vitro. Elevated elastase release by peripheral blood monocytes as they enter the lung in response to chemotactic stimuli may contribute to lung destruction in CF.
Previous studies have shown that antibodies to Pseudomonas aeruginosa mucoid exopolysaccharide (MEP) can be divided into two types on the basis of their functional activity. One type is able to mediate opsonic killing in conjunction with leukocytes and complement, and the other type is not. We investigated, in mice, the properties of this antigen associated with elicitation of opsonic killing antibody. We found that smaller-sized material (Kav = 0.26), which has been tested as a human vaccine, elicited opsonic killing antibody in mice at low doses (1 to 10 micrograms) and at doses of greater than or equal to 40 micrograms, only nonopsonic killing antibody was produced. A similar dose effect was seen with heat-killed mucoid P. aeruginosa cells. After immunization with high doses of this MEP or the heat-killed cells, the mice were refractory to induction of opsonic killing antibody no matter what dose of MEP was used as a booster. In contrast, a larger-molecular-weight preparation (Kav = 0.05) elicited opsonic killing antibody over a wide dose range (1 to 400 micrograms). Additionally, 50 micrograms of the larger-sized preparation could overcome the suppression induced by 50 micrograms of the smaller material. Suppression elicited by 50 micrograms of the smaller material could be adoptively transferred to nonimmune mice with the T-cell fraction of spleen cells. These results indicate that both molecular size and dose are critical determinants for eliciting opsonic killing antibody to mucoid P. aeruginosa after immunizing with MEP.
Previous work has described small molecular weight neutral polysaccharides from isolates of Pseudomonas aeruginosa that appear to be associated with the lipopolysaccharide (LPS) and distributed across serologic barriers defined by antibody to the O side chain. We have isolated and characterized another of these structures obtained from culture supernatants of an immunotype 3 strain of P. aeruginosa. The isolated neutral polysaccharide has a tetrasaccharide repeat unit: (formula; see text) where Rha is rhamnose. The structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments, acid hydrolysis, methylation analysis, Smith degradation, and optical rotation determinations. Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 degrees C, 2 h) LPS from the seven Fisher immunotype strains of P. aeruginosa bound well to the neutral polysaccharide. Antibodies affinity purified from these sera using immobilized neutral polysaccharide as well as a neutral polysaccharide-specific monoclonal antibody, E87, reacted with an antigenically similar structure found among many isolates of different LPS serotypes in a colony blot and with LPS from the seven Fisher immunotypes in an immunoblot. In an immunoblot assay, the neutral polysaccharide inhibited binding of the monoclonal antibody, E87, to material present in LPS preparations from a variety of serotypes. This structure may represent another P. aeruginosa neutral polysaccharide variant found associated with the LPS.
We evaluated the in vitro opsonophagocytic killing activity of monoclonal human immunoglobulin G (IgG), IgM, and IgA specific for Pseudomonas aeruginosa lipopolysaccharide and the in vivo protective capacity in neutropenic mice of both monoclonal and purified polyclonal IgG, IgM, and IgA. Monoclonal IgM was efficacious in mediating opsonophagocytic killing only in conjunction with complement, whereas monoclonal IgG opsonic killing was potentiated by complement, and monoclonal IgA opsonic killing was independent of complement. These findings are similar to those previously reported for purified polyclonal IgM, IgG, and IgA. The monoclonal and polyclonal immunoglobulins had comparable 50% protective doses in neutropenic mice (range, 0.28 to 0.46 microgram per mouse). The protective activity of IgM in neutropenic mice was abolished by cobra venom factor treatment, whereas IgG and IgA maintained efficacy in cobra venom factor-treated mice. These data indicate that all three major human serum immunoglobulin isotypes have opsonophagocytic and protective activities against P. aeruginosa, with a critical role for complement in the function of IgM.
A high-molecular-weight, immunogenic form of the lipopolysaccharide O side chain of Pseudomonas aeruginosa Fisher immunotype 4 (type 4, International Antigenic Typing System 1, Lanyi O:6) was isolated and characterized. Analysis by nuclear magnetic resonance spectroscopy confirmed the structural similarity of this high-molecular-weight polysaccharide and the type 4 O side chain. The polysaccharide was immunogenic in rabbits and mice, eliciting opsonophagocytic killing antibodies. Immunization with the polysaccharide produced significant protection against homologous challenge in both burned and granulocytopenic mice. Naturally acquired opsonic killing antibodies to type 4 polysaccharide were present in sera from unimmunized normal adults at levels comparable to postimmunization levels achieved after immunization with other type-specific polysaccharides. The specificity of the naturally occurring antibodies for the O side chain was documented by immunoblot analysis and inhibition studies. Naturally occurring polysaccharide-specific antibodies were comparable in their protective activity against live challenge in neutropenic animals to immunization-induced murine antibodies with similar specificity. These data suggest that naturally occurring serum antibody to P. aeruginosa type 4 lipopolysaccharide O side chains in most adults is not distinguishable in quantity or quality from immunization-induced antibodies in mice; evaluation of type 4-specific vaccines in humans may be complicated by this finding.
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We previously demonstrated that immunization with low (10 micrograms) doses of high m.w. polysaccharide from the gram-negative bacterium Pseudomonas aeruginosa generates T cells that suppress the ability of antibacterial T cells (Tab) to protect against bacterial infection. The current studies indicate that Ts cells with properties identical to those elicited by low dose polysaccharide immunization can be generated by immunization with Tab. Tab-elicited Ts cells can abrogate in vivo induction and in vitro and in vivo expression of antibacterial T cell activity. Tab-elicited Ts are Ag-specific and H-2 restricted in their suppressor activity. Non-immune T cells fail to elicit suppressor activity. These studies provide additional evidence that the protective T cell response to P. aeruginosa is controlled by a network of T cells that are probably recognizing idiotypic determinants on P. aeruginosa-immune T and B cells.
A neutral small molecular mass (approximately 6.5 kDa) polysaccharide comprising a pentasaccharide repeat unit was isolated from culture supernatants of Pseudomonas aeruginosa immunotype 4. The polysaccharide had a pentasaccharide repeating unit as follows (formula; see text) where Rha is rhamnose. The structure was determined using acid hydrolysis, solvolysis with anhydrous hydrogen fluoride, methylation analysis, and 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments. The polysaccharide bound antibody raised to the lipopolysaccharide of the seven P. aeruginosa Fisher-Devlin immunotype strains. Inhibition assays demonstrated the presence of a serologically similar polysaccharide in supernatants of these strains. Affinity-purified antibody to the polysaccharide bound to lipopolysaccharide and whole cells of the immunotype strains of P. aeruginosa in a Western immunoblot and colony blot assay, respectively. This polysaccharide seems to contain an antigenic determinant present in the core of the P. aeruginosa lipopolysaccharide or may represent another minor polysaccharide substituent on the lipopolysaccharide in addition to the O side chain.
The cellular interactions involved in the development of T cell-mediated immunity to Pseudomonas aeruginosa have been examined. T cell immunity can be generated by immunizing mice with 10 micrograms of P. aeruginosa polysaccharide (PS) plus the antimitotic agent vinblastine sulfate. Vinblastine is required to inactivate a population of Ts cells generated by immunization with 10 micrograms of PS alone. Immunization with either live bacteria or with higher dose (50 micrograms) of PS without vinblastine also generates T cell immunity; these protocols activate a population of Lyt-1+, 2-, I-J+ T cells which, like vinblastine, counteract the effect of Ts cells. Immunization with 10 micrograms PS alone fails to activate this T cell subpopulation. When administered at the time of immunization, this subpopulation can render the tolerogenic 10-micrograms immunization protocols immunogenic. Like previously described contrasuppressor T cells, this T cell subpopulation exhibits an affinity for the lectin Vicia villosa. We have determined, however, that the T cells that act as contrasuppressor cells in this system are directly activated by PS-immune B cells and not by PS Ag. Furthermore, their activity can be removed by adsorption to PS-specific B cell hybridomas. Our studies indicate an important role for B cells in the development of T cell immunity to P. aeruginosa and suggest that a complex idiotype network controls the development of this response.
We isolated a polysaccharide adhesin from Staphylococcus epidermidis strain RP-62A. The adhesin was composed of a complex mix of monosaccharides (with galactose and glucosamine predominating), bound well to silastic catheter tubing, inhibited adherence of strain RP-62A to catheters, and elicited antibodies that both blocked adherence and stabilized an extracellular structure (visualized by transmission electron microscopy) that appeared to be a capsule. Two of three heterologous, highly adherent strains of coagulase-negative staphylococci also produced this adhesin, and their adherence to catheters was inhibited by both purified adhesin and antibody to adhesin. In contrast, the adherence of one highly adherent and two poorly adherent heterologous strains was unaffected by the RP-62A purified adhesin or antibody, a result suggesting the expression of alternate adhesins by these strains. We conclude that the capsular polysaccharide of some strains of coagulase-negative staphylococci is an important factor in adherence to catheter tubing.