Stress,Relaxation and Saliva: A Follow-up Study Involving Clinical Endodontic Patients

Abstract

Dentally induced stress and relaxation-induced anxiety reduction recently have been correlated with salivary changes in private patients treated by a solo endodontist. Hypnosis was shown to be more effective than local anesthesia in anxiety reduction. Dental students and clinic patients were employed in this study in an attempt to replicate the previous findings. One additional salivary variable (pH) was examined. Twentynine endodontic clinic patients were treated, each by a different dental student. The patients completed dental anxiety questionnaires and had salivary samples taken prior to, and at the conclusion of, their initial endodontic treatments. Pain and anxiety were managed using local anesthesia, hypnosis, or nitrous oxide-oxygen, either alone or in combination. There were significant anxiety-reduction changes by the conclusion of the visits (p < 0.001) as measured by increased salivary volume, increased salivary translucency, reduced salivary protein, increased salivary pH and reduced questionnaire-determined anxiety level. Hypnosis and nitrous oxide-oxygen were significantly more effective (p < 0.05) than local anesthesia in anxiety reduction as measured by salivary changes and questionnaires. It can be concluded that saliva is an easily obtained fluid that can be used to determine levels of stress and relaxation.     

Reductions in Salivary Cortisol Are Associated with Mood Improvement During Relaxation Training Among HIV-Seropositive Men

This study examined salivary cortisol and mood during relaxation training in 30 symptomatic, HIV+ gay men participating in a 10-week, group-based cognitive-behavioral stress management intervention. Cortisol levels and mood were assessed within these sessions just before and after 45-min relaxation exercises given as part of each session. Participants also recorded their stress level and compliance with daily home relaxation practice. Presession cortisol levels decreased across the 10-week period and were related to decreases in global measures of total mood disturbance and anxious mood. Reductions in presession cortisol levels were also associated with decreases in self-reported stress level during home practice. Greater reductions in cortisol during the first three sessions were associated with more frequent relaxation practice at home. These findings suggest that salivary cortisol represents an objective neuroendocrine marker for changes in anxiety and distress observed during relaxation training in symptomatic, HIV-seropositive men.     

Salivary changes and dental caries as potential oral markers of autoimmune salivary gland dysfunction in primary Sjogren's syndrome

BACKGROUND: the classification criteria for primary Sjogren's syndrome (pSS) include a number of oral components. In this study we evaluated if salivary flow and composition as well as dental caries are oral markers of disease severity in pSS. METHODS: in 20 patients fulfilling the American-European Consensus criteria for pSS and 20 age-matched healthy controls whole and parotid saliva flow rates and composition, measures of oral dryness, scores of decayed, missing and filled tooth surfaces (DMFS), periodontal indices, oral hygiene, and dietary habits were examined. RESULTS: in pSS, salivary flow rates, pH, and buffer capacities were lower, and DMFS, salivary sodium and chloride concentrations higher than in the healthy controls. DMFS also correlated inversely to salivary flow rates and positively to oral dryness. Apart from slightly increased gingival index, and more frequent dental visits in pSS, the periodontal condition, oral hygiene or sugar intake did not differ between these two groups. In pSS, findings were correlated to labial salivary gland focus score (FS) and presence of serum-autoantibodies to SSA/SSB (AB). The patients having both presence of AB and the highest FS (>2) also had the highest salivary sodium and chloride concentrations, the lowest salivary phosphate concentrations, lowest salivary flow rates, and highest DMFS compared to those with normal salivary concentrations of sodium and chloride at a given flow rate. CONCLUSION: the salivary changes observed in some pSS patients reflect impaired ductal salt reabsorption, but unaffected acinar transport mechanisms, despite low salivary secretion. Our results suggest that changes in salivary flow and composition as well as dental caries may serve as potential markers of the extent of autoimmune-mediated salivary gland dysfunction in pSS. The study also indicates that the ductal epithelium is functionally affected in some pSS patients, which calls for future pathophysiological studies on the mechanisms underlying this impaired salt reabsorption.     

A Vaccine against Dental Caries

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.     

Characterization of preparations enriched for Streptococcus mutans fimbriae: salivary immunoglobulin A antibodies in caries-free and caries-active subjects.

The ability of bacteria to adhere to salivary pellicle-coated enamel tooth surfaces is a critical step in oral bacterial colonization. Oral bacteria adhere to receptors of host origin in salivary pellicle. Streptococcus mutans has been identified as the major etiological agent of human dental caries and composes a significant proportion of the oral streptococci in carious lesions. Bacterial fimbriae are small (100 to 300 nm) hairlike appendages emanating from the cell surface. Preparations enriched for S. mutans fimbriae were isolated by a shearing technique and alternating high- and low-speed centrifugations. A representative fimbrial preparation had two distinct double bands comprising four proteins of approximately 100 to 200 kDa and one faint band at 40 kDa on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblots and had demonstrable glucosyltransferase activity. Rabbit antisera raised against the preparation specifically stained the fuzzy coat of S. mutans, demonstrating short fimbria-like structures protruding 100 to 200 nm from the cell surface. Controls without antifimbria antibody did not exhibit this staining. There were significantly higher (P < or = 0.05) levels of salivary immunoglobulin A, but not serum immunoglobulin G, antibodies to the enriched S. mutans fimbria preparation by enzyme-linked immunosorbent assay from caries-free subjects than from caries-active subjects. The results suggest that S. mutans fimbriae may be an important adherence factor to which caries-free subjects mount a protective salivary immune response.     

The impact of abbreviated progressive muscle relaxation on salivary cortisol

The purpose of this study was to examine whether acute relaxation training, conducted on two separate occasions, would be associated with reliable reductions in subjective and physiological indices of stress. Forty-six experimental subjects were led through Abbreviated Progressive Relaxation Training (APRT) exercises during two laboratory sessions spaced exactly 1 week apart. Fifteen control subjects experienced two laboratory sessions where they sat quietly for an equal amount of time. Results indicated that a brief relaxation exercise led to experimental subjects having significantly lower levels of post-intervention heart rate, state anxiety, perceived stress, and salivary cortisol than control subjects, as well as increased levels of self-report levels of relaxation. The results of this study may have implications for the use of relaxation training in enhancing immune function.     

Effect of neonatal thymectomy on dental caries in rats.

The effect of T-cell depletion on susceptibility to dental caries after infection with Streptococcus mutans was studied. Rats were neonatally thymectomized (Tx) and infected with S. mutans 6715 or locally immunized with the homologous organism before infection. The Tx rats uniformly exhibited a higher level of infection with S. mutans and subsequently showed a greater extent of carious activity. Correlation studies were performed comparing the level of salivary and serum anti-S. mutans antibodies and the relative amount of dental caries. The results demonstrated that salivary immunoglobulin A antibody after immunization and infection, or infection only, showed a significant negative correlation with dental caries. Also, after local immunization, serum immunoglobulin G antibody showed a negative correlation with dental caries in the rats. These findings further support a major protective role for salivary immunoglobulin A in experimental dental caries in rats.     

Intranasal immunization against dental caries with a Streptococcus mutans-enriched fimbrial preparation

Streptococcus mutans has been identified as the major etiological agent of human dental caries. The first step in the initiation of infection by this pathogenic bacterium is its attachment (i.e., through bacterial surface proteins such as glucosyltransferases, P1, glucan-binding proteins, and fimbriae) to a suitable receptor. It is hypothesized that a mucosal vaccine against a combination of S. mutans surface proteins would protect against dental caries by inducing specific salivary immunoglobulin A (IgA) antibodies which may reduce bacterial pathogenesis and adhesion to the tooth surface by affecting several adhesins simultaneously. Conventional Sprague-Dawley rats, infected with S. mutans at 18 to 20 days of age, were intranasally immunized with a mixture of S. mutans surface proteins, enriched for fimbriae and conjugated with cholera toxin B subunit (CTB) plus free cholera toxin (CT) at 13, 15, 22, 29, and 36 days of age (group A). Control rats were either not immunized (group B) or immunized with adjuvant alone (CTB and CT [group C]). At the termination of the study (when rats were 46 days of age), immunized animals (group A) had significantly (P < 0.05) higher salivary IgA and serum IgG antibody responses to the mixture of surface proteins and to whole bacterial cells than did the other two groups (B and C). No significant differences were found in the average numbers of recovered S. mutans cells among groups. However, statistically fewer smooth-surface enamel lesions (buccal and lingual) were detected in the immunized group than in the two other groups. Therefore, a mixture of S. mutans surface proteins, enriched with fimbria components, appears to be a promising immunogen candidate for a mucosal vaccine against dental caries.     

Antigenic characterization of fimbria preparations from Streptococcus mutans isolates from caries-free and caries-susceptible subjects.

The adhesion of pathogenic bacteria to the host surface is an essential step in the development of numerous infections, including dental caries. Attachment of Streptococcus mutans, the main etiological agent of human dental caries, to the tooth surface may be mediated by glucan synthesized by glucosyltransferase (GTF) and by cell surface proteins, such as P1, which bind to salivary receptors. Fimbriae on the surfaces of many microorganisms are known to function in bacterial adhesion. Previous studies in this laboratory have initially characterized the fibrillar surface of S. mutans. The purpose of this investigation was the comparison of the antigenic properties of fimbria preparations of S. mutans isolates from five caries-resistant (CR) and six caries-susceptible (CS) subjects. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of S. mutans fimbrial preparations revealed five major protein bands at 200, 175, 157, 86, and 66 kDa in preparations from CR and CS subjects. Immunoblot analysis indicated the presence of the same major bands recognized by anti-S. mutans fimbria antisera. Furthermore, the 175- and 157-kDa bands were recognized by antibodies to P1 and GTF, respectively. Immunoblot analysis with antisera to the fimbria preparation, to P1, or to GTF indicated that the levels of fimbria-reactive components and P1 and GTF antigens were higher in S. mutans fimbria preparations from CS subjects than in those from CR individuals. For example, four of six fimbria preparations from CS patients had demonstrable P1, and all had GTF. In contrast, only two of five CR fimbrial preparations exhibited P1 and GTF. Enzyme-linked immunosorbent assay demonstrated similar results for levels of GTF antigen in the fimbrial preparations from CR and CS subjects. The results suggest that differences between the compositions of S. mutans fimbriae in CR and CS individuals may play an important role in the virulence of this microorganism in dental caries.     

Passive transfer of immunoglobulin Y antibody to Streptococcus mutans glucan binding protein B can confer protection against experimental dental caries

Active immunization with Streptococcus mutans glucan binding protein B (GBP-B) has been shown to induce protection against experimental dental caries. This protection presumably results from continuous secretion of salivary antibody to GBP-B, which inhibits accumulation of S. mutans within the oral biofilm. The purpose of this study was to explore the influence of short-term (9- or 24-day) passive oral administration of antibody to S. mutans GBP-B on the longer-term accumulation and cariogenicity of S. mutans in a rat model of dental caries. Preimmune chicken egg yolk immunoglobulin Y (IgY) or IgY antibody to S. mutans GBP-B was supplied in lower (experiment 1) and higher (experiment 2) concentrations in the diet and drinking water of rats for 9 (experiment 1) or 24 (experiment 2) days. During the first 3 days of IgY feeding, all animals were challenged with 5 x 10(6) streptomycin-resistant S. mutans strain SJ-r organisms. Rats remained infected with S. mutans for 78 days, during which rat molars were sampled for the accumulation of S. mutans SJ-r bacteria and total streptococci. Geometric mean levels of S. mutans SJ-r accumulation on molar surfaces were significantly lower in antibody-treated rats on days 16 and 78 of experiment 2 and were lower on all but the initial (day 5) swabbing occasions in both experiments. Relative to controls, the extent of molar dental caries measured on day 78 was also significantly decreased. The decrease in molar caries correlated with the amount and duration of antibody administration. This is the first demonstration that passive antibody to S. mutans GBP-B can have a protective effect against cariogenic S. mutans infection and disease. Furthermore, this decrease in infection and disease did not require continuous antibody administration for the duration of the infection period. This study also indicates that antibody to components putatively involved only in cellular aggregation can have a significant effect on the incorporation of mutans streptococci in dental biofilm.     

Hydrophobic interaction chromatography and capillary zone electrophoresis to explore the correlation between the isoenzymes of salivary alpha-amylase and dental caries

High-performance hydrophobic interaction chromatography (HIC) and capillary zone electrophoresis (CZE) were utilized in an effort to find the correlation between the composition of some isoenzymes of human salivary alpha-amylase (HSA) and dental caries. The mixture of more than three isoenzymes of HSA, fractionated from human parotid saliva with HIC, was further separated by CZE at the optimum pH 6.50. The composition and relative quantity of these isoenzymes were compared between two groups of individuals with different caries-susceptibility. It is found that the present frequency of peak II on CZE in the caries-free group was higher than that in the caries-active group and the relative quantities of peak III and peak IV showed remarkable differences (p < 0.05) between the two groups. These results may indicate that the composition of HSA isoenzymes is related to the occurrence of dental caries. However, more work should be done to further affirm this correlation between the isoenzymes of salivary alpha-amylase and dental caries.     

Intranasal Immunization against Dental Caries with a Streptococcus mutans-Enriched Fimbrial Preparation

Streptococcus mutans has been identified as the major etiological agent of human dental caries. The first step in the initiation of infection by this pathogenic bacterium is its attachment (i.e., through bacterial surface proteins such as glucosyltransferases, P1, glucan-binding proteins, and fimbriae) to a suitable receptor. It is hypothesized that a mucosal vaccine against a combination of S. mutans surface proteins would protect against dental caries by inducing specific salivary immunoglobulin A (IgA) antibodies which may reduce bacterial pathogenesis and adhesion to the tooth surface by affecting several adhesins simultaneously. Conventional Sprague-Dawley rats, infected with S. mutans at 18 to 20 days of age, were intranasally immunized with a mixture of S. mutans surface proteins, enriched for fimbriae and conjugated with cholera toxin B subunit (CTB) plus free cholera toxin (CT) at 13, 15, 22, 29, and 36 days of age (group A). Control rats were either not immunized (group B) or immunized with adjuvant alone (CTB and CT [group C]). At the termination of the study (when rats were 46 days of age), immunized animals (group A) had significantly (P < 0.05) higher salivary IgA and serum IgG antibody responses to the mixture of surface proteins and to whole bacterial cells than did the other two groups (B and C). No significant differences were found in the average numbers of recovered S. mutans cells among groups. However, statistically fewer smooth-surface enamel lesions (buccal and lingual) were detected in the immunized group than in the two other groups. Therefore, a mixture of S. mutans surface proteins, enriched with fimbria components, appears to be a promising immunogen candidate for a mucosal vaccine against dental caries.     

Salivary immunoglobulin A and serum antibodies to Streptococcus mutans ribosomal preparations in dental caries-free and caries-susceptible human subjects

Caries-free subjects or individuals with low caries susceptibility exhibited significantly higher (P less than 0.001) levels of naturally occurring salivary immunoglobulin A (IgA) and serum IgG, IgA, and IgM antibodies to a Streptococcus mutans ribosomal preparation than subjects with high caries susceptibility. Absorption of saliva and serum samples with S. mutans ribosomal preparations, but not with other S. mutans antigens or with Escherichia coli and Neisseria gonorrhoeae ribosomal preparations, removed the antibody activity. Absorption with Streptococcus sanguis ribosomes and NH4Cl-washed S. mutans ribosomes partially removed the anti-S. mutans ribosome antibody activity. These results provide evidence that naturally occurring salivary and serum antibodies to the S. mutans ribosomal preparation correlate with susceptibility to dental caries.     

Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay.

To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S-nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91-kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.     

Molecular analysis of bacterial species associated with childhood caries

Although substantial epidemiologic evidence links Streptococcus mutans to caries, the pathobiology of caries may involve more complex communities of bacterial species. Molecular methods for bacterial identification and enumeration now make it possible to more precisely study the microbiota associated with dental caries. The purpose of this study was to compare the bacteria found in early childhood caries (ECC) to those found in caries-free children by using molecular identification methods. Cloning and sequencing of bacterial 16S ribosomal DNAs from a healthy subject and a subject with ECC were used for identification of novel species or uncultivated phylotypes and species not previously associated with dental caries. Ten novel phylotypes were identified. A number of species or phylotypes that may play a role in health or disease were identified and warrant further investigation. In addition, quantitative measurements for 23 previously known bacterial species or species groups were obtained by a reverse capture checkerboard assay for 30 subjects with caries and 30 healthy controls. Significant differences were observed for nine species: S. sanguinis was associated with health and, in order of decreasing cell numbers, Actinomyces gerencseriae, Bifidobacterium, S. mutans, Veillonella, S. salivarius, S. constellatus, S. parasanguinis, and Lactobacillus fermentum were associated with caries. These data suggest that A. gerencseriae and other Actinomyces species may play an important role in caries initiation and that a novel Bifidobacterium may be a major pathogen in deep caries. Further investigation could lead to the identification of targets for biological interventions in the caries process and thereby contribute to improved prevention of and treatment for this significant public health problem.     

Protective salivary immunoglobulin A responses against Streptococcus mutans infection after intranasal immunization with S. mutans antigen I/II coupled to the B subunit of cholera toxin.

The B subunit of cholera toxin (CTB) has been shown to augment mucosal responses to microbial virulence antigens, including those of Streptococcus mutans, which is the principal etiologic agent of dental caries. In the present study, the surface fibrillar protein antigen of S. mutans, antigen I/II (Ag I/II), was chemically coupled to CTB (Ag I/II-CTB), and the conjugate was examined for its effectiveness in inducing salivary immune responses protective against S. mutans infection. Weanling Fischer rats were given Ag I/II-CTB (50 micrograms) by the intranasal route and then orally infected with a virulent strain of S. mutans. Gnotobiotic or conventional rats were given two or three additional immunizations, respectively, at about 2-week intervals. One week after each immunization, individual serum, saliva, and fecal samples were collected and stored frozen until assayed for antibody activity to Ag I/II and cholera toxin (CT) by an enzyme-linked immunosorbent assay. The rats were sacrificed 1 week after the last immunization, when mandibles were also collected from individual rats for assessment of S. mutans levels in plaque and caries activity. Rats immunized only or both immunized and infected showed a salivary immunoglobulin A (IgA) anti-Ag I/II response which reached significantly (P < 0.05) higher levels than those seen in nonimmunized, infected controls. A salivary IgA anti-Ag I/II response was also seen in rats infected only with S. mutans. Essentially no salivary antibody activity to CT was detected. Some serum anti-Ag I/II and anti-CT responses were seen in immunized animals. Serum IgG anti-Ag I/II responses were seen in immunized, infected rats and also in infected-only rats, suggesting that the responses were a result of infection with S. mutans. The immunized and infected rats had significantly (P < 0.05) lower levels of S. mutans in plaque and lower caries activity than nonimmunized, infected rats. These results indicated that intranasal immunization of rats with Ag I/II-CTB induced a protective salivary immune response which was associated with a reduction in S. mutans colonization and S. mutans-induced dental caries.     

Is a drill-less dental filling possible?

Dental caries, a bacterial process that results in the acidic destruction of tooth structure, has historically been managed by the mechanical excavation of diseased tooth structure and then restoration with a synthetic material. The mechanical excavation of the infected site is most commonly achieved by a dental handpiece, or “drill”; this handpiece may induce stress and anxiety in many patients. Alternatively, a drill-less filling will involve the utilization of silver diamine fluoride (38%) to arrest and prevent dental caries, followed by restoration with a bonded filling material to achieve adequate seal at the lesion margins. This is a minimally invasive procedure that addresses both microbial and mechanical issues posed by dental caries.     

Associations of MHC genes with levels of caries-inducing organisms and caries severity in African-American women

The aim of this investigation was to evaluate the relationship between MHC alleles at the HLA-DRB1, DQB1 and TNFa microsatellite loci and levels of oral bacteria that play a role in the etiology of dental caries, and the DMFS index in 186 AA primparous women. The average age of the cohort was 20.8+/-3.7 years. The median DMFS index was 9 (range 0-68). High levels of S. mutans were positively associated with DRB1*3 and DRB1*4 presence (p < or = 0.005). DRB1*8 was positively associated with higher levels of S. mutans as a percentage of total Streptococci (p = 0.04). DRB1*1 was positively associated with high levels L. casei (p = 0.04). DQB1 alleles were not observed associated with oral bacterial levels. TNFa allele 103 was negatively associated (p = 0.04), and TNFa 117 was positively associated (p = 0.007), with high levels of L. acidophilus. No significant associations were observed between any DRB1, DQB1 or TNFa allele and the DMFS index. These results support an hypothesis of an association between host HLA class II and TNFa genetic profile and colonization of S. mutans, L. casei, and L. acidophilus thought to be pathogens involved in the etiology of dental caries.     

Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: dose response and induction of protective humoral responses in rats.

An attenuated, recombinant Salmonella typhimurium mutant, chi 4072(pYA2905), expressing the surface protein antigen A (SpaA) of Streptococcus sobrinus was investigated for its effectiveness in inducing protective immune responses against S. sobrinus-induced dental caries in an experimental caries model. Fischer rats were orally immunized with either 10(8) or 10(9) CFU of S. typhimurium chi 4072(pYA2905). Persistence of salmonellae in Peyer's patches and spleens and the induction of immune responses were determined. Maximum numbers of salmonellae were recovered from Peyer's patches of rats within the first week of immunization, with higher numbers recovered from rats given 10(9) CFU than from those given 10(8) CFU. Serum anti-Salmonella and anti-SpaA responses increased more rapidly in rats given 10(9) CFU than in rats given 10(8) CFU. The salivary antibody response to SpaA increased with time, but the response varied in the two groups. In a separate study, rats were orally immunized with the recombinant Salmonella mutant and then challenged with cariogenic S. sobrinus 6715. The levels of serum and salivary antibody and caries activity were assessed at the termination of the experiment. Higher levels of salivary immunoglobulin A antibody to SpaA and Salmonella carrier were detected in rats given 10(9) CFU than in those given 10(8) CFU, and these responses were higher than those in nonimmunized controls. Mandibular molars from immunized rats had lower numbers of recoverable streptococci and less extensive carious lesions than those from nonimmunized, control rats. These data indicate that oral immunization with an attenuated recombinant S. typhimurium expressing SpaA of S. sobrinus induces the production of antigen-specific mucosal antibody and confers protection against dental caries.     

Diepitopic construct of functionally and epitopically complementary peptides enhances immunogenicity, reactivity with glucosyltransferase, and protection from dental caries

Coimmunization with peptide constructs from catalytic (CAT) and glucan-binding (GLU) domains of glucosyltransferase (GTF) of mutans streptococci has resulted in enhanced levels of antibody to the CAT construct and to GTF. We designed and synthesized a diepitopic construct (CAT-GLU) containing two copies of both CAT (B epitope only) and GLU (B and T epitope) peptides. The immunogenicity of this diepitopic construct was compared with that of individual CAT and GLU constructs by immunizing groups of Sprague-Dawley rats subcutaneously in the salivary gland vicinity with the CAT-GLU, CAT, or GLU construct or by treating rats by sham immunization. Levels of serum immunoglobulin G (IgG) antibody to GTF or CAT in the CAT-GLU group were significantly greater than in GLU- or CAT-immunized groups. Immunization with CAT-GLU was compared to coimmunization with a mixture of CAT and GLU in a second rodent experiment under a similar protocol. CAT-GLU immunization resulted in serum IgG and salivary IgA responses to GTF and CAT which were greater than after coimmunization. Immunization with the diepitopic construct and communization with CAT and GLU constructs showed proliferation of T lymphocytes to GTF. Immunization with either the CAT or GLU construct has been shown to elicit significant protection in a rodent dental caries model. Similarly in this study, the enhanced response to GTF after immunization with the CAT-GLU construct resulted in protective effects on dental caries. Therefore, the CAT-GLU diepitopic construct can be a potentially important antigen for a caries vaccine, giving rise to greater immune response than after immunization with CAT, GLU, or a mixture of the two.