Gene E. Watson

Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque Biofilms In Vivo

ABSTRACT Streptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived β1,3-glucans contribute to the EPS matrix structure, while fungal mannan and β-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.

Influence of cranberry proanthocyanidins on formation of biofilms by streptococcus mutans on saliva-coated apatitic surface and on dental caries development in vivo

Cranberry crude extracts, in various vehicles, have shown inhibitory effects on the formation of oral biofilms in vitro. The presence of proanthocyanidins (PAC) in cranberry extracts has been linked to biological activities against specific virulence attributes of Streptococcus mutans, e.g. the inhibition of glucosyltransferase (Gtf) activity. The aim of the present study was to determine the influence of a highly purified and chemically defined cranberry PAC fraction on S. mutans biofilm formation on saliva-coated hydroxyapatite surface, and on dental caries development in Sprague-Dawley rats. In addition, we examined the ability of specific PAC (ranging from low-molecular-weight monomers and dimers to high-molecular-weight oligomers/polymers) to inhibit GtfB activity and glycolytic pH drop by S. mutans cells, in an attempt to identify specific bioactive compounds. Topical applications (60-second exposure, twice daily) with PAC (1.5 mg/ml) during biofilm formation resulted in less biomass and fewer insoluble polysaccharides than the biofilms treated with vehicle control had (10% ethanol, v/v; p < 0.05). The incidence of smooth-surface caries in rats was significantly reduced by PAC treatment (twice daily), and resulted in less severe carious lesions compared to the vehicle control group (p < 0.05); the animals treated with PAC also showed significantly less caries severity on sulcal surfaces (p < 0.05). Furthermore, specific A-type PAC oligomers (dimers to dodecamers; 0.1 mg/ml) effectively diminished the synthesis of insoluble glucans by GtfB adsorbed on a saliva-coated hydroxyapatite surface, and also affected bacterial glycolysis. Our data show that cranberry PAC reduced the formation of biofilms by S. mutans in vitro and dental caries development in vivo, which may be attributed to the presence of specific bioactive A-type dimers and oligomers.

Apigenin and tt-Farnesol with Fluoride Effects on S. mutans Biofilms and Dental Caries:

Apigenin (Api) and tt-farnesol (Far) are two naturally occurring agents that affect the development of cariogenic biofilms. Fluoride (F) interferes physicochemically with caries development and also exhibits antibacterial activity. We examined whether the association of Api and Far enhance the anti-caries properties of F by acting cooperatively on the expression of virulence of Streptococcus mutans. The biological effects of each of the agents were greatly enhanced when used in combination with F. In general, biofilms treated with Api and/or Far in combination with F displayed less biomass and fewer insoluble glucans and iodophilic polysaccharides than did those treated with the test agents alone (P < 0.05). The combination of the test agents with F was highly effective in preventing caries development in rats, especially Api+Far+F, and results were comparable with those observed with chlorhexidine + F (positive control). Results from these studies showed that apigenin and tt-farnesol may enhance the cariostatic effectiveness of fluoride.

The Chemical Nature of Mercury in Human Brain Following Poisoning or Environmental Exposure

Methylmercury is among the most potentially toxic species to which human populations are exposed, both at high levels through poisonings and at lower levels through consumption of fish and other seafood. However, the molecular mechanisms of methylmercury toxicity in humans remain poorly understood. We used synchrotron X-ray absorption spectroscopy (XAS) to study mercury chemical forms in human brain tissue. Individuals poisoned with high levels of methylmercury species showed elevated cortical selenium with significant proportions of nanoparticulate mercuric selenide plus some inorganic mercury and methylmercury bound to organic sulfur. Individuals with a lifetime of high fish consumption showed much lower levels of mercuric selenide and methylmercury cysteineate. Mercury exposure did not perturb organic selenium levels. These results elucidate a key detoxification pathway in the central nervous system and provide new insights into the appropriate methods for biological monitoring.

Fish consumption, mercury exposure, and their associations with scholastic achievement in the Seychelles Child Development Study §

Studies of neurodevelopmental outcomes in offspring exposed to MeHg from maternal consumption of fish have primarily measured cognitive abilities. Reported associations have been subtle and in both adverse and beneficial directions. Changes in functional outcomes such as school achievement and behavior in exposed children and adolescents have not been examined. We undertook an assessment of school success of children in the Seychelles Child Development Study (SCDS) main cohort to determine if there were any associations with either prenatal or recent postnatal MeHg exposure. The primary endpoints were Seychelles nationally standardized end-of-year examinations given when the cohort children were 9 and 17 years of age. A subgroup (n=215) from the main cohort was also examined at 9 years of age using a regional achievement test called SACMEQ. Prenatal MeHg exposure was 6.8 ppm in maternal hair; recent postnatal exposure was 6.09 ppm at 9 years and 8.0 ppm at 17 years, measured in child hair. Multiple linear regression analyses showed no pattern of associations between prenatal or postnatal exposure, and either the 9- or 17-year end-of-year examination scores. For the subgroup of 215 subjects who participated in the SACMEQ test, there were significant adverse associations between examination scores and postnatal exposure, but only for males. The average postnatal exposure level in child hair for this subgroup was significantly higher than for the overall cohort. These results are consistent with our earlier studies and support the interpretation that prenatal MeHg exposure at dosages achieved by mothers consuming a diet high in fish are not associated with adverse educational measures of scholastic achievement. The adverse association of educational measures with postnatal exposure in males is intriguing, but will need to be confirmed by further studies examining factors that influence scholastic achievement.

Dose-ranging analgesic study of Prosorb diclofenac potassium in postsurgical dental pain.

BACKGROUND ProSorb diclofenac potassium (K) is a novel, liquid-filled rapid-dispersion formulation of the nonsteroidal anti-inflammatory drug diclofenac, placed into soft gelatin capsules. Its time to maximal plasma drug concentration has been shown to be approximately half, and its maximal plasma drug concentration nearly twice, that of immediate-release diclofenac K tablets. OBJECTIVE This study compared the analgesic dose-response relationship and tolerability of 3 doses of ProSorb diclofenac K and placebo in the treatment of pain after dental impaction surgery. METHODS This randomized, double-blind, double-dummy, placebo-controlled parallel-group study was conducted at 6 centers across the United States. Patients aged 18 to 65 years with moderate or severe pain after the removal of > or =1 impacted mandibular third molar were randomly assigned to receive a single dose of ProSorb diclofenac K 25, 50, or 100 mg or placebo. Pain intensity and relief were assessed up to 6 hours after dosing. Rescue treatment was allowed after 1 hour. Efficacy end points included the summed pain intensity difference over 3 and 6 hours (SPID3 and 6); total pain relief at 3 and 6 hours (TOTPAR3 and 6); median times to onset of perceptible and meaningful relief (analgesic onset) and rescue medication use (analgesic duration); and cumulative percentage of patients using rescue medication. Tolerability was assessed using vital sign measurements and spontaneous reporting of adverse events. RESULTS A total of 265 patients (154 women, 111 men; mean age, 23.3 years) were enrolled. All 3 ProSorb diclofenac K groups showed higher SPID6 and TOTPAR6 scores and longer median times to rescue medication use than the placebo group (all, P < 0.001). For these end points, a dose-response relationship was evident between the 100-mg dose and the 25- and 50-mg doses (P < or = 0.05); the 25- and 50-mg doses were similar. In the diclofenac groups, median onset times for first perceptible (< or =22.5 min) and meaningful (< or =53.0 min) relief were significantly more rapid than placebo (P < or = 0.01). Proportions of patients requiring rescue analgesic were < or =50.8% with diclofenac compared with 79.4% with placebo. Proportions of patients assigning a global evaluation of good or better was > or =68% with diclofenac compared with 21% for placebo. Tolerability was similar across all treatment groups. CONCLUSION In this study of patients treated for pain following dental impaction surgery, single doses of ProSorb diclofenac K 25, 50, and 100 mg were more efficacious than placebo with respect to reduction of pain. All 3 doses provided a rapid analgesic onset and were well tolerated.

A longitudinal analysis of prenatal exposure to methylmercury and fatty acids in the Seychelles

BACKGROUND Maternal fish consumption during pregnancy exposes the fetus simultaneously to methylmercury (MeHg) and long chain polyunsaturated fatty acids (LCPUFA). Data from the Seychelles Child Development Nutrition Study (SCDNS) showed a negative association of MeHg with child development when children were 30 months of age, only when controlling for LCPUFA. Concomitantly, n-3 LCPUFA were found to have a significant positive association only at 9 months. These findings suggest that the effects of MeHg and LCPUFA may vary with age over the first few years of life. We address this by including outcomes at two ages and adjusting for the childs age at testing. METHODS A longitudinal analysis utilizing linear mixed models was performed to assess the associations of maternal hair total mercury (THg, a biomarker for MeHg) and maternal LCPUFA with childrens Bayley Scales of Infant Development Psychomotor Developmental Index (BSID-II PDI) at 9 and 30 months of age, and to determine whether these associations change over time. Data from 228 children were included. RESULTS Maternal hair MeHg had a negative effect on BSID PDI, while maternal n-3 LCPUFA had a positive effect. These effects did not change significantly from 9 to 30 months in this analysis. CONCLUSIONS The longitudinal analysis provides increased power for estimating the relationships of prenatal MeHg and LCPUFA exposures during child development. Significant associations of these exposures in opposite directions confirm the importance of LCPUFA in development and the need to adjust for maternal nutrition when studying prenatal MeHg exposure.

Prenatal exposure to methyl mercury from fish consumption and polyunsaturated fatty acids: associations with child development at 20 mo of age in an observational study in the Republic of Seychelles

BACKGROUND Fish is a rich source of n-3 polyunsaturated fatty acids (PUFAs) but also contains the neurotoxicant methyl mercury (MeHg). PUFAs may modify the relation between prenatal MeHg exposure and child development either directly by enhancing neurodevelopment or indirectly through the inflammatory milieu. OBJECTIVE The objective was to investigate the associations of prenatal MeHg exposure and maternal PUFA status with child development at 20 mo of age. DESIGN The Seychelles Child Development Study Nutrition Cohort 2 is an observational study in the Republic of Seychelles, a high-fish-eating population. Mothers were enrolled during pregnancy and their children evaluated at 20 mo of age by using the Bayley Scales of Infant Development II (BSID-II), the MacArthur Bates Communicative Development Inventories (CDI), and the Infant Behavior Questionnaire-Revised. There were 1265 mother-child pairs with complete data. RESULTS Prenatal MeHg exposure had no direct associations with neurodevelopmental outcomes. Significant interactions were found between MeHg and PUFAs on the Psychomotor Developmental Index (PDI) of the BSID-II. Increasing MeHg was associated with lower PDI but only in children of mothers with higher n-6/n-3. Among mothers with higher n-3 PUFAs, increasing MeHg was associated with improved PDI. Higher maternal docosahexaenoic acid (DHA) was associated with improved CDI total gestures (language development) but was significantly adversely associated with the Mental Development Index (MDI), both with and without MeHg adjustment. Higher n-6:n-3 ratios were associated with poorer scores on all 3 CDI outcomes. CONCLUSIONS We found no overall adverse association between prenatal MeHg exposure and neurodevelopmental outcomes. However, maternal PUFA status as a putative marker of the inflammatory milieu appeared to modify the associations of prenatal MeHg exposure with the PDI. Increasing DHA status was positively associated with language development yet negatively associated with the MDI. These findings may indicate the existence of an optimal DHA balance with respect to arachidonic acid for different aspects of neurodevelopment.

Autism Spectrum Disorder Phenotypes and Prenatal Exposure to Methylmercury

Background: There continues to be public concern that mercury exposure and autism spectrum disorder (ASD) may be associated. The primary source of exposure to organic mercury in humans is to methylmercury from fish consumption. We evaluated the association between prenatal methylmercury exposure and ASD phenotype in children and adolescents in the Republic of Seychelles, where fish consumption is high. Methods: We administered the Social Communication Questionnaire to parents of a cohort of 1784 children, adolescents, and young adults. The Social Responsiveness Scale was administered to teachers of 537 cohort subjects at about 10 years of age. Prenatal exposure to methylmercury was measured in maternal hair samples collected at or near the time of birth. Multivariable regression models evaluated the relationship between prenatal methylmercury exposure and ASD phenotypic scores, adjusting for relevant covariates. Results: The mean prenatal methylmercury exposure for subjects in the analysis was 8.4 ppm (standard deviation [SD] = 5.7). The mean Social Communication Questionnaire score was 8.0 (SD = 4.4). The mean prenatal methylmercury exposure for subjects with Social Responsiveness Scale scores was 6.7 ppm (SD = 4.4) and the mean Social Responsiveness Scale score was 57.6 (SD = 26.8). No consistent association between prenatal methylmercury exposure and ASD screening instrument was found, using linear and nonlinear regression analyses. Conclusions: Prenatal exposure to methylmercury was not associated with ASD phenotypic behaviors in our cohort of high fish consumers. Our findings contribute to the growing literature suggesting that exposure to methylmercury does not play an important role in the development of ASD phenotypic behavior.

Choline status and neurodevelopmental outcomes at 5 years of age in the Seychelles Child Development Nutrition Study

Choline is an essential nutrient that is found in many food sources and plays a critical role in the development of the central nervous system. Animal studies have shown that choline status pre- and postnatally can have long-lasting effects on attention and memory; however, effects in human subjects have not been well studied. The aim of the present study was to examine the association between plasma concentrations of free choline and its related metabolites in children and their neurodevelopment in the Seychelles Child Development Nutrition Study, an ongoing longitudinal study assessing the development of children born to mothers with high fish consumption during pregnancy. Plasma concentrations of free choline, betaine, dimethylglycine (DMG), methionine and homocysteine and specific measures of neurodevelopment were measured in 210 children aged 5 years. The childrens plasma free choline concentration (9·17 (sd 2·09) μmol/l) was moderately, but significantly, correlated with betaine (r 0·24; P= 0·0006), DMG (r 0·15; P= 0·03), methionine (r 0·24; P= 0·0005) and homocysteine (r 0·19; P= 0·006) concentrations. Adjusted multiple linear regression revealed that betaine concentrations were positively associated with Preschool Language Scale – total language scores (β = 0·066; P= 0·04), but no other associations were evident. We found no indication that free choline concentration or its metabolites, within the normal physiological range, are associated with neurodevelopmental outcomes in children at 5 years of age. As there is considerable animal evidence suggesting that choline status during development is associated with cognitive outcome, the issue deserves further study in other cohorts.