Correlating Streptococcus mutans counts in saliva with plaque amount, gingival inflammation and caries experience in school children

The DNA methyltransferases, M.HhaI and M.TaqI, and catechol O-methyl-transferase (COMT) catalyze the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to carbon-5 of cytosine, to nitrogen-6 of adenine, and to a hydroxyl group of catechol, respectively. The catalytic domains of the bilobal proteins, M.HhaI and M.TaqI, and the entire single domain of COMT have similar folding with an alpha/beta structure containing a mixed central beta-sheet. The functional residues are located in equivalent regions at the carboxyl ends of the parallel beta-strands. The cofactor binding sites are almost identical and the essential catalytic amino acids coincide. The comparable protein folding and the existence of equivalent amino acids in similar secondary and tertiary positions indicate that many (if not all) AdoMet-dependent methyltransferases have a common catalytic domain structure. This permits tertiary structure prediction of other DNA, RNA, protein, and small-molecule AdoMet-dependent methyltransferases from their amino acid sequences.     

Tonsillar application of killed Streptococcus mutans induces specific antibodies in rabbit saliva and blood plasma without inducing a cross-reacting antibody to human cardiac muscle

When Streptococcus mutans cells are injected into the skeletal muscle of rabbits, an antibody against human cardiac muscle, as well as an anti-S. mutans antibody, is induced in blood plasma. Our previous study showed that when sheep erythrocytes are applied to palatine tonsils, an antibody against the applied cells is induced both in blood plasma and saliva. This antibody has no activity against cardiac muscle. It is not clear, however, if S. mutans application to the tonsils evokes an antibody response against cardiac muscle. In this study, we immunized rabbits against S. mutans or Streptococcus sobrinus by tonsillar application or by intramuscular injection every 3 days for 6 weeks. Tonsillar applications of formalin-killed cells of S. mutans induced saliva immunoglobulin A (IgA) and blood plasma IgG to the applied cells. In contrast, intramuscular injection of such cells induced only blood plasma IgG. When the route of immunization was intramuscular injection, antibodies in blood plasma cross-reacted with cardiac muscle. By enzyme-immunohistochemistry and Ouchterlony immunodiffusion tests, no cross-reaction to cardiac muscle was observed with the antibody in saliva or in blood plasma after the tonsillar applications. Western blotting of the S. mutans antigen showed that blood plasma from rabbits injected with S. mutans reacted with antigens of 46, 52, 62, and 85 kDa, while that from rabbits subjected to tonsillar application of S. mutans did not react with these bands. Similar results were obtained for S. sobrinus applications. Thus, tonsillar applications of mutants group streptococci induce antibodies differing in antigen specificity and do not induce any cross-reacting antibody to cardiac muscle.     

Effective immunity to dental caries: passive transfer to rats to antibodies to Streptococcus mutans elicits protection

Rat dams, given intravenous injections of heat-killed Streptococcus mutans 6715, mutant C211 demonstrated significant agglutinin activity to the homologous S. mutans in colostrum, milk, and serum. This antibody activity was associated with the immunoglobulin G (IgG) class. High titers of anti-S. mutans antibody associated with the IgG class were also exhibited in the sera and saliva of the offspring that suckled these dams. After challenge with the homologous, live S. mutans, these offspring developed significantly fewer caries on all molar surfaces than did nonimmunized infected controls. A secretory immune response (manifested by the presence of specific IgA antibody to S. mutans in colostrum and milk) was elicited (i) in rat dams locally injected, in the region of the mammary gland, with heat-killed S. mutans antigen, and (ii) in other rat dams that were provided formalin-killed S. mutans in their drinking water. Offspring suckling these dams were challenged with virulent S. mutans before weaning and developed significantly fewer caries than did their infected controls. These findings clearly suggest that passively derived IgG or IgA antibodies to S. mutans are protective against dental caries.     

Generation of bovine immune colostrum against Streptococcus mutans and Streptococcus sobrinus and its effect on glucose uptake and extracellular polysaccharide formation by mutans streptococci

Due to potential side-effects of active immunization by cariogenic mutans streptococci, oral administration of passively-derived antibodies could be a more acceptable way to reduce colonization and virulence of these microorganisms in human dentition. The aim of this study was to produce antistreptococcal immunoglobulins into bovine colostrum and explore the possible antibacterial mechanisms of these immunoglobulins against mutans streptococci. Specific serum IgG antibodies to whole cell antigens of both Streptococcus mutans and Streptococcus sobrinus increased rapidly in cows during immunization and were high also in the final whey-product. Low concentration (0.5% w/v) of bovine immune preparation inhibited significantly the incorporation of [14C]glucose by both S. mutans and S. sobrinus. Higher concentration (> 1%) was needed to inhibit the glucosyltransferase or fructosyltransferase activities of these bacteria. No such inhibitory effects were observed with the control preparation from the non-immunized cows. Our results indicate that bovine immune colostrum has a significant inhibitory potential against mutans streptococci, apparently dependent on the presence of specific IgG antibodies against S. mutans and S. sobrinus.     

Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants

In the present study, we compared the ability of the soluble adjuvants concanavalin A (ConA), muramyl dipeptide (MDP), and peptidoglycan (PG) to enhance immune responses to orally administered particulate antigens of Streptococcus mutans 6715 in gnotobiotic rats. The isotype and levels of antibody in saliva and in serum from experimental rats were determined by an enzyme-linked immunosorbent assay using S. mutans whole cells (WC) as the coating antigen. The specificities of salivary and serum immunoglobulin A (IgA) antibodies to particulate S. mutans antigens, lipoteichoic acid, S. mutans serotype g carbohydrate, and dextran were also determined. When 50 micrograms of ConA was used as the oral adjuvant with S. mutans 6715 WC immunogen, a slight enhancement of immune responses was obtained. A higher dose of ConA suppressed humoral responses to the immunogen. Enhanced immune responses, especially of the IgA isotype, in both serum and saliva were induced in gnotobiotic rats given MDP and either S. mutans 6715 WC or purified cell walls (CW) by gastric intubation. Elevated IgA antibody levels to CW, lipoteichoic acid, and carbohydrate were observed in rats given S. mutans WC and MDP by gastric intubation, whereas oral immunization with S. mutans CW and MDP resulted in higher antibody levels to CW and carbohydrate and lower levels to lipoteichoic acid when compared with the antibody levels in rats given antigen alone. Rats orally immunized with either S. mutans WC or CW and MDP and challenged with virulent S. mutans 6715 exhibited significantly (P less than or equal to 0.05) lower plaque scores, numbers of viable S. mutans in plaque, and caries scores than did rats immunized with antigen alone or in infected-only controls. In another series of experiments, a PG fraction derived from S. mutans 6715 CW was assessed for adjuvant properties. The oral administration of PG and either S. mutans WC or CW induced good salivary and serum IgA antibody responses. The specificity of the antibodies was similar to that obtained in rats given antigen and MDP. Rats receiving either S. mutans WC or CW and PG and challenged with virulent S. mutans 6715 had lower plaque scores, fewer numbers of viable S. mutans in plaque, and lower caries activity than did infected rats receiving S. mutans WC or CW immunogen alone. These results provide evidence that soluble adjuvants derived from the gram-positive bacterial CW, e.g., MDP and PG, are effective oral adjuvants and augment IgA immune responses to particulate S. mutans antigens which are protective against the mucosally associated disease, dental caries.     

Detection of Streptococcus mutans by PCR amplification of the spaP gene in teeth rendered caries free

OBJECTIVES: To investigate the degree of association between tactile and optical criteria as used to assess the carious status of the enamel-dentine junction (EDJ) during cavity preparation, assessment with a caries detector dye and detection of Streptococcus mutans using culture and polymerase chain reaction (PCR) techniques. METHODS: Twenty-nine teeth, extracted within the previous 30 min, and 15 teeth prepared under rubber dam in vivo, were clinically assessed at the EDJ after the removal of evident carious tissue. Demineralisation was then assessed using a caries detector dye (1% acid red in propylene glycol; Cavex). A rosehead bur was used to remove tissue at the EDJ for culture and PCR analysis. Culture was carried out on a tryptone yeast cystine sucrose bacitracin selective medium, and PCR used to amplify a sequence (192 bp) of the spaP gene, which encodes the surface protein antigen I/II of S. mutans. RESULTS: Demineralised tissue at the EDJ, as shown using the dye, was found in 52% of teeth. Removed tissue was culture and PCR positive for S. mutans in 2 and 47% of teeth, respectively. A highly significant association (77% of cases; P < 0.001) was shown between dye and PCR assessment methods. No association was found between any other combination of assessment methods. CONCLUSIONS: Culture methods may underestimate the presence of S. mutans. Removal of sufficient dye-stained tissue is therefore recommended to prevent further carious assault from residual S. mutans.     

Influence of secretory immunoglobulin A and purified secretory component on dextran-sucrase activity of Streptococcus mutans

The net stimulation of dextransucrase EC 2.4.1.5) activity from Streptococcus mutans HS6 by dextran, secretory immunoglobulin A, or secretory component was investigated. Approximately equal stimulation resulted from treatment with these three components.     

The clinician''s ability to identify caries risk subjects without saliva tests--a pilot study

The results of many studies on the effects of mutagens on germ cells of the testes indicate that the recommendation of Carson et al. (Hum. Reprod., 6, 992-994, 1989) that semen samples collected during chemotherapy and cryopreserved may be safely used for insemination is incorrect because of the large potential for genetic risk to the offspring. Data from experimental animals, which indicate high levels of mutagenic effects in offspring from matings during or soon after treatment of the male with chemotherapy or radiation, are reviewed. The relevance of these data to the human situation is discussed. Based on these results, I recommend that patients not only complete their semen cryopreservation prior to the initiation of chemotherapy, but that they practice reliable contraception from the time of initiation of treatment until 6 months after completion of treatment.     

Cross-reactions of Streptococcus mutans due to cell wall teichoic acid

Antisera to the whole cells of Streptococcus mutans cross-reacted with antigen extracts from four other gram-positive species, as well as with those of three other oral streptococci. Similarly, antisera to these bacteria cross-reacted with extracts from S. mutans and with those from each other. Using a purified phenol extract of the walls of S. mutans, which was identified by chemical, immunochemical, and enzymatic analyses as glycerol teichoic acid, the cross-reactions were shown to be specific for a determinant of the teichoic acid backbone. Results were confirmed in immunodiffusion tests where clear bands of identify were shown. These observations point out the need for caution in sereological research empolying extracts of gram-positive bacteria and may be of interest in investigations of periodontal disease.     

Expression of the PAC gene of S. mutans in S. lactis

Expression of the pac gene in S. lactis were analyzed by dot immunoblotting with rabbit anti-PAc serum. The amount of PAc produced by S. lactis HL107 (pLF107) was measured. This result indicated that a small amount of rPAc was detected in the culture supernatant and cell homogenate of S. lactis HL107 (pLF107) but not in the control strain S. lactis LM0230. The amount of rPAc in S.lacits HL107 (pLF107) was six times lower than that in S. mutans Ingbritt. Expression of cloned pac gene in S. lactis was also confirmed in this result.     

The effect of fluoride on Streptococcus sanguis 7863 IgA1 protease production and activity

Fluoride was found to affect the production of the bacterial IgA1 protease but to have no effect on IgA1 protease activity. The concentrations of fluoride that do affect Streptococcus sanguis growth and IgA1 protease production are higher than those normally seen in vivo under normal circumstances. The concentrations of fluoride in dental plaque following use of a fluoride rinse or dentifrice would be sufficient to reduce Strep. sanguis IgA1 protease production.     

Tonin activity in rat saliva: effect of sympathomimetic and parasympathomimetic drugs

The effects of carbachol, propranolol and isoproterenol on tonin concentrations in rat saliva have been investigated. The results suggest that the activity of this enzyme in rat saliva is dependent on activity of beta-adrenergic receptors.     

Combined inhibitory effect of bovine immune whey and peroxidase-generated hypothiocyanite against glucose uptake by Streptococcus mutans

A case of chronic ulcer of the lower lip, which persisted for three years and resisted all locals treatments, is presented. Recovery was achieved with excision. The specimen was examined macroscopically, and showed a prominent inferior labial artery or calibre-persistent artery, an uncommon entity. Patient-literature was reviewed.     

Concentrated bovine colostral whey proteins from Streptococcus mutans/Strep. sobrinus immunized cows inhibit the adherence of Strep. mutans and promote the aggregation of mutans streptococci

The aim of this study was to examine the effect of bovine colostral whey proteins from cows immunized with Streptococcus mutans/Strep. sobrinus on the adherence and aggregation of caries-inducing bacteria, i.e., mutants streptococci. Both adherence and aggregation are important phenomena in the bacterial colonization of the human oral cavity. In all adherence experiments there was a significant difference between treatments by immune product (IP; from immunized cows) and a control product (CP; a similar product from non-immunized cows). The adherence of 35S-labelled Strep. mutans cells (serotype c) to parotid saliva-coated hydroxyapatite (SHA) was dose-dependently inhibited by both IP and CP if SHA was coated with either product before exposure to bacteria, but markedly lower concentrations of IP than CP were effective. When instead of SHA the bacterial cells were pretreated with IP or CP, only IP strongly and dose-dependently inhibited streptococcal adherence. When bacteria, IP or CP, and SHA were incubated simultaneously, a significant difference between IP and CP treatments was again found. Further, IP effectively aggregated both Strep. mutans and Strep. sobrinus cells, whereas hardly any effect was seen with CP. Both IP and CP aggregated the control bacterium Strep. sanguis, which affected the adherence of the pretreated bacteria.     

Effects of surfactants on glucosyltransferase production and in vitro sucrose-dependent colonization by Streptococcus mutans

OBJECTIVE: To study the distribution of myocardial structural elements in a univentricular experimental model (Halobatrachus didactylus) and the changes induced by acute exposure to a sublethal concentrations of cadmium. DESIGN: 15 specimens of H. didactylus (marine teleost) were divided in three groups: CTRL: the control group, the fish were injected with a saline solution; 24 H: 1 mg/kg of cadmium chloride was injected and fish were sacrificed after 1 day post injection; 7 D: fish where subjected to the same cadmium concentration and sacrificed after 7 days post injection. INTERVENTIONS: Histological sections (5 microns) of ventricle were coloured by PicroSirius method and used to determine the fraction area occupied by the several myocardium structural elements, using specific software Optimas Bioscan 5.2. MEASUREMENTS AND RESULTS: The different structural elements of H. didactylus myocardium displayed in different ways and its distribution along the ventricular wall remained unchanged on exposure to cadmium. The structural elements of H. didactylus myocardium showed a characteristic pattern: collagen type I revealed a heterogeneous distribution, with fractional area values greater in epicardial and endocardial layers; collagen type III, has a homogeneous distribution with the myocardial wall thickness with lesser fractional area values than collagen I; cardiac muscle tissue showed a tendency to occupy a greater fractional area in the epicardial-endocardial region. Cadmium toxicity resulted in an increase in the fractional area occupied by muscle tissue to the detriment of the area occupied by the intertrabecular spaces (lacunae), with no alterations in the other structural components. CONCLUSIONS: On our H. didactylus fish model, cadmium may have induced a myocardial edema which resulted in an increase in the fractional area occupied by muscle tissue, with no alterations in other structural components of the myocardium or in the relative ventricular mass.     

Identification of essential amino acids in the Streptococcus mutans glucosyltransferases

A comparison of the amino acid sequences of the glucosyltransferases (GTFs) of mutans streptococci with those from the alpha-amylase family of enzymes revealed a number of conserved amino acid positions which have been implicated as essential in catalysis. Utilizing a site-directed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp413, Trp491, and His561, as being important in enzymatic activity. Mutagenesis of Asp413 to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp491 or His561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes.     

HLA-DR4 and number of mutans streptococci in saliva among dental students and staff

OBJECTIVE: To evaluate the ratio of peak systolic to least diastolic flow velocity (S/D), pulsatile index (PI) and resistance index (RI) of the umbilical arterial doppler flow at 20-40 gestational weeks in twin pregnancy for the surveillance of twin transfusion syndrome. METHODS: The fetal hemodynamics in 36 cases of twin pregnancy were monitored by Combison A-ZIPE 4871 with spectral doppler ultrasound. RESULTS: The value of S/D ratio and PI in 4 patients with twin transfusion syndrome at 32-34 weeks were significantly higher than those of normal twin (P < 0.01). Two cases of twin reversed arterial pressure (TRAP), i.e. artery to artery transfusion in one of the twin with acardius, were found at 26 and 26 + 4 weeks respectively. CONCLUSION: The S/D ratio and PI of umbilical artery might be useful indices for the surveillance of twin transfusion syndrome.     

Detection of IgG antibody to Epstein-Barr virus viral capsid antigen in saliva by antibody capture radioimmunoassay

In order to characterize the cochlear transducer nonlinearities which are involved in the generation of the summating potential (SP), we investigated the effect of a change in the electrical operating point of the cochlear transducer on the SP. The electrical operating point of the cochlear transducer was affected by suppressing reversibly the endocochlear potential (EP). This was realized by intravenous injection of furosemide in guinea pig. A differential recording technique was used in the basal turn of the cochlea to measure locally generated even-order distortion products: the SP and the second harmonic component (2F0) of the cochlear microphonics (CM). These potentials were evoked by 2 and 8 kHz stimuli presented at 60 dB SPL. Following furosemide injection, the SP changed polarity twice over time. The zero crossings of the SP coincided with a minimum in the amplitude of 2F0. Concomitantly, the phase of 2F0 shifted about 120 degrees. The changes in the electrical even-order products were comparable to the changes that occurred in a mechanical even-order intermodulation distortion product (the difference tone F2-F1 otoacoustic emission) after furosemide application (Mills et al., J. Acoust. Soc. Am. 94 (1993) 2108-2122). The combined results suggest that only one sigmoidal transfer function may account for the SP, 2F0, and the emission of the difference tone F2-F1, and that shifts in the operating point of the transfer function would be the major cause behind the furosemide-induced changes in the even-order distortion products. The sigmoidal transfer function is likely associated with the mechano-electrical transducer channel at the apical pole of the outer hair cell.