Immunization using a purified protein antigen from P. gingivalis inhibits alveolar bone destruction in a ligature-induced periodontitis model in M. fascicularis.
ActinobaciUus actinomycetemcomitans has been closely associated with early-onset, severe periodontitis, and such patients often have serum immunoglobulin G (IgG) antibodies reactive with antigens of this gram-negative pathogen. We examined the functionality and potential importance of these antibodies. The opsonic activity against A. actinomycetemcomians of sera from 30 patients with rapidly progressive periodontitis (RPP) and from 28 periodontally normal subjects was tested by using polymorphonuclear leukocyte (PMN) chemiluminescence and bactericidal assays. Peak chemiluminescence values correlated strongly with kllUing observed in the PMN-dependent bactericidal assay (r = 0.88; P < 0.001). Neither the mean IgG titer nor the mean peak chemiluminescence differed significantly between the two groups. However, when the relationship between chemiluminescence and titer was examined, regression analysis showed that antibodies present in low-titer normal sera were significantly more effective at opsonizing A. actinomycetemcomians than antibodies present in low-titer RPP patient sera (P = 0.04). Thus, periodontally normal individuals may be better able than RPP patients to dear A. actinomycetemcomitans in early stages of colonization, and anti-A. actinomycetemcomitans antibodies in RPP patients may be relatively ineffective in preventing infection by this organism.
These findings suggest that a small proportion (20-30%) of individuals in each sub-group are more predisposed to TLR activity, which may be related to bacterial composition, diversity and abundance in the sinuses.
Analogs of 3-deoxy-D-manno-octulosonate (KDO) were designed to inhibit CTP:CMP-KDO cytidylyltransferase (CMP-KDO synthetase). Since these analogs lacked whole-cell antibacterial activity, a permeabilized-cell method was developed to measure intracellular compound activity directly. The method employed a mutant of Salmonella typhimurium defective in KDO-8-phosphate synthetase (kdsA), which accumulated lipid A precursor at 42°C. Cells permeabilized with 1% toluene were used to evaluate inhibitor effect on 133H]KDO incorporation into preformed lipid A precursor. KDO incorporation proceeded through the enzymes CMP-KDO synthetase and CMP-KDO:lipid A KDO transferase. Optimum KDO incorporation occurred between pH 8 and 9 and required CTP, prior lipid A precursor accumulation, and a functional kdsB gene product, CMP-KDO synthetase. The apparent Km for KDO in this coupled system at pH 7.6 was 1.38 mM. The reaction products isolated and characterized contained 1 and 2 KDO residues per lipid A precursor molecule. Several KDO analogs produced concentration-related reductions of KDO incorporation in toluenized cells with 50% inhibitory concentrations comparable to those obtained in purified CMP-KDO synthetase systems. Two compounds, 8-amino-2-deoxy-KDO (A-60478) and 8-aminomethyl-2-deoxy-KDO (A-60821), competitively inhibited KDO incorporation, displaying K,s of 4.2 ,uM for A-60478 and 2.5 ,uM for A-60821. These data indicated that the inactivity of the KDO analogs on intact bacteria was the result of poor permeation into cells rather than intracellular inactivation.The lipopolysaccharide (LPS) of gram-negative bacteria contains 3-deoxy-D-manno-octulosonate (KDO), which links lipid A with the polysaccharide chain (6). The pathway for LPS biosynthesis has been studied through the isolation of mutants defective in KDO biosynthesis. One such mutant of Salmonella typhimurium contains a temperature-sensitive defect in the kdsA gene product, KDO-8-phosphate (KDO-8-P) synthetase, which results in the accumulation of lipid A precursor lacking KDO and ester-linked lauric and myristic fatty acid residues when cells are shifted to the nonpermissive temperature of 42°C (12, 13, 28, 30). Lipid A precursor accumulated in the inner membrane, and this was proposed to result in growth stasis (13,22,30 sensitive mutant in CMP-KDO synthetase]) were used in this study. Strains were constructed after insertion of a TnJO transposon near the kdsA and kdsB genes by methods reported previously (8). The genes were then transduced into RG111 by using phage P22. Transductants were selected for tetracycline resistance and screened for temperaturesensitive kdsA and kdsB alleles. Transduced strains were confirmed by enzymatic analysis of and CMP-KDO synthetase (7) isolated after growth at 30°C and after the shift to 42°C. Lipid A precursor accumulation in both strains at 42°C was verified by thin-layer chromatography on Silica Gel H as previously described (25 Isolation and characterization of reaction products. Immediately after a shift to 42°C, a 400-m...
Actinobacillus actinomycetemcomitans has been associated with early-onset periodontitis, including the localized juvenile and rapidly progressive forms. The immunodominant antigens of A. actinomycetemcomitans recognized by rapidly progressive periodontitis patients remain unidentified. Sera from 22 patients with rapidly progressive periodontitis and 20 periodontally normal subjects were tested by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G antibodies to whole-cell sonicate, protein, purified lipopolysaccharide and lipopolysaccharide fractions of A. actinomycetemcomitans. The median titers of rapidly progressive periodontitis patients and control subjects to whole-cell sonicate were 25.0 and 14.5 ELISA units, respectively (not significantly different). Binding of antibody from patient sera occurred to both the lipopolysaccharide and the protein fractions, with greater binding to lipopolysaccharide than to protein. We show for the first time that patient sera contain antibodies that bind specifically to antigenic epitopes in lipid A and in the core carbohydrate of lipopolysaccharide that were previously considered to be inaccessible and unavailable, as well as to epitopes in the O side chains. Sera manifesting antibody titers 2-fold or greater than the median titer for control sera were judged to be seropositive. More patients were seropositive for lipid A than for any of the other antigen preparations studied, and the median titer for patient sera to lipid A but to none of the other purified lipopolysaccharide fractions was significantly elevated relative to control values. Of 22 patients, 10 were seropositive to whole-cell sonicate, 7 to protein, 8 to lipopolysaccharide, 7 to the high-molecular-weight lipopolysaccharide-polysaccharide fraction rich in O side chains, and 16 to lipid A. The core carbohydrate did not adhere to the test plate surface, and this precluded ELISA measurements. However, when the core carbohydrate was used in the ELISA inhibition assay, it reduced antibody binding to lipopolysaccharide-coated plates by up to 45%, thereby demonstrating antibody binding to core carbohydrate. The core carbohydrate fraction from the Re mutant of Salmonella minnesota known to contain no O-side chains also inhibited binding of specific antibody to plates coated with A actinomycetemcomitans lipopolysaccharide. Overall, there was extreme variation in responses among patients to the various antigen preparations, with no single pattern dominating. Lipopolysaccharide and its components appear to be the immunodominant epitopes, since most rapidly progressive periodontitis patients are seropositive for lipopolysaccharide and/or its components and they have titers relative to those for proteins.
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