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Objective The effects of probiotics on cariogenic biofilms remain controversial. Our aim was to characterise two probiotic Lactobacillus reuteri strains, ATCC PTA 5289 and ATCC 55730 from a cariogenic standpoint in vitro . These strains are used in commercial products designed for oral health purposes. Design The adhesion and biofilm formation were studied on saliva-coated hydroxyapatite. The effects of glucose or sucrose on the biofilm formation were also tested. Arginine metabolism was assessed by measuring the pH in the presence of glucose and arginine. The degradation of hydroxyapatite was measured in three different growth media. Streptococcus mutans strains Ingbritt and MT 8148 were used as positive controls for bacterial adhesion and degradation of hydroxyapatite. Results Strain ATCC PTA 5289 adhered on saliva-coated hydroxyapatite and formed detectable biofilm, but strain ATCC 55730 was poor in both adhesion and biofilm formation. Both strains were arginolytic and raised the pH in the presence of arginine. The amount of dissolved calcium from hydroxyapatite correlated with bacterial growth rate and the final pH of the growth medium. Conclusion L. reuteri strains ATCC PTA 5289 and ATCC 55730 differed in their adhesion, biofilm formation and arginine metabolism in vitro . Thus, these probiotic lactobacilli are likely to differ in their behaviour and cariogenic potential also in an oral environment.

Serum and salivary total IgA, IgG and IgM as well as salivary innate non-immunoglobulin antimicrobial factors (lysozyme, lactoferrin, salivary and leukocyte peroxidase systems) were measured in 13 children prone to recurrent respiratory infections and compared to their age-matched healthy controls. Serum IgG and IgM levels were significantly elevated and salivary IgA remarkably low in infection-prone children as compared to the controls. However, the levels of secretory piece-bearing IgA were about the same in both groups. There were no significant differences between the two groups in serum IgA levels or in any of the non-immunoglobulin factors. The results indicate that low salivary IgA is associated with recurrent respiratory infections.

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340.

Introduction: Most probiotic products are consumed orally and hence it is feasible that the bacteria in these products may also attach to oral surfaces; however, the effects of these bacteria on the oral ecosystem are mostly unknown. Our aim was to evaluate the oral colonization potential of different probiotic, dairy, and fecal Lactobacillus and Bifidobacterium strains in vitro. Methods: The binding of 17 Lactobacillus and seven Bifidobacterium strains to hydroxyapatite and microtitre wells coated with human saliva was tested. Binding of selected strains to human buccal epithelial cells and co-adherence with Fusobacterium nucleatum were also investigated. In addition, the survival in sterilized human whole saliva was examined. Results: There was a large variation in binding to saliva-coated surfaces and buccal epithelial cells but all strains survived in saliva. The binding pattern of the probiotics did not differ from the binding of the fecal strains. F. nucleatum altered the binding of both the low-binding bifidobacteria and the high-binding lactobacilli. Conclusion: The differences in binding in vitro may indicate that there are also differences in the persistence of the different probiotic strains in the oral cavity in vivo.

Human whole saliva contains two peroxidases, salivary peroxidase (hSPO) and myeloperoxidase (hMPO), which are part of the innate host defence in oral cavity. Both hSPO as well as human milk lactoperoxidase (hLPO) are coded by the same gene, but to what extent the different producing glands, salivary and mammary glands, affect the final conformation of the enzymes is not known. In human saliva the major function of hSPO and hMPO is to catalyze the oxidation of thiocyanate (SCN−) in the presence of hydrogen peroxide (H2O2) resulting in end products of wide antimicrobial potential. In addition cytotoxic H2O2 is degraded. Similar peroxidation reactions inactivate some mutagenic and carcinogenic compounds, which suggests another protective mechanism of peroxidases in human saliva. Although being target of an active antimicrobial research, the structure–function relationships of hSPO are poorly known. However, recently published method for recombinant hSPO production offers new tools for those investigations.

Helicobacter pylori has frequently been isolated from human dental plaque, and oral spread via saliva is thought to be one of its principal modes of transmission. Among other innate defence systems human saliva contains peroxidase enzymes and lysozyme. The sensitivity of H. pylori to physiological concentrations of lactoperoxidase and its salivary substrate thiocyanate, and different amounts of hydrogen peroxide (H(2)O(2)) was investigated in buffer and in human whole saliva. The effect of lysozyme was also studied in saliva. All tested H. pylori strains, ATCC 43504(T) and five clinical isolates, were efficiently inhibited by the peroxidase system with high concentrations of H(2)O(2) in buffer. The inhibition was stronger at lower pH. However, in human saliva these high concentrations of H(2)O(2) generated less hypothiocyanite, the antibacterial product of the peroxidase system and the effects of the peroxidase system were weaker. Physiological concentration of lysozyme was not bacteriocidal against H. pylori, nor did it enhance the effect of the peroxidase system in saliva. Thus, further studies are needed to enhance the efficacy of peroxidase systems in human saliva to make it more beneficial not only against dental but also against gastric pathogens.

Actinobacillus actinomycetemcomitans has an important aetiological role in localized juvenile periodontitis and in progressive periodontitis in adults. A. actinomycetemcomitans is found mainly in periodontal pockets but also in whole saliva, a potential transmission medium. It is sensitive to peroxidase–halide systems, but the differences between periodontitis associated clinical isolates and type strains are unclear. The sensitivities of these 2 strain groups to lactoperoxidase (LP)–iodide (I − )–hydrogen peroxide (H 2 O 2 ) combinations were investigated, and the sensitivities were compared with the susceptibilities to four antibiotics. There was great variation between the sensitivities of different strains, but the 2 strain groups responded similarly. The LP (75 μg)–I − (100 nmol)–H 2 O 2 (1000 nmol) combination produced a similar degree of inhibition as 2 μg ampicillin. The LP–I − system might be a potential antimicrobial agent against A. actinomycetemcomitans transmission via saliva.

Our aim was to study the effects of mildly flavoured sodium lauryl sulphate (SLS)-containing and detergent-free toothpastes with and without betaine (BET) on subjective symptoms of dry mouth in a randomised clinical trial. BET is an osmoprotectant that reacts with molecules to supply the surface with a water coating that protects cells from surfactants. Twenty-seven xerostomic patients and 18 healthy controls took part in the randomised, double-blind clinical trial with a crossover design. Three mildly flavoured toothpastes: (1) 4% BET, (2) 1% SLS and 4% BET, and (3) 1% SLS were used for six weeks each. The reference or washout paste contained neither SLS nor BET. The subjects’ dental appointments were at the beginning of the trial and before and after the use of each toothpaste. At each appointment, the subjects were interviewed about subjective sensations of dry mouth (Visual Assessment Scoring (VAS) Index). The subjects did not report any adverse effects in connection with the use of the toothpastes. The VAS scores for lip dryness and eating difficulties were significantly lower for the BET paste (lip dryness: BET Citation Rantanen I, Tenovuo J, Pienihäkkinen K, et. al. Effects of a betaine-containing toothpaste on subjective symptoms of dry mouth, a randomised clinical trial. J Contemp Dent Pract 2003 May;(4)2:011-023.

The concentration of total mercury in stimulated saliva was studied in humans with dental amalgam fillings and in 2 nonamalgam groups. The probability of exceeding the limits of mercury permitted in wastewater increased proportionally as the number of amalgam-filled surfaces increased. The mercury limit for sewage is 0.05 mg/l (= 250 nmol/l) effluent, according to the Council of European Communities directive 84/156/EEC. In neither of the nonamalgam groups was this limit exceeded, but 20.5% in the amalgam group exceeded the limit (p < .001). The risk of exceeding the limit increased 2-fold for every 10 additional amalgam-filled surfaces (odds ratio = 2.0; 95% confidence interval = 1.3, 3.3). These results demonstrate that humans, especially in populated areas, can be a significant source of mercury pollutants. As a consequence of mercury release, bacteria may acquire mercury resistance, as well as resistance to other antimicrobial agents, thus resulting in failure of antibiotic treatment.

Passive local immunization against dental caries is a promising approach to its prevention, as clinical evidence of active oral or nasal immunization is still limited and controversial. By means of systemic immunization of pregnant cows with a multivalent vaccine, high titres of IgG antibodies against human cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus, were produced in bovine colostrum. The purified immune product (IP) of this preparation has a number of anticariogenic properties, such as inhibition of streptococcal adherence to saliva-coated hydroxyapatite and inhibition of glucosyltransferase enzymes. This study investigated whether IP antibodies remained active and functional when added to ultra-high temperature (UHT)-treated milk or to Lactobacillus rhamnosus GG (LGG)-fermented milk stored for an extended time. LGG was chosen because of its widely known health benefits in humans and animals. A commercial UHT toddler's milk was supplemented with IP and stored for 2 months at 5, 21 and 30 degrees C. The antistreptococcal titres in UHT milk did not decline at any temperature during storage, and UHT-IP inhibited the adherence of S. mutans for up to 2 months. This was not the case with UHT toddler's milk without IgG antibodies. Milk was fermented with live LGG cells in the presence or absence of 5% IP. The antistreptococcal titres declined to about 30% of the original titres after storage. Fresh milk alone slightly enhanced streptococcal adhesion but fresh milk with IP inhibited the adherence of S. mutans by over 50%. LGG-positive fermented milk without antibodies also inhibited (P < 0.05) the adhesion by about 40%. In both LGG-fermented and UHT immune milk, the activity of antibodies against cariogenic streptococci was maintained during the expected shelf-life of these products. From the anticariogenic point of view it may be beneficial to add bovine-specific antibodies against mutans streptococci to probiotic LGG-containing milk products.

This study examines the possible effect of the antimicrobial peroxidase system on the activity of streptococcal glucosyltransferases B, C and D (GtfB, GtfC and GtfD), either in solution (GtfB and GtfC) or when adsorbed to hydroxyapatite (GtfC and GtfD) at pH 6.5. The lactoperoxidase (LP) system (LP, H2O2, SCN–) had no effect on the activity of dissolved GtfC, but the activity of dissolved GtfB was enhanced. The LP system, however, strongly inhibited the activities of both GtfC and GtfD in their adsorbed form. LP enzyme, without its substrates, inhibited all three Gtf enzymes: GtfB and GtfC in concentrations between 10 and 100 µg/ml in liquid phase and adsorbed GtfC and GtfD in concentrations between 25 and 50 µg/ml. This inhibition was in part abolished in liquid phase, but not in solid phase, if the substrates of LP were added. This study shows that the lactoperoxidase system can exert inhibitory activity against streptococcal Gtfs without generating oxidizing agents.

We studied differences in the amounts of organic and inorganic mercury in saliva samples between amalgam and nonamalgam human study groups. The amount of organic and inorganic mercury in whole saliva was measured in 187 adult study subjects. The mercury contents were determined by cold–vapor atomic absorption spectrometry. The amount of organic and inorganic mercury in paraffin–stimulated saliva was significantly higher (pe (organic mercury) was linearly related to loge (inorganic mercury, r2 = 0.52). Spearman correlation coefficients of inorganic and organic mercury concentrations with the number of amalgam–filled tooth surfaces were 0.46 and 0.27, respectively. Our results are compatible with the hypothesis that amalgam fillings may be a continuous source of organic mercury, which is more toxic than inorganic mercury, and almost completely absorbed by the human intestine.

Previous studies have shown that the peroxidase system with iodide is particularly effective against Actinobacillus actinomycetemcomitans. In the present study, the effects of iodide, chloride and thiocyanate in combinations with lactoperoxidase (LP) and myeloperoxidase (MP) on the viability of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and S. rattus were analysed. Bacteria were incubated in buffer solution containing peroxidase, substrate(s) and H2O2 (all in oral physiological concentrations), and plated after 0, 0.5 and 1 h. The oxidation product of iodide was the most bactericidal against all the bacteria tested. The effect was significantly weaker on mutans streptococci. Physiological concentrations of thiocyanate abolished the effects of LP-H2O2-iodide and MP-H2O2-iodide/chloride combinations. Thiocyanate-peroxidase systems have already been used in oral hygiene products. The incorporation of iodide into these products could make them much more potent against periodontal pathogens, and also help to prevent transmission of these pathogens from person to person via saliva.

Xylitol is effective as a noncariogenic or even cariostatic sugar substitute. Habitual xylitol consumption appears to select for mutans streptococci (MS) which shed easily into saliva from plaque. We have earlier shown that habitual xylitol consumption of mothers was associated with a statistically significant reduction in the probability of mother–child transmission of MS assessed at 2 years of age. The aim of the present study was to assess the children’s MS counts 1 and 4 years after the maternal xylitol consumption had been discontinued. At baseline, during pregnancy, all mothers (n = 195) showed high salivary levels of MS. The mothers were randomly assigned to xylitol, fluoride (F) and chlorhexidine (CHX) groups. In the xylitol group, the mothers chewed xylitol–sweetened gum, for 21 months, starting 3 months after delivery. In the two control groups, the mothers received CHX or F varnish treatments at 6, 12 and 18 months after delivery. At the 2–year examination, 169 mother–child pairs participated. At the 3–year and 6–year examinations, there were 159 and 147 children in the study, respectively. For children’s MS analyses, visible plaque was collected using toothpicks at the age of 3 and paraffin–stimulated saliva at the age of 6. The persons involved in the collection and analysis of the microbiological samples were blinded as to the study design and group. Both the plaque and salivary MS were cultured on Mitis salivarius agars containing bacitracin. In all groups, the colonization percentages increased during the follow–up. At the 3–year examination, the children’s risk of having MS colonization was 2.3–fold in the F group (95% CI 1.3–4.2) compared to the xylitol group. This difference was statistically significant. Even at 6 years of age, the salivary MS levels were significantly lower in the xylitol group than in the other groups (ANOVA, p

UNLABELLED We studied the effects of glycopyrrolate on oral mucous host defenses. Single IV doses of glycopyrrolate (4 microg/kg) or placebo were administered to 12 healthy volunteers in a randomized, double-blinded, cross-over study. Salivary flow rates and the concentrations/activities of total protein, amylase, and nonimmunologic (lysozyme, lactoferrin, myeloperoxidase, total salivary peroxidase, and thiocyanate) and immunologic (total immunoglobulin A, immunoglobulin G, and immunoglobulin M) mucous host defense factors were determined for paraffin-stimulated whole saliva before and 1, 3, 6, 12, 24, and 48 h after drug administration. Glycopyrrolate serum concentrations were determined before and 2, 4, 6, 10, 15, and 30 min and 1, 2, 3, 6, 12, and 24 h after IV drug injection. Salivary flow rates were decreased significantly for 12 h after glycopyrrolate injection, compared with saline injection. The concentrations of immunologic and nonimmunologic defense factors were increased in the glycopyrrolate group, and differences between the groups were found for all factors (P < 0.05-0.001) except lysozyme and total salivary peroxidase. In contrast, because of the reduced flow rate, the output of all defense factors into the saliva was decreased after glycopyrrolate injection, compared with saline injection. Glycopyrrolate thus decreases the output of salivary host defense factors into the oral cavity. IMPLICATIONS Glycopyrrolate induces long-lasting hyposalivation and decreases the secretion of salivary immunologic and nonimmunologic defense factors in healthy volunteers.

Xylitol is effective as a non-cariogenic sugar substitute. Habitual xylitol consumption appears to select for mutans streptococci (MS) with impaired adhesion properties, i.e., they shed easily to saliva from plaque. One hundred sixty-nine mother-child pairs participated in a two-year study exploring whether the mothers' xylitol consumption could be used to prevent mother-child transmission of mutans streptococci. All mothers showed high salivary levels of mutans streptococci during pregnancy. The mothers in the xylitol group (n = 106) were requested to chew xylitol-sweetened gum (65% w/w) at least 2 or 3 times a day, starting three months after delivery. In the two control groups, the mothers received either chlorhexidine (n = 30) or fluoride (n = 33) varnish treatments at 6, 12, and 18 months after delivery. The children did not chew gum or receive varnish treatments. MS were assessed from the mothers' saliva at half-year intervals and from the children's plaque at the one- and two-year examinations. The MS were cultured on Mitis salivarius agars containing bacitracin. The salivary MS levels of the mothers remained high and not significantly different among the three study groups throughout the study. At two years of age, 9.7% of the children in the xylitol, 28.6% in the chlorhexidine, and 48.5% in the fluoride varnish group showed a detectable level of MS. In conclusion, therefore, habitual xylitol consumption by mothers was associated with a statistically significant reduction of the probability of mother-child transmission of MS assessed at two years of age. The effect was superior to that obtained with either chlorhexidine or fluoride varnish treatments performed as single applications at six-month intervals.

A novel slow-release administration device, the "Fall-Asleep Pacifier" (FAP), was studied as a prophylactic measure against mutans streptococcal oral infection and dental caries in a risk group of 1-year-old children by comparing the test (T, n = 34) and control (C, n = 88) groups in a prospective cohort study. In the T group the children received their fluoride tablets (Fludent, containing NaF corresp. 0.25 mg F0- , xylitol 159 mg and sorbitol 153 mg) in the evenings in FAP. In the C group the children received the same dose of Fludent crushed in food in the evenings. The proportion of children, whose plaque samples from the upper incisors were mutans streptococcus positive at the age of 24 months, was significantly (P < 0.05) greater in group C (25%) than in group T (9%). The children in the T group developed significantly (P < 0.001) less (none) new dentinal carious lesions in their primary dentitions than the children in the C group between 2 and 3 1/2 years of age. Fifty-four percent of the children to whom the FAP was offered complied with regular use of it. The beneficial effect observed in the T group compared with the C group was apparently mostly due to the administration mode via FAP, which could prolong the intra-oral bioavailability of the prophylactic preparation.

Passive immunisation, based on bovine colostral preparations, is an area of active research. Specific bovine antibodies inhibit the virulence factors of target pathogens but the interactions between whey preparations and human immune defence cells are not well known. Bovine colostrum inhibits the phagocytic activity of bovine leucocytes and this may reflect the biological activity of immunoglobulins in it. Therefore, this study aimed to examine the effects of bovine whey protein preparations from the colostrum of Streptococcus mutans/S. sobrinus-immunised and sham-immunised cows on binding, ingestion and killing of these bacteria by human leucocytes. Binding and ingestion of FITC-labelled bacteria were estimated by flow cytometry and leukocyte activation was measured as chemiluminescence. Killing rate was estimated by plate counting and by measuring bioluminescence from S. mutans- containing the insect luciferase gene. Colostral whey protein preparation from hyperimmunised cows activated human leucocytes by opsonising specific bacteria. Neutrophils, eosinophils and monocytes weakly phagocytosed non-opsonised bacteria and bacteria opsonised with control product. On the contrary, binding and ingestion were efficient in the presence of the preparation from immunised cows. Thus, these results show that bovine colostral whey proteins are able to support the activation of human phagocytes against pathogenic microbes and that this property is related to specific antibodies in whey preparations. These whey proteins may also be clinically useful, especially in preventing the colonisation of newly erupted teeth by mutans streptococci.

Colostral products from non-immunized cows (CP) and cows immunized with mutans streptococci (IP) were used as mouth rinses in a short-term human study. The acidogenic potential of the products was tested and found to be negligible in vivo before application to subsequent rinsing tests. At first, all the participants received a professional tooth cleaning, after which they rinsed with one of the solutions (IP; CP; water) three times per day for 3 d. After each rinsing period, the resting pH and decrease in plaque pH after sucrose challenge were determined, the amount of plaque was estimated, and all available plaque was collected. No significant differences were recorded in the composition or in the amounts of accumulated plaque. The resting pH values of plaques with low "innate" pH were increased after the IP rinsing period. Surprisingly, the lowest pH values after the sucrose challenge were recorded in IP plaques. The number of cultivable facultative flora or total streptococci were not affected by different rinsings, but the relative number of mutans streptococci significantly decreased after the IP rinsing period when compared to the CP period. Thus, the short term rinsing indicates favourable effects of bovine immune whey on human dental plaque.

Objective. The purpose of this investigation was to study the effect of a potent topical steroid, fluticasone propionate, on patients with early signs and symptoms of the common cold. To characterize the mucosal inflammatory response, salivary defense factors and flow rate in these patients were analyzed. Study Design. Forty patients with symptoms of the common cold were randomized into 2 groups to receive either high-dose fluticasone propionate (100 μg per nostril) or placebo 4 times daily for 6 days. Paraffin-stimulated whole saliva was collected on day 1 (before the onset of medication), day 7 (posttreatment), and day 21 (follow-up). Results. Salivary flow rate, innate host defense factors, and total protein content were not affected by the common cold. IgA increased between day 7 and day 21 ( P ≤ .01; Student 2-tailed t test), and the relative proportions of salivary peroxidase and IgA increased on day 7 ( P = .01) and day 21 ( P = .05). In patients receiving fluticasone, saliva flow rate was lower on day 21 ( P ≤ .05) than on days 1 and 7. The innate salivary defense factors were not affected, but IgA increased both on day 7 ( P ≤ .001) and on day 21 ( P ≤ .001) in comparison with day 1. Conclusions. Of the oral mucosal defense factors, only IgA is activated during the common cold. Intranasally administrated fluticasone propionate does not have a suppressive effect on salivary antimicrobial capacity. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:695-9)

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. mutans 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.

We compared the effects of propofol-based (n = 15) and isoflurane-based anaesthesia (n = 15) on mucous host defences by measuring the salivary flow and the concentrations/activities of salivary total protein and amylase, and of salivary immunological (IgA, IgG and IgM) and nonimmunoglobulin defence factors (lysozyme, myeloperoxidase, total salivary peroxidase and thiocyanate) in patients undergoing elective abdominal hysterectomy. The saliva samples were collected pre-operatively and on the first and fourth postoperative days. The concentrations of salivary protein and amylase as well as those of immunological and nonimmunological defence factors were significantly increased on the first postoperative day. The secretion rate of total protein, amylase, lysozyme, total peroxidase, thiocyanate and IgG, however, decreased owing to a marked decrease in the salivary flow, but no alterations were found in secretion rate of myeloperoxidase, IgA and IgM. The changes were similar in both groups. These findings show that nonimmunological oral mucous host defences are altered after major surgery, but immunoglobulin responses are better maintained. Both types of anaesthesia induce marked short-term hyposalivation.

The oral bacterium Streptococcus mutans was transformed by electroporation with a shuttle vector (pCSS945) containing insect luciferase gene from a click beetle ( Pyrophorus plagiophthalamus ) resulting in a bioluminescent phenotype. This S. mutans strain was used in experiments in which light emission was used as a rapid and, compared to conventional CFU counting, more convenient means of estimating the effects of various antimicrobial treatments. The basic parameters affecting in vivo light production by the strain were studied. It was found that pH 6.0 was optimal for incorporation of the substrate d -luciferin for the luciferase reaction. The optimum concentration of d -luciferin was approximately 150 μM at room temperature. Under optimum conditions the light emission in vivo increased rapidly to a constant level and thereafter had a decay of 0.6%/min when logarithmic-growth-phase cells were used. The light emission closely paralleled the numbers of CFU, giving a detectable signal from 30,000 cells and having a dynamic measurement range over 4 log CFU/relative light unit. The cells were treated with various antimicrobial agents, and the emitted bioluminescence was measured. With the bioluminescent measurements, the results were obtained within hours compared to the days required for agar plates, and also, the kinetics of the antibacterial actions could be followed. Thus, the light emission was found to be a reliable, sensitive, and real-time indicator of the bacteriostatic actions of the antimicrobial agents tested.

Previous studies of the possible associations of salivary antimicrobial agents with dental caries have given controversial results, obviously mainly because almost all studies have been cross-sectional. Our aim was to find out, in a two-year longitudinal follow-up study, the associations among selected salivary non-immune and immune antimicrobial variables, cariogenic bacteria, and caries increment. The study population was comprised of 63 subjects, all of whom had their 13th birthday during the first study year. In addition to a comprehensive dental examination at baseline and after 2 yrs, paraffin-stimulated whole saliva samples were collected in a standardized way at six-month intervals. Saliva samples were analyzed for flow rate, buffer effect, lysozyme, lactoferrin, total peroxidase activity, hypothiocyanite, thiocyanate, agglutination rate, and total and specific anti-S. mutans IgA and IgG, as well as for numbers of total and mutans streptococci, lactobacilli, and total anaerobic bacteria. Cluster analysis and Spearman-Rank correlation coefficients were used to explore possible associations between and among the studied variables. During the two-year period, a statistically significant increase was observed in flow rate, thiocyanate, agglutination rate, anti-S. mutans IgA antibodies, lactobacilli, and total anaerobes, whereas lysozyme, lactoferrin, and total and anti- S.mutans IgG antibodies declined significantly. Based on various analyses, it can be concluded that, at baseline, total IgG and hypothiocyanite had an inverse relationship with subsequent two-year caries increment, anti-S. mutans IgG antibodies increased with caries development, and mutans streptococci and lactobacilli correlated positively with both baseline caries and caries increment. Total anaerobic microflora was consistently more abundant among caries-free individuals. In spite of the above associations, we conclude that none of the single antimicrobial agents as such has sufficiently strong power to have diagnostic significance in vivo with respect to future caries.

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.

The human mouth is an important route of viral transmission and evidence exists that human saliva can neutralize some viruses, e.g. herpes simplex type 1 (HSV-1) and human immunodeficiency virus (HIV) in vitro. However, little is known of the actual antiviral agents in saliva. We have analyzed how hypothiocyanite (HOSCN/ − OSCN) ions, present in human saliva and generated by salivary peroxidase systems, affect the viability of three different types of viruses: HSV-1 (capable of inducing oral lesions), respiratory syncytial virus (RSV, respiratory infections), and echovirus 11 (EV 11, enteric diseases). Viral suspensions were pretreated (30 min) with HOSCN/ − OSCN concentrations up to 180 μM both at pH 6.0 and 7.1 and inoculated into human gingival fibroblasts. The cultures were incubated at 37°C for 18–48 h, fixed and the infected cells were counted after immunoperoxidase staining. HSV-1 was most sensitive to HOSCN/ − OSCN with an IC 50 of 8.5 μM at pH 6.0 and an IC 50 of 20 μM at pH 7.1, respectively. RSV was inhibited by HOSCN/ − OSCN only at pH 6.0 with an IC 50 of 8.0 μM. EV 11 was also resistant at neutral pH, but sensitive at pH 6.0 with an IC 50 of 68 μM. In contrast to HSV-1 and RSV, the inhibition of EV 11 was not dependent on the concentration of HOSCN/ − OSCN. The inhibition was in all cases stronger at pH 6.0 than at neutral pH. Our results suggest that hypothiocyanite, a normal component of human whole saliva, in physiological concentrations effectively inhibits HSV-1 and RSV at acidic pH, whereas EV 11 is more resistant in vitro.

Effects of three different types of short-term applications (1-3 times during 1 week) of chlorhexidine (1 or 40%) on the susceptibility of 863 clinical isolates of Streptococcus mutans and 53 isolates of Streptococcus sobrinus from 58 subjects were studied. Chlorhexidine-resistant isolates were not found either before or after the treatment. The minimum inhibitory concentrations (MICs) to chlorhexidine of all isolates of S. mutans were < or = 1 microgram/ml, and of S. sobrinus < or = 2 micrograms/ml. S. mutans and S. sobrinus were also susceptible to ampicillin, penicillin, cefuroxime, and tetracycline. In conclusion, different short-term chlorhexidine regimens do not induce resistance in S. mutans or S. sobrinus and, furthermore, these species have so far retained their susceptibility to common antibiotics.

The aim of the study was to compare the efficacy of a 40% chlorhexidine (CHX) varnish (EC40, Certichem, Nijmegen, The Netherlands) with a 1% CHX-0.2% NaF gel in decreasing the level of salivary mutans streptococci (MS). The subjects were screened for a high level of MS using a Dentocult-SM strip method (Orion Diagnostica, Finland). In varnish groups with fluoride (VCHXF, n = 20) and without fluoride (VCHX, n = 19), the CHX varnish was applied on dry teeth using an ampoule and an anesthetic syringe with blunt needle, and removed after 15 min. In group VCHXF an additional 2.26% fluoride varnish (Duraphat, Woelm Pharma GmbH, Eschwege, Germany) was applied. The CHX-NaF gel treatment included the application of the gel with rubber cups and dental tape for 5 min on three occasions during a week in group GCHXF (n = 21). The level of MS (MSB agar) was significantly lower after 4 weeks than at baseline in VCHX (p0.001) and VCHXF (p0.05), but not after 12 weeks. In GCHXF a significant decrease (p = 0.001) was observed after 4 weeks only with the strip method. In VCHX and VCHXF the strip values for MS were still reduced after 12 weeks. In VCHX and GCHXF a small, although statistically significant, increase was observed in the total number of microorganisms after 4 and 12 weeks. Opinions on taste sensations associated with the treatments were generally negative, but least negative in the VCHXF group; fewer side effects were also reported in the VCHXF group.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentrations of salivary antimicrobial factors are well documented in children and young adults, but little information is available on such defense factors in healthy elderly persons. We determined the levels of total IgA, total IgG, lysozyme, lactoferrin, myeloperoxidase, salivary peroxidase, amylase, sialic acid, and total protein in a group of 71 subjects aged 76, 81, and 86 yr, as well as their correlations to paraffin-wax-stimulated salivary flow rate. Participants were either unmedicated (n = 67) or using medicines with no oral significance (n = 4). Statistically significant negative correlations existed between flow rate and total IgA, lysozyme, lactoferrin, sialic acid, and total protein. Concentrations of sialic acid and salivary peroxidase were highest in the oldest age group. Total IgA concentration was higher in women than in men, although men showed higher concentrations of sialic acid and higher sialic acid/total protein ratios. Subjects with poor gingival health had higher concentrations of total protein than did those with no need for periodontal treatment. Edentulous subjects with complete dentures showed significantly lower concentrations of IgG, lactoferrin, and myeloperoxidase than did dentate subjects. Our results suggest that, when compared with data from previous studies, concentrations of salivary antimicrobial agents do not decline with age in unmedicated elderly people. However, defense factors which are derived also from gingival crevicular fluid are decreased in the absence of teeth.

Fluoride (F-) ions at concentrations present in vivo at the plaque/enamel interface (0.05-10 mM) inhibited the activities of lactoperoxidase (LP), myeloperoxidase (MP) and total salivary peroxidase (TSP) in a pH- and dose-dependent way. The inhibition was observed only at pHor = 6.5 and with F- concentrationsor = 0.1 mM. At pH 5.5 LP activity was inhibited by 85% and MP by 34% with 10 mM F-. TSP activity was also inhibited only at low pH (5.5) by approximately 25%. Furthermore, the generation of the actual antimicrobial agent in vivo, hypothiocyanite (HOSCN/OSCN-), of the oral peroxidase systems was inhibited by F-, again at low pH (5.0-5.5) both in buffer (by 45%) and in saliva (by 15%). This inhibition was observed only with the highest F- concentrations studied (5-10 mM). Fluoridated toothpaste (with 0.10 or 0.14% F) mixed with saliva did not inhibit TSP or HOSCN/OSCN- generation. This may have been due to the 'buffering' effect of toothpaste which did not allow salivary pH to drop below 5.9. We conclude that the F- ions in acidic fluoride products, e.g. in gels or varnishes (but not in toothpastes), may have the potential to locally inhibit the generation of a nonimmune host defense factor, HOSCN/OSCN/SCN-, produced by oral peroxidase systems. The possible clinical significance of this finding remains to be shown.

The author describes his involvement in the discovery of antimicrobial agents in the human mouth.

Because smoking negatively affects oral health, we investigated whether this could be due to adverse effects of smoking per se on human oral microflora. Faecal microflora was also analysed for comparison. Samples of whole saliva, dental plaque, pharynx and faeces were collected from 48 healthy young military conscripts, 24 smokers and 24 non-smokers, and studied by gas–liquid chromatography of bacterial cellular fatty acids. The method has proved to be fast and sensitive in detecting microfloral differences and their magnitudes. The results showed significant differences in microflorae between the different sample types (saliva, dental plaque, pharyngeal or faecal). However, there were no significant differences within any sample type between smokers and non-smokers. When oral health is good, smoking history short (mean 5.4 years) and groups otherwise uniform, moderate daily smoking does not seem to change human microbial florae.Keywords: smoking, gas–liquid chromatography, bacterial fatty acids, microbial flora.

Androgen receptors were demonstrated in human salivary glands by immunohistochemistry using polyclonal antibodies. Fresh, clinically healthy salivary gland samples (two from minor, seven from parotid and eight from submandibular glands) of both sexes were used. Frozen tissue sections were incubated with the antibody against human androgen receptor and visualized by an indirect immunoperoxidase technique. Androgen receptors could be detected in all salivary tissues studied. Positive staining was confined to nuclei of almost all acinar cells as well as to the majority of nuclei in ductal cells. Very few of the nuclei of connective tissue and endothelial cells stained positively. The presence of androgen receptors in human salivary glands suggests possible direct effects of androgens on these tissues.

The ability of various forms of human lactoferrin (LF) to agglutinate oral Streptococcus mutans, Strep. sobrinus, Strep. rattus, Strep. sanguis, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans cells was studied spectrophotometrically. Fe3+ saturated LF was unable to agglutinate these bacteria, whereas iron-free LF (apo LF) effectively agglutinated Strep. mutans cells but not the other bacteria. The efficiency and rate of agglutination of Strep. mutans were somewhat lower with apo LF than with human whole saliva. However, secretory IgA, phosphate and whole saliva almost totally abolished the apo LF-mediated agglutination of Strep. mutans, suggesting binding to the same target sites on bacterial cell surfaces, or to each other. The presence of exogenous iron (Fe2+, Fe3+), lactoperoxidase or serum albumin did not affect the agglutination by apo LF. Low Ca2+ (50-100 microns) slightly enhanced the agglutination by apo LF but higher concentrations (0.5-1.0 mM) totally blocked the apo LF-mediated agglutination of Strep. mutans. Both saliva and apo LF significantly delayed the rapid autoaggregation of P. gingivalis cells. Aggregation of P. gingivalis is considered a potential virulence factor and a protective mechanism against the host's cellular defences in the gingival crevice. These findings show a novel, strain-specific antibacterial mechanism for LF against Strep. mutans and P. gingivalis and adds a new compound to the group of agglutinating proteins in human saliva.

Lactoperoxidase (LPO)/H2O2/SCN(-)-system-generated hypothiocyanite ions (OSCN-) and hypothiocyanous acid (HOSCN) are inhibitory against a number of oral bacteria, including mutans streptococci. A commercially available toothpaste (Biotene) comprises the complete LPO system. Generation of HOSCN/OSCN- by Biotene was studied in vitro both in sterilized and nonsterilized saliva of 10 healthy subjects. The HOSCN/OSCN- yield ranged from 100 to 300 microM with Biotene, while the salivary levels of HOSCN/OSCN- before the addition of Biotene were 30.1 +/- 25.1 microM. Two in vivo trials were carried out. In the first study, resting saliva was collected from 12 individuals before, immediately after, and 2, 5, 10 and 20 min after brushing with Biotene to evaluate the in vivo generation and decomposition of HOSCN/OSCN-. In the second study, 26 healthy individuals attended a 1-month crossover trial with Biotene and a control toothpaste, Vademecum (no LPO system), both containing F- and xylitol. The salivary counts of total streptococci, mutans streptococci (MS), lactobacilli and the total flora (TF), as well as the peroxidase, thiocyanate ion and HOSCN/OSCN- levels were determined before and after 2 and 4 weeks daily use of the toothpastes. Twice-a-day use of Biotene for 1 month resulted in an elevation of 'resting levels' of HOSCN/OSCN-. No such effect was found with the control toothpaste. No significant changes were found in the salivary levels of total streptococci, MS, lactobacilli or TF after 1-month use of either toothpaste. The results show the capability of the LPO-system-containing toothpaste to elevate the salivary levels of HOSCN/OSCN-, although no bactericidal effect was observed.

Both intra- and inter-individual variation in salivary flow rate, buffer effect, and the levels of salivary mutans streptococci and lactobacilli were analyzed in 128 11-year-old children. The follow-up period was 9 months, with six saliva samplings done at regular intervals. Inter-individual variation was relatively large in paraffin-stimulated salivary flow rate: low (1.0 ml/min) and high (or = 2.0 ml/min) flow rates were measured in 18% and 13% of the children, respectively. Intraindividual variation during the follow-up period was found in 63% of the boys and in 73% of the girls. The buffer effect stayed stable in all samplings in 59% of the boys and in 42% of the girls. Buffer effect was significantly (p0.001) lower in girls than in boys. Mutans streptococci were analyzed by a chair-side method (Strip mutans test) and by cultivation on mitis-salivarius-bacitracin (MSB) agar plates. The results of the two methods correlated highly significantly (r = 0.79, p0.001). With the Strip mutans test no variation in test scores occurred in 49% of all subjects in all six samplings, whereas the respective percentage for MSB scores was only 19%. No variation in salivary lactobacilli occurred in only 18% of the subjects, and in 13% the intraindividual variation was as high asor = 3 logs. These results show that in young teenagers with a developing dentition, simultaneous changes in behavioral, hormonal, and dietary factors make single-point measurements of salivary factors too unreliable for caries-diagnostic or predictive purposes.

The frequent oral shedding of herpes simplex virus type 1 (HSV-1) in the absence of clinical disease suggests that symptomatic HSV-1 recurrences may be inhibited by the mucosal environment. Indeed, saliva has been shown to contain substances with anti-HSV activity. In the current study, we investigated the anti-HSV-1 activity of human lactoferrin (hLf) and lysozyme (hLz), two highly cationic polypeptides of the mucosal innate defence system. HLf blocked HSV-1 infection at multiple steps of the viral replication cycle, whereas lysozyme displayed no anti-HSV-1 activity. Preincubation of HSV-1 virions and presence of hLf during or after viral absorption period or for the entire HSV-1 infection cycle inhibited HSV-1 infection by reducing both the plaque count and plaque size in a dose- and virus strain-dependent manner. Cell-to-cell spread of wild-type HSV-1 and the strain gC-39, deleted of glycoprotein C, was dramatically reduced, but the cell-to-cell spread of HSV-1 Rid1, harboring a mutated gD and thus unable to react with the cellular HVEM receptor, remained unchanged. This suggests that the inhibition of cell-to-cell spread is mediated by effects on gD or its cellular counterparts. Our results show that the cationic nature is not a major determinant in the anti-HSV action of mucosal innate cationic polypeptides, since whereas hLf inhibited HSV-1 infection efficiently, hLz had no HSV-1 inhibiting activity. Our results show that in addition to inhibiting the adsorption and post-attachment events of HSV-1 infection, hLf is also able to neutralize HSV-1 and that the inhibition of cell-to-cell spread involves viral gD. These results suggest that Lf may have a significant role in the modulation of HSV-1 infection in the oral cavity as well as in the genital mucosa, the major sites of HSV-1 infection.

Forty-five young adults were divided into three groups of equal size and given a professional toothcleaning (3 times during 1 wk) with dental gels containing either chlorhexidine (CHX, 1%), or a SnF2-amine fluoride (AmF) combination (F- content 1.20%). The control group received toothcleaning with a placebo gel. The study was done double blind. The number of salivary mutans streptococci was monitored for 11 wk after the gel treatment using both mitis-salivarius-bacitracin agar plates and the chairside method based on the adhesion of mutans streptococci on plastic strips (Dentocult-SM Strip mutans). Professional toothcleaning with a CHX gel was clearly most effective (P less than 0.001) but the baseline levels of streptococci returned in 11 wk. SnF2-AmF gel also reduced significantly (P less than 0.001) mutans streptococci but recolonization occurred already in 7 wk. Placebo gel, i.e. professional toothcleaning as such, did not show any statistically significant effect on the numbers of salivary mutans streptococci. None of the treatments affected the levels of lactobacilli or the total aerobic flora in saliva samples. Professional toothcleaning with a CHX gel, or with a SnF2-AmF gel, can be regarded as an alternative to the commonly used, but more patience demanding, application of gel in mouth trays at home for individuals with high caries activity.

We have studied the effects of iron-free lactoferrin (apo LF) and lactoperoxidase system (lactoperoxidase, LP/SCN-/H2O2), separately and together, on the viability of Streptococcus mutans (serotype c) in vitro. The bacteria were incubated in buffered KCl (pH 5.5) with and without the above components which were used at concentrations normally present in human saliva. Both apo LF and LP-system had a bactericidal effect against S. mutans at low pH. Together they showed an additive, but not a synergistic, antibacterial effect against S. mutans. Apo LF enhanced the LP enzyme activity but decreased the yield of the antimicrobial component, hypothiocyanite (HOSCN/OSCN-), when incorporated into the reaction mixtures. This decrease, which was most pronounced at low pH, was due to an LP-independent reaction between apo LF and HOSCN/OSCN-. Our study indicates that the LP-system and apo LF can be combined to combat oral S. mutans.

The effect of 4 to 6 years of habitual use of xylitol gum (greater than or equal to 2 times per day) on the viable counts of mutans streptococci in both plaque and saliva was evaluated in a cross-sectional study of participants of the Ylivieska xylitol study (Finland, 1982-1984). Paraffin-stimulated saliva was collected first, whereafter the remaining plaque was collected from all available tooth surfaces and pooled. The habitual xylitol consumers (n = 15) had less plaque than the controls (n = 21). The total viable counts of mutans streptococci in the plaque collected from the habitual xylitol users were about 10% of those determined in controls (p less than 0.01). The salivary mutans streptococci levels were relatively high in both habitual xylitol consumers and controls, as determined using a plate count and with an adhesion-based assay; no significant differences were detected between these groups. Thus, habitual xylitol consumption appeared not to be associated with a reduction in the total numbers of mutans streptococci in saliva. The plaque and the mutans streptococci were, however, more loosely bound to the tooth surfaces of the habitual xylitol users. The results of the plate count and the adhesion-based assay for salivary mutans streptococci were in good agreement for both habitual xylitol consumers and the controls. This suggests that it was the accumulation of plaque rather than the adhesiveness of mutans streptococci that was affected by the habitual consumption of xylitol.

We have studied the antibacterial effect of human lactoferrin and lysozyme (LZ), separately and together, on the viability of Streptococcus mutans , serotype c, in vitro . Both proteins were purified from human colostrum and lactoferrin was used in iron-free (apo LF), Fe 3+ -saturated (sat LF), and partly saturated (LF) forms. In incubation experiments at pH 5.5, both LZ and apo LF at physiological human salivary concentrations (25-50 mg/l) inhibited streptococcal viability in a time- and dose-dependent way. In all experiments LF and sat LF were clearly least effective. Interestingly, LZ and apo LF together displayed an enhanced bactericidal activity against S. mutans (serotype c), whereas a combination of sat LF and LZ did not have such an effect. This is a new finding to the previously described interactions between innate antimicrobial agents against cariogenic bacteria. An understanding of these interactions may be important in engineering approaches to selectively control specific oral bacteria, such as mutans streptococci. Keywords: Streptococcus mutans ; Lactoferrin; Lysozyme; Antimicrobial factors; Saliva.

Effects of human lysozyme (HLZ) combined with thiocyanate (SCN-) ions on mutans streptococci, both in physiologic salivary concentrations, were studied. The bacteria were incubated for 75 min either in HLZ-supplemented sterilized human whole saliva (pH 5 and 7) or in neutral buffer in the presence or absence of HLZ (30 mg/l)-SCN- (1-5 mM) combinations. HLZ had no inhibitory effect on the viability of Streptococcus mutans, serotype c, either in saliva or in buffer, not even at pH 5, in the presence of salivary bicarbonate or in higher (up to 240 mg/l) concentrations of HLZ. In contrast, HLZ significantly decreased the viability of S. rattus in both media. HLZ also effectively blocked the lactic acid production of S. rattus but not that of S. mutans. Thiocyanate ions, which have been proposed to enhance the antimicrobial activity of lysozyme, did not affect the antibacterial activity of HLZ or HLZ-HCO3- combinations. It is concluded that the in vivo levels of SCN- ions, which constitute an integral part of the peroxidase antimicrobial system in saliva, may not be high enough to trigger the lysis of S. mutans by lysozyme in human saliva. The very low prevalence of S. rattus compared with S. mutans in human populations may be associated with their different susceptibility to lysozyme-mediated inhibition in saliva.

Streptococcus mutans, Strep. sobrinus and Lactobacillus casei were grown in glucose-supplemented, sterilized, human whole saliva, adjusted to pH 5, 6 or 7. Components of the antibacterial peroxidase system--hypothiocyanous acid (HOSCN) and hypothiocyanite ions (OSCN-)--were generated by adding exogenous H2O2 to sterilized saliva containing endogenous peroxidases and thiocyanate (SCN-) ions. HOSCN/OSCN- generation was proportional to the amount of H2O2 added, and more HOSCN/OSCN- was detected in saliva at pH 7 than at pH 5. However, the growth of mutans streptococci and L. casei was inhibited at pH 5 by HOSCN/OSCN-, whereas no inhibition was found at pH 7. The findings show that (a) sufficient amount of HOSCN/OSCN- will inhibit the growth of cariogenic bacteria in human saliva at pH 5; (b) this amount of HOSCN/OSCN- can be generated in saliva by exogenously added H2O2; and (c) peroxidase systems have stronger antistreptococcal effects in human whole saliva than in phosphate buffer.

The relationship between the caries status and the microbiological status [mutans streptococci (MS)] in the proximal areas of lower right and upper left molars was investigated in subjects of the Ylivieska xylitol study in Finland (1982-1985) in a cross-sectional follow-up in 1988. Those children who had used xylitol chewing gum regularly since 1982 showed significantly lower caries indices in 1988, including the proximal caries scores. The presence of MS (expressed in log10 colony-forming units) in the proximal areas studied was significantly lower in habitual xylitol consumers. Carious interproximal areas of all subjects had significantly higher total levels of MS than clinically and radiographically sound interproximal areas. The present study supports the idea of a close association between MS and dental caries, and shows that the total level of MS in the human dentition can be influenced by xylitol. It is likely that the long-term (between 1982 and 1988) habitual xylitol consumption had decreased the prevalence of MS in the interproximal areas of the present subjects and, subsequently, decreased the proximal and overall caries indices.