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1.  Plausibility of HIV-1 Infection of Oral Mucosal Epithelial Cells 
Advances in Dental Research  2011;23(1):38-44.
The AIDS pandemic continues. Little is understood about how HIV gains access to permissive cells across mucosal surfaces, yet such knowledge is crucial to the development of successful topical anti-HIV-1 agents and mucosal vaccines. HIV-1 rapidly internalizes and integrates into the mucosal keratinocyte genome, and integrated copies of HIV-1 persist upon cell passage. The virus does not appear to replicate, and the infection may become latent. Interactions between HIV-1 and oral keratinocytes have been modeled in the context of key environmental factors, including putative copathogens and saliva. In keratinocytes, HIV-1 internalizes within minutes; in saliva, an infectious fraction escapes inactivation and is harbored and transferable to permissive target cells for up to 48 hours. When incubated with the common oral pathogen Porphyromonas gingivalis, CCR5− oral keratinocytes signal through protease-activated receptors and Toll-like receptors to induce expression of CCR5, which increases selective uptake of infectious R5-tropic HIV-1 into oral keratinocytes and transfer to permissive cells. Hence, oral keratinocytes—like squamous keratinocytes of other tissues—may be targets for low-level HIV-1 internalization and subsequent dissemination by transfer to permissive cells.
PMCID: PMC3144042  PMID: 21441479
HIV/AIDS; epithelia; oral epithelium; infectious disease; mucosal immunity; vaccines
2.  Calprotectin Expression In Vitro by Oral Epithelial Cells Confers Resistance to Infection by Porphyromonas gingivalis 
Infection and Immunity  2001;69(7):4242-4247.
Calprotectin, an S100 calcium-binding protein with broad-spectrum antimicrobial activity in vitro, is expressed in neutrophils, monocytes, and gingival keratinocytes. In periodontitis, calprotectin appears upregulated and is detected at higher levels in gingival crevicular fluid and tissue specimens. How calprotectin contributes to the pathogenesis of periodontal diseases is unknown. To isolate the effects of calprotectin, a calprotectin-negative oral epithelial cell line was transfected with calprotectin genes to enable expression. Porphyromonas gingivalis was permitted to bind and invade transfected cells expressing calprotectin and sham transfectants. Rates of invasion into both cell lines were compared using the antibiotic protection assay. Transfected cells expressing calprotectin showed 40 to 50% fewer internalized P. gingivalis than sham transfectants. Similarly, binding to calprotectin expressing cells was reduced approximately twofold at all time points (15, 30, 45, and 60 min) as estimated by immunofluorescence analysis. Independent of invasion, however, prolonged exposure to P. gingivalis induced epithelial cell rounding and detachment from the substratum. These morphological changes were delayed, however, in cells expressing calprotectin. Using P. gingivalis protease-deficient mutants, we found that Arg-gingipain and Lys-gingipain contributed to epithelial cell rounding and detachment. In conclusion, expression of calprotectin appears to protect epithelial cells in culture against binding and invasion by P. gingivalis. In addition, cells expressing calprotectin are more resistant to detachment mediated by Arg-gingipain and Lys-gingipain. In periodontal disease, calprotectin may augment both the barrier protection and innate immune functions of the gingival epithelium to promote resistance to P. gingivalis infection.
PMCID: PMC98457  PMID: 11401960
3.  Specificity of salivary-bacterial interactions: role of terminal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans. 
Infection and Immunity  1978;19(1):107-115.
Four highly purified salivary glycoproteins were used to study salivary-bacterial interactions. One pair of glycoproteins was mucin-like in composition, whereas the second pair was not. By an agglutination assay, it was found that only the mucin-glycoproteins agglutinated Streptococcus sanguis and S. mutans. Removal of sialic acid from these molecules resulted in a loss of agglutination of S. sanguis but not of S. mutans. The agglutination phenomenon was shown to require a salivary macromolecule of at least 150,000 daltons.
PMCID: PMC414055  PMID: 415001
4.  A Streptococcal Adhesion System for Salivary Pellicle and Platelets 
Infection and Immunity  1998;66(11):5388-5392.
A Streptococcus sanguis 133-79 adhesin identified by the monoclonal antibody 1.1 (MAb 1.1) binds both saliva-coated hydroxylapatite (sHA) and platelets. The complementary binding site(s) for the adhesin was identified by the anti-idiotypical MAb 2.1. To learn if this adhesion system, marked by the antiadhesin MAb 1.1 and anti-binding site MAb 2.1, is commonly used by strains within the sanguis group and other viridans group streptococci, 42 strains from seven species were tested. Strains that bind to both sHA and platelets use the same adhesin and binding site epitopes. Strains that do not adhere to platelets rely on other adhesin specificities to bind to sHA.
PMCID: PMC108674  PMID: 9784548
5.  Streptococcus sanguis expresses a 150-kilodalton two-domain adhesin: characterization of several independent adhesin epitopes. 
Infection and Immunity  1997;65(9):3815-3821.
Streptococcus sanguis binds to saliva-coated hydroxylapatite (sHA), an in vitro model of the enamel pellicle. To learn if more than one adhesin functions during adhesion, 12 reactive monoclonal antibodies (MAbs) were isolated by screening against both adhesive and nonadhesive strains. Two of these MAbs, 1.1 and 1.2, inhibited adhesion in a dose-dependent fashion, although maximum inhibition with either was only 37%. When these two MAbs plus a polyclonal antibody to P1-like adhesin were combined, the inhibition was additive to about 82%. These data indicated that there were at least three distinct, functional adhesion epitopes on the surface of S. sanguis. Western blot analyses of S. sanguis surface macromolecules showed antigens at 36 and 56 (with MAb 1.2), 87 and 150 (with both MAb 1.1 and MAb 1.2), and 100, 130, and 170 kDa (with anti-P1 antibody). The antigens were eluted from gels. Isolated antigens and corresponding antibodies inhibited adhesion similarly. Additivity experiments suggested the distinct epitopes were in three groups: (i) 36/56 kDa, (ii) 87/150 kDa, and (iii) 100/130/170 kDa. The 150-kDa antigen reacting with both MAbs was isolated from gels and digested with trypsin. The digestion revealed a series of tryptic bands. A band at 38 kDa reacted with MAb 1.1 whereas a band at 54 kDa reacted with MAb 1.2 in Western blot analysis, indicating two distinct adhesive epitopes on the 150-kDa antigen. These data strongly suggest that S. sanguis adhesion to sHA is maximized when several adhesin epitopes are coexpressed on surface antigens of different sizes.
PMCID: PMC175544  PMID: 9284157
6.  Platelet receptors for the Streptococcus sanguis adhesin and aggregation-associated antigens are distinguished by anti-idiotypical monoclonal antibodies. 
Infection and Immunity  1995;63(9):3628-3633.
Platelets aggregate in response to an adhesin and the platelet aggregation-associated protein (PAAP) expressed on the cell surfaces of certain strains of Streptococcus sanguis. We sought to identify the corresponding PAAP receptor and accessory adhesin binding sites on platelets. Since the adhesion(s) of S. sanguis for platelets has not been characterized, an anti-idiotype (anti-id) murine monoclonal antibody (MAb2) strategy was developed. First, MAb1s that distinguished the adhesin and PAAP antigens on the surface of S. sanguis I 133-79 were selected. Fab fragments of MAb1.2 (immunoglobulin G2b [IgG2b]; 70 pmol) reacted with 5 x 10(7) cells of S. sanguis to completely inhibit the aggregation of human platelets in plasma. Under similar conditions, MAb1.1 (IgG1) inhibited the adhesion of S. sanguis cells to platelets by a maximum of 34%, with a comparatively small effect on platelet aggregation. Together, these two MAb1s inhibited S. sanguis-platelet adhesion by 63%. In Western immunoblots, both MAb1s reacted with S. sanguis 133-79 87- and 150-kDa surface proteins and MAb1.2 also reacted with purified type I collagen. The hybridomas producing MAb1.1 and MAb1.2 were then injected into BALB/c mice. Enlarged spleens were harvested, and a panel of MAb2 hybridomas was prepared. To identify anti-ids against the specific MAb1s, the MAb2 panel was screened by enzyme-linked immunosorbent assay for reaction with rabbit polyclonal IgG antibodies against the 87- and 150-kDa antigens. The reactions between the specific rabbit antibodies and anti-ids were inhibited by the 87- and 150-kDa antigens. When preincubated with platelets, MAb2.1 (counterpart of MAb1.1) inhibited adhesion to platelets maximally by 46% and MAb2.2 (anti-MAb1.2) inhibited adhesion to platelets maximally by 35%. Together, both MAb2s inhibited the adhesion of S. sanguis to platelets by 81%. MAb2.2 also inhibited induction of platelet aggregation. MAb2.2 immunoprecipitated a biotinylated platelet membrane antigen of 170 kDa (unreduced); MAb2.1 precipitated membrane antigens of 175- and 230-kDa (unreduced). Therefore, platelet binding sites and the receptor for the S. sanguis adhesin and PAAP, respectively, are distinguished by the anti-id MAb2s.
PMCID: PMC173503  PMID: 7642300
7.  Altered expression of the platelet aggregation-associated protein from Streptococcus sanguis after growth in the presence of collagen. 
Infection and Immunity  1995;63(3):1084-1088.
Certain strains of Streptococcus sanguis adhere selectively to human platelets (Adh+) and, in plasma, induce them to aggregate into in vitro thrombi (Agg+). The induction of aggregation is mediated by the platelet aggregation-associated protein (PAAP) expressed on the cell surface of the streptococcus. In endocarditis, expression of PAAP may be regulated by association with host proteins on damaged heart valves. To begin to test this hypothesis, three strains of S. sanguis were each cultured in the presence or absence of collagens (types I to X), laminin, or PAAP-derived peptide preparations. After harvesting and washing, the platelet-interactive phenotype of strains 133-79 (Adh+ Agg+), L74 (Adh+ Agg-), and 10556 (Adh- Agg-) was unchanged. The cells from each culture were then digested mildly with trypsin to isolate PAAP. PAAP isolated from strain 133-79 (Adh+ Agg+) grown in the absence of added collagen, other proteins, or peptides inhibited platelet aggregation in response to untreated cells of S. sanguis. Platelet aggregation was induced immediately, however, by PAAP from strain 133-79 isolated after growth in the presence of 300 nM type I collagen, while lower concentrations yielded protein fragments that potentiated the response to intact cells. Aggregation-inducing PAAP could be removed by anti-PAAP (PGEQGPK) immunoaffinity chromatography, but only inhibitory activity could be recovered. The agonist effect of PAAP was not associated with collagen itself, since the PAAP preparations did not contain detectable amounts of hydroxyproline. PAAP antigens isolated from cells grown in the presence and absence of collagen had similar apparent molecular weights, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. When electrophoresis was performed under nondenaturing conditions, however, PAAP isolated from cells grown in type I collagen migrated more slowly. Strain L74 grown with type I collagen yielded tryptic fragments of proteins that inhibited aggregation significantly better than control peptides (no collagen in the medium). Strain 10556 was apparently unaffected by growth in type I collagen. The effect of type I collagen was somewhat unique. Growth in the presence of collagen types II to VI (300 nM) yielded protein fragments that potentiated without inducing platelet aggregation, while other collagens, laminin, and PAAP-derived peptides did not affect platelet aggregation. These results suggest that growth in the presence of type I collagen and, perhaps, collagens II to VI alters the expression and conformation of PAAP in certain strains of S. sanguis.
PMCID: PMC173113  PMID: 7868231
8.  ADP-like platelet aggregation activity generated by viridans streptococci incubated with exogenous ATP. 
Infection and Immunity  1983;40(1):120-125.
To explore the possibility that Streptococcus sanguis aggregation of platelet-rich plasma (PRP) might be mediated by soluble agents, we tested cell-free S. sanguis supernatant for aggregation activity. The supernatant of untreated S. sanguis was without measurable PRP aggregation activity. In contrast, the cell-free supernatant of ATP-incubated S. sanguis produced an immediate wave of PRP aggregation. The supernatant with PRP aggregating activity contained insufficient protease to produce a response. The response increased with the time of incubation with ATP. Active supernatant was desalted and chromatographed on nucleotide-calibrated columns of Dowex 1-X8. An active ADP function was identified. The activity was insensitive to dicyclohexylcarbodiimide, but was sensitive to both Ca2+ and Ca2+-Mg2+ chelating agents, cold (4 degrees C), heat (80 degrees C), pH (optimum between pH 7 and 8), apyrase, and sodium molybdate. In addition, preincubation of PRP with adenosine inhibited activity. Strains of viridans streptococci were screened for activity. Aggregation-promoting strains showed two times more activity than did other strains. Although it was not vigorously excluded that the ADP was discharged from the microbes, the existence of an exogenous ATPase on S. sanguis was strongly suggested. The expression of the activity was associated with the lag time to onset of PRP aggregation with intact S. sanguis. This activity could, therefore, be a synergistic promoter of PRP aggregation and an additional virulence factor in endocarditis.
PMCID: PMC264825  PMID: 6219955
9.  The platelet interactivity phenotype of Streptococcus sanguis influences the course of experimental endocarditis. 
Infection and Immunity  1992;60(11):4809-4818.
A strain of Streptococcus sanguis that induced rabbit platelets to aggregate in vitro (Agg+ phenotype) was hypothesized to be a more virulent pathogen than an Agg- strain in experimental endocarditis in rabbits. A left ventricular catheter was implanted, and then an Agg+ or Agg- strain was inoculated intravenously. Vegetations formed on the aortic semilunar valves but were unaffected by the duration of implantation of the catheter. Vegetations enlarged by accumulating platelets and their mass increased directly with the duration of endocarditis. Inoculation of the Agg+ strain consistently caused endocarditis with significantly larger vegetations, a more severe clinical course (including febrile episodes, hematological changes, and signs of myocardial ischemia), more gross lesions in major organs, and greater mortality than inoculation with the Agg- strain, saline, or the Agg+ strain pretreated with monospecific rabbit immunoglobulin G or Fab fragments against its platelet aggregation-associated protein (PAAP; class II). In experimental endocarditis, PAAP expressed by Agg+ S. sanguis appeared to be an important virulence factor.
PMCID: PMC258235  PMID: 1398992
10.  Platelet-interactive products of Streptococcus sanguis protoplasts. 
Infection and Immunity  1990;58(12):4117-4125.
To isolate a more native, platelet-interactive macromolecule (class II antigen) of Streptococcus sanguis, cultured protoplasts were used as a source. Protoplasts were optimally prepared from fresh washed cells by digestion with 80 U of mutanolysin per ml for 75 min at 37 degrees C while osmotically stabilized in 26% (wt/vol) raffinose. Osmotically stabilized forms were surrounded by a 9-nm bilaminar membrane, as shown by transmission electron microscopy. Protoplasts were cultured in chemically defined synthetic medium and osmotically stabilized by ammonium chloride. Spent culture media were harvested daily for 7 days. Each day, soluble proteins were isolated from media, preincubated with platelet-rich plasma, and tested for inhibition of platelet aggregation induced by S. sanguis cells. Products released from S. sanguis protoplasts and reactive with an anti-class II antigen immunoaffinity matrix were able to inhibit S. sanguis-induced platelet aggregation. As resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, anti-class II-reactive protoplast products included silver-stained bands of 67, 79, 115, 216, and 248 kDa. The 115-kDa protein fraction was isolated by gel filtration and ion-exchange chromatography. This form of the class II antigen contained N-formylmethionine at its amino terminus. Rhamnose constituted 18.2% of the total residual dry weight and nearly half of its carbohydrate content. Diester phosphorus constituted 1% of this fraction. After trypsinization of the protoplast products from either preparation, a 65-kDa protein fragment was recovered. This protoplast protein fragment and the S. sanguis cell-derived 65-kDa class II antigen, previously implicated in the induction of platelet aggregation, were shown to be functionally and immunologically identical.
PMCID: PMC313784  PMID: 2254032
11.  Phenotypic characterization of Streptococcus sanguis virulence factors associated with bacterial endocarditis. 
Infection and Immunity  1990;58(2):515-522.
Certain strains of Streptococcus sanguis adhere (Adh+) selectively to human platelets and, in plasma, induce them to aggregate (Agg+) into in vitro thrombi. In this study, we examined 18 recent endocarditis and dental plaque isolates of microorganisms that were biotyped as S. sanguis for coexpression of platelet interactivity phenotypes with another possible virulence factor in bacterial endocarditis, dextran synthesis. Detectable production of extracellular glucosyltransferase ranged from 0.2 to 66 mU/mg of culture fluid for 10 representative strains tested. Production of extracellular or cell-associated glucosyltransferase, fructosyltransferase, and soluble or insoluble dextrans was not necessarily coexpressed with platelet interactivity phenotypes, since the levels of production of soluble and insoluble dextrans varied among representative Adh+ Agg+ and Adh- Agg- strains. Analysis of a second panel of 38 fresh dental plaque isolates showed that S. sanguis distributes in a reproducible manner into the possible phenotype groups. Strains with different platelet interactivity phenotypes were distinguished with a panel of four murine monoclonal antibodies (MAbs) raised against Adh+ Agg+ strain 133-79 and screened to rule out artifactual reactions with antigenic components in culture media. The MAbs reacted selectively with Adh+ Agg+ strains in a direct-binding, whole-cell, enzyme-linked immunosorbent assay and also inhibited their interactions with platelets. Analysis of minimal tryptic digests of many strains, including variants that failed to bind the MAbs, suggested that some noninteractivity phenotypes possess cryptic surface determinants. Since the ability to adhere to platelets and induce them to aggregate is relatively stable, these traits may be useful in a phenotyping scheme for these Lancefield nontypeable streptococci.
PMCID: PMC258487  PMID: 2137112
12.  Cell-free released components of Streptococcus sanguis inhibit human platelet aggregation. 
Infection and Immunity  1983;42(1):394-401.
To study the role of surface components in the selective binding and aggregation of platelet-rich plasma (PRP) by strains of viridans streptococci, we treated the binding, aggregation strain Streptococcus sanguis I 2017-78 by sonication or trypsinization. Morphologically identifiable electron-dense fibrils were released from the cell wall, apparently from an inner electron-dense layer, under conditions that left cells intact. These controlled conditions were determined to cause submaximal loss in adhesion to platelet ghosts and PRP aggregation by treated, washed S. sanguis. Soluble components were recovered from the controlled sonic or L-(tosylamido 2-phenyl)ethyl chloromethyl ketone-trypsin treatments. Each showed dose-response inhibition of aggregation when preincubated with PRP before challenge with fresh, untreated S. sanguis. The time to onset of PRP aggregation was inhibited by 50% with 0.2 mg of TPCK-trypsin peptides or 1.0 mg of the sonicate per ml per 2 X 10(8) platelets. Components of both preparations were immunologically cross-reactive, but lipoteichoic acid was not a major antigen of either. By weight, the TPCK-trypsin peptides were virtually all protein; the sonicate residues identified were about 50% protein and 7% hexose. Each was a complex mixture of components as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 8 TPCK-trypsin peptides and 16 sonicate components were so identified. In contrast, at least four or five components from either preparation were recognized as surface determinants by a rabbit antiserum to whole homologous microbes. Platelet-binding ligands of S. sanguis could be among these determinants.
PMCID: PMC264570  PMID: 6618669
13.  Aggregation of human platelets and adhesion of Streptococcus sanguis. 
Infection and Immunity  1983;39(3):1457-1469.
Platelet vegetations or thrombi are common findings in subacute bacterial endocarditis. We investigated the hypothesis that human platelets selectively bind or adhere strains of Streptococcus sanguis and Streptococcus mutans and aggregate, as a result, into an in vitro thrombus. Earlier ultrastructural studies suggested that aggregation of platelets over time by Staphylococcus aureus was preceded in order by adhesion and platelet activation. We uncoupled the adhesion step from activation and aggregation in our studies by incubating streptococci with platelet ghosts in a simple, quantitative assay. Adhesion was shown to be mediated by protease-sensitive components on the streptococci and platelet ghosts rather than cell surface carbohydrates or dextrans, plasma components, or divalent cations. The same streptococci were also studied by standard aggregometry techniques. Platelet-rich plasma was activated and aggregated by certain isolates of S. sanguis. Platelet ghosts bound the same strains selectively under Ca2+- and plasma-depleted conditions. Fresh platelets could activate after washing, but Ca2+ had to be restored. Aggregation required fresh platelets in Ca2+-restored plasma and was inducible by washed streptococcal cell walls. These reactions in the binding and aggregometry assays were confirmed by transmission electron microscopy. Surface microfibrils on intact S. sanguis were identified. These appendages appeared to bind S. sanguis to platelets. The selectivity of adhesion of the various S. sanguis strains to platelet ghosts or Ca2+- and plasma-depleted fresh washed platelets was similar for all donors. Thus, the platelet binding site was expressed widely in the population and was unlikely to be an artifact of membrane aging or preparation. Since selective adhesion of S. sanguis to platelets was apparently required for aggregation, it is suggested that functionally defined receptors for ligands on certain strains of S. sanguis may be present on human platelets. Some differences in the selectivity and rate of the aggregation response were noted among platelet donors, although the meaning of the variability requires further study. Nonetheless, these interactions may contribute to platelet accretion in the initiation and development of vegetative lesions in the subacute bacterial endocarditis.
PMCID: PMC348116  PMID: 6188697

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