Dietary Nitrite Drives Disease Outcomes in Oral Polymicrobial Infections.

Streptococcus mutans resides in the oral polymicrobial biofilm and is a major contributor to the development of dental caries. Interestingly, high salivary nitrite concentrations have been associated with a decreased prevalence of dental caries. Moreover, the combination of hydrogen peroxide-producing oral commensal streptococci and nitrite has been shown to mediate the generation of reactive nitrogen species, which have antimicrobial activity. The goal of this study was to examine whether nitrite affects S. mutans virulence during polymicrobial infections with the commensal Streptococcus parasanguinis. Here, we report that the combination of S. parasanguinis and nitrite inhibited S. mutans growth and biofilm formation in vitro. Glucan production, which is critical for S. mutans biofilm formation, was also inhibited in 2-species biofilms with S. parasanguinis containing nitrite as compared with biofilms that contained no nitrite. In the in vivo caries model, enamel and dentin carious lesions were significantly reduced in rats that were colonized with S. parasanguinis prior to infection with S. mutans and received nitrite in the drinking water, as compared with animals that had a single S. mutans infection or were co-colonized with both bacteria and received no nitrite. Last, we report that S. mutans LiaS, a sensor kinase of the LiaFSR 3-component system, mediates resistance to nitrosative stress. In summary, our data demonstrate that commensal streptococci and nitrite provide protection against S. mutans pathogenesis. Modulating nitrite concentrations in the oral cavity could be a useful strategy to combat the prevalence of dental caries.

Targeting of Streptococcus mutans Biofilms by a Novel Small Molecule Prevents Dental Caries and Preserves the Oral Microbiome.

Dental caries is a costly and prevalent disease characterized by the demineralization of the tooth's enamel. Disease outcome is influenced by host factors, dietary intake, cariogenic bacteria, and other microbes. The cariogenic bacterial species Streptococcus mutans metabolizes sucrose to initiate biofilm formation on the tooth surface and consequently produces lactic acid to degrade the tooth's enamel. Persistence of S. mutans biofilms in the oral cavity can lead to tooth decay. To date, no anticaries therapies that specifically target S. mutans biofilms but do not disturb the overall oral microbiome are available. We screened a library of 2-aminoimidazole antibiofilm compounds with a biofilm dispersion assay and identified a small molecule that specifically targets S. mutans biofilms. At 5 µM, the small molecule annotated 3F1 dispersed 50% of the established S. mutans biofilm but did not disperse biofilms formed by the commensal species Streptococcus sanguinis or Streptococcus gordonii. 3F1 dispersed S. mutans biofilms independently of biofilm-related factors such as antigen I/II and glucosyltransferases. 3F1 treatment effectively prevented dental caries by controlling S. mutans in a rat caries model without perturbing the oral microbiota. Our study demonstrates that selective targeting of S. mutans biofilms by 3F1 was able to effectively reduce dental caries in vivo without affecting the overall oral microbiota shaped by the intake of dietary sugars, suggesting that the pathogenic biofilm-specific treatment is a viable strategy for disease prevention.

Immune response in humans to a nasal boost with Streptococcus mutans antigens.

We previously reported that a Streptococcus mutans enriched-glucosytransferase (E-GTF) preparation induces an immune response following intranasal, but not tonsillar, immunization of humans. In this study, we determined whether intranasal immunization of these subjects 2 years later resulted in augmented immune responses compared to those seen in control subjects. Subjects previously immunized via the intranasal (IN, n = 7) or tonsillar (IT, n = 7) route and control (n = 12) subjects were immunized via the intranasal route with E-GTF. Nasal wash, saliva, and serum were collected before immunization and then weekly for 3 months after immunization. Significant (P < 0.05) mucosal and serum immunoglobulin A (IgA) anti-E-GTF responses were observed in all three groups. Nasal and serum IgA anti-E-GTF responses were significantly higher (P < 0.05) in the IN group. The salivary responses in the three groups were, in general, similar. These results indicate that intranasal immunization primes the immune system for a localized secondary response to S. mutans antigens.

Comparative analysis of the antibacterial effects of combined mouthrinses on Streptococcus mutans.

BACKGROUND/AIMS: Chlorhexidine has been proposed as a potent chemotherapeutic agent against oral bacteria. However, there are some inconsistent results regarding the usefulness of chlorhexidine mouthrinse as an antimicrobial for Streptococcus mutans. The purpose of this study was to investigate the effectiveness of combining oral rinses to reduce S. mutans levels in human saliva. METHODS: Sixteen healthy adult subjects were randomly assigned to one of four rinse groups using a 4-cell crossover design. The groups rinsed twice a day for 7 days with one of the following: 0.12% chlorhexidine (PerioGard), 1.5% hydrogen peroxide (Peroxyl), a combined chlorhexidine+hydrogen peroxide, or water (control). Every 5 weeks, each group initiated a different rinse. Saline wash samples were collected on days 7 and 21 for assessment of S. mutans and total streptococci. RESULTS: No significant differences were seen in S. mutans levels among the groups; however, the levels of total streptococci on day 7 samples were significantly lower in the chlorhexidine and chlorhexidine+hydrogen peroxide groups than in the hydrogen peroxide and control groups. There was no additional decrease seen in S. mutans or total streptococci levels in the group receiving chlorhexidine+hydrogen peroxide compared to chlorhexidine alone. CONCLUSIONS: Sample variation was high throughout the study, with a significant trend toward lower counts as the study progressed. Adding hydrogen peroxide to the chlorhexidine mouthrinse did not result in a further decrease in S. mutans levels.

Effectiveness of sodium hypochlorite in preventing inoculation of periapical tissues with contaminated patency files.

The aim of this study was to analyze the effectiveness of 5.25% sodium hypochlorite (NaOCI) in preventing inoculation of periapical tissues with contaminated patency files. Twenty-eight extracted human permanent teeth with single canals were used in the study. Group I teeth were filled with NaOCl, and #15 stainless steel files contaminated with Streptococcus sanguis (ATCC #10556) were allowed to pass through the NaOCI into the culture medium. The teeth in group II were also filled with NaOCl, but the contaminated files used in group II canals were immersed in NaOCl for 10 s prior to being placed into the canals and cultured. The negative control group used sterile files (0% growth), the first positive control group used contaminated patency files in teeth with empty canals (100% growth), and the second positive control group placed contaminated files into broth next to teeth filled with NaOCl (to evaluate potential chlorine leakage; 100% growth). The experimental results showed no positive growth of S. sanguis for groups I and II, indicating that the NaOCl present in the canal after irrigation was sufficient to kill the test organism.

Intranasal immunization of humans with Streptococcus mutans antigens.

To evaluate the effectiveness of a low dose of soluble or liposomal (L) glucosyltransferase-enriched preparation (E-GTF) in inducing mucosal immune responses after intranasal immunization, 12 adults were immunized on days 0 and 7 by the IN route with 62.5 microg of soluble E-GTF or L-E-GTF. An increase in the mean salivary IgA anti-E-GTF response (P < 0.03) was seen in the L-E-GTF but not the soluble E-GTF group. A significant increase (P < 0.05) in the mean specific IgA antibody activity was also seen in nasal wash from both groups. Although the nasal wash responses were higher in the L-E-GTF than in the soluble E-GTF group, they were not significantly different. The soluble E-GTF immunized group showed a higher serum IgG response than the L-E-GTF immunized group on day 90 (P < 0.05). These results indicate that as little as 62.5 microg of E-GTF, when given by the intranasal route, induced an IgA response in secretions.

Effect of age on immunoglobulin A subclass distribution in human parotid saliva.

Two subclasses of immunoglobulin A (IgA) antibodies are produced in humans, IgA1 and IgA2, IgA2 being more resistant to digestion by bacterial proteases than IgA1. The amount of IgA in saliva has been shown to vary with age; however, little is known about the correlation between IgA subclass distribution in saliva and age. The purpose of this study was to determine whether differences exist in the levels and ratio of IgA subclasses in parotid saliva of children and adults. Parotid saliva was obtained from healthy children (age range 6-12 years, n = 14) and adults (age range 22-51 years, n = 20) using Schaefer cups. Samples were analyzed for levels of total IgA, IgA1, and IgA2 by ELISA. IgA and IgA1 levels were significantly higher in adults than in children. However, no differences were seen in the ratio of IgA1 and IgA2 in the two groups of subjects. These findings indicate that levels of IgA increase with age, whereas the IgA subclass ratio is established early in life.

Humans immunized with Streptococcus mutans antigens by mucosal routes.

Strategies aimed at the prevention of Streptococcus mutans infection and dental caries include mucosal immunization, which results in salivary anti-S. mutans responses. The purpose of this study was to evaluate the effectiveness of nasal vs. tonsillar immunization with S. mutans antigens in inducing salivary immune responses. Twenty-one adult subjects were immunized twice, within a seven-day interval, with a glucosyltransferase-enriched preparation (E-GTF) administered by nasal or tonsillar topical spray. Parotid saliva, nasal wash, and serum were collected prior to and at one- to two-week intervals for 3 months following immunization and were assayed by ELISA for anti-E-GTF activity. Results were analyzed by means of the mixed-models procedure with p < 0.05 level of significance. Significantly higher anti-E-GTF responses were detected in saliva and nasal wash samples from the group immunized by the nasal compared with the tonsillar route, indicating that nasal immunization was more effective in inducing mucosal responses in adults.

Differential induction of endotoxin tolerance by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli.

Exposure of mononuclear phagocytes to enterobacterial LPS induces a state of transient hyporesponsiveness to subsequent LPS exposure, termed endotoxin tolerance. In the present study, LPS derived from the oral periodontal pathogen, Porphyromonas gingivalis, was compared with that derived from the enterobacterium, Escherichia coli, for the ability to induce endotoxin tolerance. Pretreatment of the human macrophage cell line, THP-1, with E. coli LPS resulted in a severe reduction in the levels of IL-1beta, IL-6, and TNF-alpha upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a mitigation of IL-1beta, but not IL-6 and TNF-alpha production upon subsequent exposure to P. gingivalis LPS: primary or secondary stimulation with < or =100 ng/ml P. gingivalis LPS resulted in comparable levels of IL-6 and TNF-alpha, while stimulation of THP-1 cells with > or =1 microg/ml P. gingivalis LPS induced a significant enhancement in IL-6 and TNF-alpha levels upon secondary exposure. To identify possible mechanisms for these differences, changes in the expression of molecules involved in the LPS-signaling pathway were assessed. Pretreatment of THP-1 cells with E. coli LPS resulted in a significant reduction in surface Toll-like receptor 4 (TLR4) expression and an inability to degrade I-kappaB-alpha or I-kappaB-beta proteins upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a significant enhancement of both CD14 and TLR2, while maintaining the ability to degrade I-kappaB-beta only upon secondary stimulation. Thus, E. coli and P. gingivalis LPS differentially affect CD14 and TLR expression as well as secondary LPS-associated responses.

Effect of attenuated Salmonella enterica serovar Typhimurium expressing a Streptococcus mutans antigen on secondary responses to the cloned protein.

Attenuated Salmonella enterica serovar Typhimurium has been used for targeted delivery of recombinant antigens to gut- and nose-associated lymphoid tissues. Contradictory reports have described the effect of preexisting immunity to the antigen delivery vehicle. We decided to examine this discrepancy by studying the effect of immunizing mice by the intranasal (i.n.) route with Salmonella expressing an insoluble protein and to study the ability to augment recall responses by boosting with either Salmonella-expressed protein or purified soluble protein alone. The glucan-binding domain (GLU) of the enzyme glucosyltransferase (GTF), which is an important virulence factor of Streptococcus mutans, was recombinantly expressed in the insoluble phase in S. enterica serovar Typhimurium, and the immunogenicity of this construct was studied in mice. We examined the induction of primary immune responses by insoluble GLU polypeptide delivered in Salmonella at week 1 (groups 1 and 2) and recall responses after a week 15 boost with either Salmonella expressing GLU (group 1) or purified GLU polypeptide (groups 2 and 3). Group 4 served as the control and received phosphate-buffered saline alone by the i.n. route. Significant anti-GLU serum immunoglobulin G (IgG) levels were seen in groups 1, 2, and 3 at week 18 (P < 0.001), i.e., 3 weeks after the booster immunization. Mice in group 2, who received Salmonella followed by GLU, had the highest GLU-specific IgG levels among all groups. The serum IgG levels persisted in all responding groups for at least 7 weeks after the boost (week 22). The IgG2a/IgG1 subclass ratio of serum anti-GLU antibodies in group 1 significantly increased after the boost. These results support the induction of a type 1-like immune response to GLU after primary and booster immunizations with Salmonella expressing GLU. On the other hand, group 2 mice, which received Salmonella expressing GLU as the primary dose and soluble protein as the booster dose, exhibited a shift from a type 1-like to a more type 2-like immune response to GLU following the boost. These results indicate that S. enterica serovar Typhimurium is an excellent delivery vehicle for the insoluble and recombinantly expressed GLU of GTF and that this construct was especially effective in priming the host for a secondary response to soluble GLU polypeptide.

Human salivary immunoglobulin and antigen-specific antibody activity after tonsillectomy.

The importance of the lymphoid tissue collectively known as Waldeyer's ring, which includes the palatine, lingual and nasopharyngeal tonsils, in the induction and contribution of specific antibody responses in human saliva is not clear. The purpose of this study was to determine whether salivary immunoglobulin A (IgA) levels differ in quantity and quality between subjects who have had a tonsillectomy and age, sex and race-matched controls. Parotid saliva, whole saliva, and blood serum samples were collected from 25 volunteer children who had undergone tonsillectomy (T-) within 6-14 months of sampling and from 25 age, sex and race-matched controls. The levels of total IgA (and subclasses) in saliva, and of antigen-specific salivary IgA and serum IgA and IgG antibodies to 4-9 relevant antigens were analyzed by ELISA. No significant difference was observed in the mean total IgA and IgA subclass levels in parotid and whole saliva, although the mean levels for children with a T- were slightly lower. Children with a T- had significantly higher parotid salivary IgA and IgA1 specific/total activity than controls. The total and specific whole saliva IgA and the specific serum IgA or IgG activities were not significantly different from controls. These results indicate an association between the removal of tonsils and increased levels of specific IgA activity in parotid saliva within the first year after a T-.

A vaccine against dental caries: an overview.

Dental caries continues to be a costly and prevalent oral disease. Research efforts towards developing a well tolerated and effective vaccine against dental caries were initiated following the demonstration of a specific bacterial aetiology for this disease. The cariogenic mutans streptococci are the principal bacteria causing this disease. Specific immune defence against these bacteria is provided mainly by secretory immunoglobulin (Ig) A antibodies present in saliva, which are generated by the common mucosal immune system. Progress in the development of a vaccine against dental caries has increased due to both advancements in molecular biology and our understanding of the mucosal immune system and mucosal vaccines. Advancements in molecular biology have facilitated the cloning and functional characterisation of virulence factors of the mutans streptococci, including the cell-surface fibrillar proteins, which mediate adherence to the tooth surface, and the glucosyltransferase enzymes, which synthesise adhesive glucans and allow microbial accumulation on the teeth. Current strategies for immunisation against dental caries are using these virulence factors as key antigens and incorporating them into novel mucosal vaccine systems and delivering them with or without adjuvants to mucosal IgA inductive sites. The most popular routes of mucosal immunisation are via the oral or nasal route. The mucosal immune system is functional in newborn infants, who develop salivary IgA antibodies as they become colonised by oral micro-organisms. Mucosal immunisation strategies result in the induction of salivary IgA antibody responses and pose fewer problems than parenteral injection of antigen. Therefore, mucosal immunisation of infants prior to the appearance of their first teeth may be a well tolerated and effective way to induce immunity against the colonisation of teeth by mutans streptococci and protection against subsequent dental caries. The purpose of this article is to provide an overview of the recent progress on the development of a vaccine against infection by Streptococcus mutans for the prevention of dental caries, with emphasis on the mucosal immune system and vaccine design.

Distinct cytokine regulation by cholera toxin and type II heat-labile toxins involves differential regulation of CD40 ligand on CD4(+) T cells.

Cholera toxin (CT) and the type II heat-labile enterotoxins (HLT) LT-IIa and LT-IIb act as potent systemic and mucosal adjuvants and induce distinct T-helper (Th)-cell cytokine profiles. In the present study, CT and the type II HLT were found to differentially affect cytokine production by anti-CD3-stimulated human peripheral blood mononuclear cells (PBMC), and the cellular mechanisms responsible were investigated. CT suppressed interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-alpha), and IL-12 production by PBMC cultures more than either LT-IIa or LT-IIb. CT but not LT-IIa or LT-IIb reduced the expression of CD4(+) T-cell surface activation markers (CD25 and CD69) and subsequent proliferative responses of anti-CD3-stimulated T cells. CT but not LT-IIa or LT-IIb significantly reduced the expression of CD40 ligand (CD40L) on CD4(+) T cells. In a coculture system, CT-treated CD4(+) T cells induced significantly less TNF-alpha and IL-12 p70 production by both autologous monocytes and monocyte-derived dendritic cells than either LT-IIa- or LT-IIb-treated CD4(+) T cells. These findings demonstrate that CT, LT-IIa, and LT-IIb differentially affect CD40-CD40L interactions between antigen-presenting cells and T cells and help explain the distinct cytokine profiles observed with type I and type II HLT when used as mucosal adjuvants.

Induction of protective immunity against Streptococcus mutans colonization after mucosal immunization with attenuated Salmonella enterica serovar typhimurium expressing an S. mutans adhesin under the control of in vivo-inducible nirB promoter.

The purpose of the present study was to evaluate the effectiveness of an attenuated Salmonella enterica serovar Typhimurium vaccine strain expressing the saliva-binding region (SBR) of the Streptococcus mutans antigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2 and B subunits (CTA2/B) and under the control of the anaerobically inducible nirB promoter, in inducing a protective immune response against S. mutans infection. BALB/c mice were immunized by either the intranasal or the intragastric route with a single dose of 10(9) or 10(10) Salmonella CFU, respectively. The Salmonella vaccine strain expressing an unrelated antigen (fragment C of tetanus toxin [TetC]) was also used for immunization as a control. Samples of serum and secretion (saliva and vaginal washes) were collected prior to and following immunization and assessed for antibody activity by enzyme-linked immunosorbent assay. Anti-SBR antibodies were detected in the serum and saliva of experimental animals by week 3 after immunization. A booster immunization at week 17 after the initial immunization resulted in enhanced immune responses to the SBR. The serum immunoglobulin G subclass profiles were indicative of T helper type 1 responses against both the vector and the SBR antigen. To determine the effectiveness of these responses on the protection against S. mutans infection, mice were challenged after the second immunization with a virulent strain of S. mutans which was resistant to tetracycline and erythromycin. Prior to the challenge, mice were treated for 5 days with tetracycline, erythromycin, and penicillin. S. mutans was initially recovered from all of the challenged mice. This bacterium persisted at high levels for at least 5 weeks in control TetC-immunized or nonimmunized mice despite the reappearance of indigenous oral organisms. However, mice immunized with Salmonella clones expressing SBR or SBR-CTA2/B demonstrated a significant reduction in the number of S. mutans present in plaque compared to the control groups. These results provide evidence for the effectiveness of the Salmonella vector in delivering the SBR antigen for the induction of mucosal and systemic immune responses to SBR. Furthermore, the induction of a salivary anti-SBR response corresponded with protection against S. mutans colonization of tooth surfaces.

Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages.

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.

Recombinant antigen-enterotoxin A2/B chimeric mucosal immunogens differentially enhance antibody responses and B7-dependent costimulation of CD4(+) T cells.

The ADP-ribosylating enterotoxins, cholera toxin (CT) and the Escherichia coli heat-labile toxin (LT-IIa), have been shown to enhance mucosal and systemic antibody (Ab) responses to coadministered antigens. The purpose of the present study was to compare the ability of the nontoxic A2/B subunits of these toxins, which have distinct targeting properties, to augment the immunogenicity of a genetically coupled protein antigen. Structurally similar chimeric proteins were generated by genetically replacing the toxic A1 subunit of CT or LT-IIa with the saliva-binding region (SBR) from the streptococcal adhesin AgI/II. Intranasal immunization of BALB/c mice with either chimeric protein induced significantly higher plasma and mucosal anti-SBR immunoglobulin A (IgA) and IgG Ab responses than SBR alone. Moreover, compared to SBR-LT-IIaA2/B, SBR-CTA2/B elicited significantly higher levels of plasma IgG1 and salivary IgA anti-SBR Ab responses. Ex vivo and in vitro experiments revealed that SBR-CTA2/B selectively up-regulated B7-2 expression on murine B cells isolated from both the nasal associated lymphoid tissue, cervical lymph nodes, and spleen. In contrast, SBR-LT-IIaA2/B had little effect on B7-1 or B7-2 expression on B220(+), CD11b(+), or CD11c(+) cells. Analysis of the functional costimulatory activity of SBR-CTA2/B-treated B cells revealed a significant enhancement in anti-CD3-stimulated CD4(+) T-cell proliferative responses, and this proliferation was significantly reduced by treatment with anti-B7-2 but not with anti-B7-1 or isotype control Abs. Thus, SBR-CTA2/B and SBR-LT-IIaA2/B exhibit distinct patterns of antibody responses associated with differential effects on B7-2 expression and subsequent costimulatory effects on CD4(+) T cells.

Adjuvant activity of monophosphoryl lipid A for nasal and oral immunization with soluble or liposome-associated antigen.

The effectiveness of monophosphoryl lipid A (MPL) as a mucosal adjuvant was investigated following oral or intranasal (i.n.) administration of an aqueous adjuvant formulation of MPL (MPL-AF) added to soluble antigen or liposomal antigen or incorporated into liposomal antigen membranes. Groups of BALB/c female mice were immunized with 50 to 100 microg of free or liposomal Streptococcus mutans crude glucosyltransferase (C-GTF) with or without MPL-AF added to the vaccine or incorporated into the liposomal membrane. Plasma, saliva, vaginal wash, and fecal extract samples were collected biweekly following immunization and assessed for antigen-specific antibody activity by enzyme-linked immunosorbent assay (ELISA). Mice immunized by the i.n. route had higher levels of salivary, plasma, and vaginal immunoglobulin A (IgA) anti-C-GTF responses and higher levels of plasma IgG anti-C-GTF than the orally immunized groups. A second administration of the vaccine 14 weeks after the initial immunization resulted in an anamnestic response to C-GTF resulting in 10- and 100-fold increases in saliva and plasma IgA and plasma IgG, respectively (in the i.n. immunized groups). Mice receiving a second i.n. immunization with liposomal antigen and MPL-AF had higher salivary IgA anti-C-GTF responses than mice immunized with antigen plus MPL-AF or liposomal antigen (P < 0.05). Plasma IgG anti-C-GTF activity was highest in mice immunized by the i.n. route with antigen formulations containing MPL-AF (P < 0.05). These results demonstrate the effectiveness of MPL-AF as an adjuvant for potentiating mucosal and systemic immune responses to liposomal C-GTF following i.n. immunization.

Comparison of the standard and the computerized versions of the Symptom Check List (SCL-90-R): a randomized trial.

OBJECTIVE: The purpose of this study was to examine whether computer administration of the Symptom Check List (SCL-90-R) is equivalent to paper-and-pencil originals. METHOD: 282 psychosomatic outpatients were randomly assigned to computer or paper-and-pencil conditions. Statistical equivalence tests were used to examine psychometric equivalence for the means. Reliabilities and correlations were compared for the two methods of administration. RESULTS: No systematic differences were observed in group means for most of the subscales. Subjects of the computer-administered group scored higher on the SCL-90-R subscale 'Obsessive-Compulsive' and 'Anger-Hostility' than the control subjects. Gender and administration mode interaction was observed for one subscale, while age and administration interaction was observed for another subscale. CONCLUSION: Using computer-administered tests makes administration and scoring of tests more efficient. The differences between the two administration modes were small, although noticeable. Further research is needed to determine whether computer environment, computer experience and age may influence the test results.

Genetic characterization of a Streptococcus mutans LraI family operon and role in virulence.

Proteins belonging to the LraI (for "lipoprotein receptor antigen") family function as adhesins in several streptococci, as a virulence factor for endocarditis in at least one of these species, and potentially as metal transporters in many bacteria. We have identified and characterized the chromosomal locus containing the LraI family gene (designated sloC) from Streptococcus mutans, an agent of dental caries and endocarditis in humans. Northern blot analysis indicated that sloC is cotranscribed with three other genes. As with other LraI operons, the sloA and sloB genes apparently encode components of an ATP-binding cassette transport system. The product of the fourth gene, sloR, has homology to the metal-dependent regulator from Corynebacterium diphtheriae, DtxR. A potential binding site for SloR was identified upstream from the sloABCR operon and was conserved upstream from LraI operons in several other streptococci. Potential SloR homologs were identified in the unfinished genomic sequences from two of these, S. pneumoniae and S. pyogenes. Mutagenesis of sloC in S. mutans resulted in apparent loss of expression of the entire operon as assessed by Northern blot analysis. The sloC mutant was indistinguishable from its wild-type parent in a gnotobiotic rat model of caries but was significantly less virulent in a rat model of endocarditis. Virulence for endocarditis was restored by correction of the sloC mutation but not by provision of the sloC gene in trans, suggesting that virulence requires the expression of other genes in the sloC operon.

Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes.

Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that P. gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (beta1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to P. gingivalis (>10(9) bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and beta1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral P. gingivalis. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to P. gingivalis. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and beta1-integrin in lysates derived from MDCK cells exposed to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P. gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest that P. gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of P. gingivalis to initiate epithelial barrier destruction.