Faculty Bibliography

In vitro studies have demonstrated the antiplaque properties of C31G, a potent broad spectrum antimicrobial agent consisting of an equimolar mixture of alkyl dimethyl glycine and alkyl dimethyl amine oxide, buffered with citric acid. In this initial clinical study, C31G at concentrations of 0.05%, 0.1%, 0.2% and 0.5%. Listerine, and placebo were tested in a complete crossover design. Twelve subjects were evaluated, with a minimum of 2 days between treatments. Parameters monitored were salivary bacterial counts and saliva glycolysis. The 0.5% and 0.2% C31G mouthrinses significantly reduced total bacterial counts in saliva samples obtained up to and including three hours after rinsing, compared with counts obtained prerinsing or after placebo rinsing. Both 0.5%, and 0.2% C31G significantly inhibited glycolysis of salivary bacteria for up to 6 hours postrinsing, compared with pH values obtained prerinsing. 0.1% and 0.05% C31G exhibited little or no effect in either assay. Listerine showed a significant reduction in bacterial counts for up to 1 hour postrinsing, compared with prerinse counts, but the effect was less sustained. Listerine showed no significant inhibition of glycolysis at any time point. No tooth staining or altered taste sensation was noted with either product.

The interaction of a high-molecular-weight salivary glycoprotein (agglutinin) with Streptococcus sanguis M5 leads to the formation of bacterial aggregates. We have previously shown that the SSP-5 surface antigen from S. sanguis M5 binds the salivary agglutinin and therefore may be involved in the aggregation process. Here we report the transformation of a nonaggregating Enterococcus faecalis strain with the SSP-5 gene and show that the protein is expressed on the cell surface and confers an aggregation-positive phenotype. E. faecalis S161 protoplasts were transformed with pAM401 EB-5, a shuttle vector containing the S. sanguis SSP-5 gene, resulting in the isolation of E. faecalis S161EB-5. Crude cell extracts from this transformant and from S. sanguis M5 were analyzed by Western blotting. Extracts from S. sanguis M5 possessed peptides of 190 and 205 kilodaltons that reacted strongly with polyclonal antibodies against the recombinant SSP-5 antigen. E. faecalis S161EB-5 contained only the 190-kilodalton immunoreactive protein, suggesting that the antigen may be processed differently in E. faecalis S161EB-5. The parent strain, E. faecalis S161, did not react with this antibody preparation. Immunogold labeling of intact E. faecalis S161EB-5 and S. sanguis M5 with anti-SSP-5 immunoglobulin G showed that both organisms expressed similar levels of the antigen. Both organisms formed visible aggregates upon incubation with salivary agglutinin. These results suggest that the SSP-5 antigen may mediate both the binding of agglutinin to S. sanguis M5 and the subsequent formation of bacterial aggregates.

Human saliva contains a high-molecular-weight glycoprotein (agglutinin) which binds to specific streptococci in a calcium-dependent reaction leading to the formation of bacterial aggregates. We report the cloning of a gene encoding a surface antigen from Streptococcus sanguis M5 and show that the expressed protein inhibits agglutinin-mediated aggregation and specifically binds the salivary agglutinin in a calcium-dependent fashion. Clones isolated from the immunological screening of S. sanguis M5 genomic libraries with polyclonal antibodies against whole cells were assayed for the ability to compete with S. sanguis for agglutinin. One clone, pSSP-5, expressed antigens of 165 and 130 kilodaltons (kDa) possessing this activity. A 3-kilobase-pair (kbp) insert fragment from this clone was used to screen a genomic library in lambda EMBL3 which resulted in the isolation of clone SSP-5A. This clone contained an insert of 17 kb and expressed proteins of 170 to 205 kDa that reacted with the anti-S. sanguis antibodies. Subcloning of a 5.3-kbp EcoRI-BamHI fragment from SSP-5A produced pEB-5, which expressed streptococcal components that were indistinguishable from SSP-5A. The streptococcal antigen was purified by gel permeation and ion exchange chromatography and shown to potently compete with S. sanguis M5 cells for agglutinin. The antigen also bound purified salivary agglutinin in the presence of 1 mM CaCl2. This binding was inhibited by EDTA. Both the SSP-5 antigen and a 205-kDa protein in surface protein extracts from S. sanguis M5 cross-reacted with antibodies directed against antigen B from S. mutans and SpaA from S. sobrinus 6715. These results indicate that a 205-kDa surface protein that is antigenically related to SpaA and antigen B is involved in the binding of salivary agglutinin to S. sanguis M5.

C31G, an equimolar mixture of alkyl dimethyl glycine and alkyl dimethyl amine oxide, was evaluated for antimicrobial and antiadherence properties. The efficacy of C31G, its two components, and several commercial mouth rinses was determined in assays measuring inhibition of glycolysis, inhibition of bacterial adherence, and MICs. Inhibition of glycolysis was determined by using a saliva sediment model, with glycolytic activity expressed as the change in pH relative to that of a control. Adherence studies were undertaken with Streptococcus sobrinus 6715 to measure inhibition of adherence to nichrome wires. MICs were determined against selected microorganisms by standard methods. C31G demonstrated broad-spectrum antimicrobial properties, with activity against both gram-positive and gram-negative organisms and Candida albicans, a yeast. C31G inhibited both glycolysis by salivary bacteria and adherence of Streptococcus strains to wire mesh. C31G was more effective in the assays conducted than any commercial formulation tested and was as effective as chlorhexidine. A synergistic effect was demonstrated between the individual components of C31G, and no loss of activity was noted when it was formulated into a mouth rinse vehicle.

The major factor in human saliva responsible for the specific aggregation of oral streptococci is a high molecular-weight glycoprotein (agglutinin). To determine if the level of this glycoprotein in whole and parotid saliva was genetically determined, agglutinin activity for Streptococcus sanguis and Streptococcus mutans in saliva obtained from identical and fraternal twins was compared. Evidence for the heritability of agglutinin activity and also parotid flow rate and total protein was obtained. There was no evidence for a significant genetic contribution to salivary sodium concentration.

The role of saliva in sour taste perception was investigated in a series of 4 experiments. In one pair of experiments, solution pH was measured before and after acetic, citric or hydrochloric acid solutions were mixed with saliva either normally in the oral cavity or after saliva was directly added to solutions. The results showed that large increases in solution pH occurred over a wide range of acid concentrations and that the changes in pH were related to individual salivary flow rates; greater increases in solution pH occurred among those individuals with higher flow rates. The other pair of experiments measured taste threshold and suprathreshold responses to different volumes of acids. The results demonstrated that individuals with high salivary flow rates were less sensitive to the taste of acids and that large volumes of acid were more easily perceived. The pattern of findings suggest that saliva-induced changes in solution pH are important in sour taste perception.

The objective of this study was to characterize a fraction from oral streptococci containing receptor activity for salivary agglutinin molecules. Several species and strains of streptococci were disrupted in a Ribi press. The supernatant was nuclease-treated and subjected to differential centrifugation. Receptor activity in the fractions was measured by the inhibition of saliva-mediated bacterial aggregation. In addition, bacterial strains were tested for their ability to aggregate and to deplete saliva of agglutinin activity. Three patterns of activity were observed: Streptococcus sanguis M5 depleted saliva of agglutinin activity and aggregated well; Streptococcus sanguis CC5A depleted saliva of agglutinin but did not aggregate well; and Streptococcus faecalis S-161 neither depleted saliva of agglutinin nor did it aggregate. The 105,000 g supernatant fractions derived from Ribi-disrupted Streptococcus sanguis M5 and CC5A, but not from Streptococcus faecalis, showed dose-dependent inhibition of saliva-mediated aggregation. This inhibitory activity was non-dialyzable, had the same heat and trypsin sensitivity as that seen with intact bacteria, and was not due to enzymatic digestion of the salivary agglutinin. Iso-electric focusing revealed a single active region with a pI of 5.5 which was clearly separated from the bulk of the bacterial proteins.

Human blood leukocytes generate intense luminol-dependent chemiluminescence (LDCL) following stimulation by streptococci and by Gram negative rods which had been preopsonized by cationic polyelectrolytes (histone, poly L-arginine-PARG, poly L-histidine-PHSTD). Streptococci but not Gram negative rods or hyaluronic acid-rich streptococci (group C) also induced intense LDCL following opsonization with the anionic polyelectrolytes-dextran sulfate or polyanethole sulfonate (liquoid) suggesting that the outer surfaces of different bacteria bound anionic polyelectrolytes to different extents. Both normal and immune serum, synovial fluids and pooled human saliva inhibited the LDCL responses induced by streptococci preopsonized with poly cations. On the other hand, bacteria which had been first preopsonized by the various body fluids and then subjected to a second opsonization by cationic ligands ("sandwiches"), induced a very intense LDCL response in leukocytes. Streptococci which had been preopsonized by PARG, histone or by PHSTD also triggered superoxide generation by blood leukocytes, which was markedly enhanced by a series of cytochalasins. PHSTD alone induced the formation of very large amounts of superoxide. Paradoxically, the same concentrations of cytochalasins B or C which markedly boosted the generation of superoxide following stimulation of leukocytes with soluble or particulate ligands, had a strong inhibitory effect on the generation of LDCL. On the other hand cycochalasins failed to inhibit LDCL which had been induced by phorbol myristate acetate (PMA). Peritoneal macrophages which had been harvested from C. parvum-stimulated mice, generated more LDCL and superoxide following stimulation by PARG than macrophages obtained from proteose peptone-stimulated mice. Macrophages which had been activated either by proteose peptone or by C. parvum and cultivated for 2 hours on teflon surfaces, generated much more LDCL than macrophages which had been cultivated for 24 hours on teflon surfaces. Both cationic and anionic polyelectrolytes mimic the effects of antibodies as activators of the oxygen burst in blood leukocytes and in macrophages. Such polyelectrolytes can serve as models to further study leukocyte-bacteria interactions in infectious and inflammatory sites.

The objective of this investigation was to examine the effects of low levels of Hg(II) on the respiratory burst of PMNs by monitoring O2 consumption, superoxide radical formation, and chemiluminescence. Hg(II) at concentrations of 10-100 ng/ml profoundly inhibited zymosan-stimulated human cells. This inhibition was immediate in onset and occurred with minimal loss of cell viability. Effects of Hg(II) on the PMN respiratory burst were compared with those of Sn, Pb, Se, Au, Ag and Cu. Only in the case of Ag and Cu did the inhibitory effects approach those of Hg. The results indicate that Hg(II) may serve as a specific inhibitor of components of the respiratory burst.