Association of common variants in PAH and LAT1 with non-syndromic cleft lip with or without cleft palate (NSCL/P) in the Polish population

Background

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common structural malformation with a complex and multifactorial aetiology. Associations of abnormalities in phenylalanine metabolism and orofacial clefts have been suggested.

Methods

Eight single nucleotide polymorphisms (SNPs) of genes encoding phenylalanine hydroxylase (PAH) and large neutral l-amino acid transporter type 1 (LAT1), as well as the PAH mutation that is most common in the Polish population (rs5030858; R408W), were investigated in 263 patients with NSCL/P and 270 matched controls using high resolution melting curve analysis (HRM).

Results

We found that two polymorphic variants of PAH appear to be risk factors for NSCL/P. The odds ratio (OR) for individuals with the rs7485331 A allele (AC or AA) compared to CC homozygotes was 0.616 (95% confidence interval [CI] = 0.437–0.868; p = 0.005) and this association remains statistically significant after multiple testing correction. The PAH rs12425434, previously associated with schizophrenia, was borderline associated with orofacial clefts. Moreover, haplotype analysis of polymorphisms in the PAH gene revealed a 4-marker combination that was significantly associated with NSCL/P. The global p-value for a haplotype comprised of SNPs rs74385331, rs12425434, rs1722392, and the mutation rs5030858 was 0.032, but this association did not survive multiple testing correction.

Conclusion

This study suggests the involvement of the PAH gene in the aetiology of NSCL/P in the tested population. Further replication will be required in separate cohorts to confirm the consistency of the observed association.     

Sensitivity of Candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine

Objectives

Candida albicans cells form biofilms on polymeric surfaces of dentures and other prostheses introduced into the oral cavity. Many biofilm microorganisms exhibit resistance to antimicrobial agents; C. albicans cells may also develop resistance to naturally occurring antifungal peptides in human saliva including histatins (Hsts) and defensins (hBDs). Therefore, we evaluated Hst 5 activity on C. albicans biofilm cells compared to planktonic cells and measured whether surface treatment of denture acrylic with Hst 5, hBD-3, or chlorhexidine gluconate could inhibit in vitro biofilm development.

Methods

Acrylic disks were preconditioned with 500 μl saliva for 30 min, and inoculated with C. albicans cells (10⁶ cells/ml) for 1 h, at 37 °C. Non-adherent cells were removed by washing and disks and were incubated in YPD growth medium for 24, 48, and 72 h at 37 °C. Candidacidal assays were performed on 48-h-biofilms and on planktonically grown cells using Hst 5 (15.5, 31.25, and 62 μM). Cell adhesion was compared on disks pre-coated with 0.12% chlorhexidine gluconate, 50 μM Hst 5, or 0.6 μM hBD-3 after 24, 48, and 72 h growth.

Results

No significant difference was observed in sensitivity to Hst 5 of biofilm cells compared to planktonic cells (p > 0.05). Pre-coating disks with hBD-3 did not inhibit biofilm development; however, Hst 5 significantly inhibited biofilm development at 72 h, while 0.12% chlorhexidine significantly inhibited biofilm development at all time intervals (p < 0.05).

Conclusions

C. albicans biofilm cells grown on denture acrylic are sensitive to killing by Hst 5. Surface coating acrylic with chlorhexidine or Hst 5 effectively inhibits biofilm growth and has potential therapeutic application.     

Adherence in vitro of Candida albicans to plasma treated acrylic resin. Effect of plasma parameters, surface roughness and salivary pellicle

The adhesion of Candida albicans to surfaces is the prerequisite for occurrence of denture stomatitis.

Objective

Hence, this study investigated if surface modifications with plasma treatments could reduce the adherence of C. albicans to a denture base resin.

Methods

Specimens (n = 180) with roughened and smooth surfaces were made and divided into five groups: control—specimens were left untreated; experimental groups—specimens were submitted to plasma treatments to obtain surfaces with different hydrophobicity (Ar/50 W; ArO₂/70 W; AAt/130 W) or incorporation of fluorine (Ar/SF₆70 W). Contact angle measurements were performed immediately after the treatments and after immersion in water for 48 h. For each group, half of the specimens were incubated with saliva prior to the adhesion assay. The number of adherent yeasts was evaluated by XTT reduction method.

Results

For the experimental groups, there was significant change in the mean contact angle after 48 h of immersion in water. Groups ArO₂/70 W and ArSF₆/70 W showed significantly lower absorbance readings than the other groups, regardless the presence or absence of saliva and surface roughness.

Conclusions

Results demonstrated that ArO₂/70 W and ArSF₆/70 W plasma treatments showed promising potential for reducing the adherence of C. albicans to denture base resins.     

Interleukin-1 receptor gene variants are associated with aggressive periodontitis in the Japanese

Objective

Previous studies have indicated that type-1 and type-2 interleukin-1 (IL-1) receptors (IL-1R1 and IL-1R2) play important roles in periodontitis progression. We investigated the association between periodontitis and polymorphisms in the IL-1R1 and IL-1R2 genes (IL1R1 and IL1R2).

Design

We searched for genetic variants in IL1R1 and IL1R2 in 24 Japanese patients with aggressive periodontitis (AgP) and 24 periodontally healthy controls. Thirty-eight single nucleotide polymorphisms (SNPs) were identified within genomic regions containing all exons and relevant exon-intron boundaries in IL1R1 and IL1R2. Possible associations of each gene locus with AgP were investigated in 119 AgP patients and 102 periodontally healthy controls using allelotypes, genotypes, and haplotypes.

Results

Significant differences were noted in the frequencies of 3 SNPs in IL1R2 (rs3819370, rs3218974 and rs3218977) for AgPs and controls (p = 0.012, p = 0.008, and p = 0.038, respectively), after adjustment for gender and smoking status in the additive model (p = 0.016, p = 0.007, and p = 0.027, respectively) and 2 haplotypes (p = 0.010 and p = 0.011, respectively) constructed from 2 SNPs (rs3819370 and rs3218974) that showed the lowest p-values after adjustment of covariates in additive models.

Conclusion

A genetic susceptibility locus for AgP may lie within or close to the IL1R2 locus. Further studies in other populations are necessary to confirm these results.     

The anti-adherence activity and bactericidal effect of microparticulate silver additives in composite resin materials

Objective

Resin composite materials tend to accumulate microorganisms and dental plaque, which in turn may induce secondary caries around adhesive restorations. The aim of the present in vitro study was to evaluate the antibacterial activity of a resin composite material loaded with silver microparticles against Streptococcus mutans.

Design

Circular specimens (10.0 mm in diameter) of a resin composite matrix loaded with two different concentrations of a silver additive (Comp0.3: 0.3%; Comp0.6: 0.6%) and one unloaded reference composite matrix (Comp0: 0%) were made. Surface roughness R_a was assessed by perthometer measurements and hydrophobicity according to water contact angles was determined by computerized image analysis. The specimens were incubated in a S. mutans suspension (1 h, 37 °C) and adhering streptococci were quantified by using a biofluorescence assay (Alamar blue/Resazurin). Additionally, the viability of adhering bacteria was assessed by live/dead cell labelling in combination with fluorescence microscopy.

Results

Statistically significant differences between the median water contact angles of Comp0 (66.3°), Comp0.3 (76.7°), and Comp0.6 (89.4°) were observed (p < 0.001). A three- to fourfold higher amount of adhering S. mutans was found on reference Comp0 (12,093 relative fluorescence units) than on Comp0.3 (4258 rfu) and Comp0.6 (3292) (p < 0.001 for both). Significantly higher percentages of dead cells than on Comp0 (0.5%) were found on Comp0.3 (6.1%) and on Comp0.6 (10.1%) (p < 0.001 for both).

Conclusions

The addition of microparticulate silver to a resin composite material increased the surface hydrophobicity and reduced the number of adhering streptococci. Simultaneously it increased the percentage of dead and inactive cells on the composite surface. Thus, silver additives seem to demonstrate anti-adherence activity as well as a bactericidal effect.     

Transaminases in the acquired pellicle

Objective

Transaminases (AST, aspartate amino transferase; ALT, alanine amino transferase) are relevant enzymes in physiology and pathology of the human organism. The aim of the present in situ study was to demonstrate the presence of these enzymes in the enamel pellicle.

Methods

Bovine enamel slabs were fixed on buccal sites of individual upper jaw splints and worn for 3, 30 and 120 min by 5 subjects to allow pellicle formation. The in situ pellicles were tested for AST and ALT. Enzyme activities were measured photometrically via determination of the products pyruvate and oxalacetate using lactate-dehydrogenase and malate-dehydrogenase, respectively.

Results

Enzymatic AST- as well as ALT-activities are present in the acquired pellicle within 3 min. The enzyme activities exposed at the pellicles’ surfaces increased slightly with the pellicle formation time (ANOVA, AST: n.s., ALT: p = 0.021). However, the two enzymes show considerable intraindividual and interindividual variability. The mean AST-activity of the pellicle amounted to 1.07 ± 0.81 mU/cm² (ALT 1.18 ± 0.52 mU/cm²). The ALT-activity of the centrifuged saliva was 26.62 ± 11.09 mU/ml (AST 35.98 ± 29.35 mU/ml).

Conclusions

AST as well as ALT are present in the in situ pellicle layer and may contribute to the intrinsic maturation of pellicle proteins.     

Parylene coating hinders Candida albicans adhesion to silicone elastomers and denture bases resin

Objective

To investigate whether parylene coatings over denture bases and silicone elastomers can effectively reduce Candida albicans adhesion and thus to decrease the incidence of denture stomatitis.

Design

Specimens of silicone elastomers A-2186 or lucitone 199 resin were prepared, and the measurements of contact angle, assay of XTT reduction and cell count of C. albicans adhesion were taken before and after parylene treatment. Furthermore, morphology of C. albicans adhesion for 48 h was observed by scanning electron microscope (SEM), and C. albicans adhesion for 4 h was illustrated by confocal scanning laser microscopy (CLSM) in combination with fluorescent dyes FUN-1 and Concanavalin A.

Result

There was a statistical difference between mean contact angles of silicone elastomer A-2186 before and after parylene coating (P < 0.05). The amount of C. albicans adhesion to the surface of silicone elastomer A-2186 and lucitone 199 resin after parylene treatment was significantly less than before parylene treatment by cells count and XTT reduction assay (P < 0.05). In SEM and CLSM analysis, C. albicans biofilm was more apt to generate on the surface of silicone elastomer A-2186 than other three groups, and more C. albicans aggregation formed on the surface of silicone elastomer A-2186 and lucitone 199 resin before parylene treatment than after parylene treatment.

Conclusion

Parylene coating reduced C. albicans adhesion and aggregation on the surface of silicone elastomer A-2186 and lucitone 199 resin, and improved the wettability of silicone elastomer A-2186.     

Prevalence and distribution of serotype-specific genotypes of Aggregatibacter actinomycetemcomitans in chronic periodontitis Brazilian subjects

Objective

Previous studies have suggested that Aggregatibacter actinomycetemcomitans is involved in the aetiology of aggressive periodontitis as well as chronic periodontitis. In addition, some authors have also reported that serotype-specific antigens of A. actinomycetemcomitans determine the severity of disease. This study aimed to elucidate the prevalence of A. actinomycetemcomitans and the distribution of A. actinomycetemcomitans serotypes in Brazilian subjects with chronic periodontitis.

Design

A total of 486 individuals were enrolled in this survey. All patients received clinical examinations that included periodontal pocket depth, clinical attachment loss, plaque, and gingival indexes. Subgingival samples were taken for microbial analysis. The genomic DNA of A. actinomycetemcomitans was provided by PCR.

Results

Out of 486 subjects examined, A. actinomycetemcomitans was isolated in 85 (17.5%) individuals. Out of 85 positive samples, 68 were infected by at least 1 serotype, 7 by mixed infection, and 10 were non-serotyped. Serotypes d and f were not detected. Serotype c showed the highest prevalence (52.9%), followed by serotype a (31.8%).

Conclusions

Intragroup analysis revealed that, in slight/moderate periodontitis, serotypes c and a were significantly more prevalent than serotypes b and d-f; the prevalence of serotype c in severe periodontitis was significantly greater than that of serotypes a and b. Our data were similar in Asian and Eurasian populations.     

Presence of Helicobacter pylori in supragingival dental plaque of individuals with periodontal disease and upper gastric diseases

Background

Helicobacter pylori is a Gram-negative microorganism which is able to colonize the gastric mucosa and is associated with peptic ulcer, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Several studies have detected this bacterium in the oral cavity, suggesting it as a potential reservoir. The aim of this study was to investigate the presence of H. pylori in the oral cavity of individuals with periodontal disease and gastric diseases.

Methods

115 individuals, with mean age 49.6 (±5.8) years, were divided in 4 groups: (A) with gastric diseases and periodontal disease; (B) with gastric diseases and no periodontal disease; (C) without gastric diseases and without periodontal disease, (D) without gastric diseases and with periodontal disease. Supra and subgingival plaque samples were collected from posterior teeth of the individuals with sterile paper points, and prepared for Polymerase Chain Reaction analysis. Fisher's exact test was used for detecting statistical differences between groups (p < 0.05).

Results

H. pylori was detected in supragingival plaque of 9/36 (25%) of group A, 1/31 (0.3%) of group B, 0 (0%) of group C and 3/36 (8.3%) of group D. No subgingival samples were positive for H. pylori. There was a statistically higher prevalence of H. pylori in groups A and D when compared to B and C (p < 0.05).

Conclusion

H. pylori was detected in the supragingival plaque, but not in the subgingival plaque, of individuals with periodontal disease and upper gastric diseases. There was an association between the supragingival colonization of H. pylori and oral hygiene parameters such as the presence of plaque and gingival bleeding.     

Application of an active attachment model as a high-throughput demineralization biofilm model

Objectives

To investigate the potential of an active attachment biofilm model as a high-throughput demineralization biofilm model for the evaluation of caries-preventive agents.

Methods

Streptococcus mutans UA159 biofilms were grown on bovine dentine discs in a high-throughput active attachment model. Biofilms were first formed in a medium with high buffer capacity for 24 h and then subjected to various photodynamic therapies (PACT) using the combination of Light Emitting Diodes (LEDs, Biotable^®) and Photogem^®. Viability of the biofilms was evaluated by plate counts. To investigate treatment effects on dentine lesion formation, the treated biofilms were grown in a medium with low buffer capacity for an additional 24 h. Integrated mineral loss (IML) and lesion depth (LD) were assessed by transversal microradiography. Calcium release in the biofilm medium was measured by atomic absorption spectroscopy.

Results

Compared to the water treated control group, significant reduction in viability of S. mutans biofilms was observed when the combination of LEDs and Photogem^® was applied. LEDs or Photogem^® only did not result in biofilm viability changes. Similar outcomes were also found for dentine lesion formation. Significant lower IML and LD values were only found in the group subjected to the combined treatment of LEDs and Photogem^®. There was a good correlation between the calcium release data and the IML or LD values.

Conclusions

The high-throughput active attachment biofilm model is applicable for evaluating novel caries-preventive agents on both biofilm and demineralization inhibition. PACT had a killing effect on 24 h S. mutans biofilms and could inhibit the demineralization process.     

Clinical and genetic evaluation of a Chinese family with isolated oligodontia

Objectives

Oligodontia is defined as the congenital absence of 6 or more permanent teeth excluding the third molar. Tooth agenesis may be classified as syndromic/non-syndromic and as familial/sporadic. To date, more than 300 genes have been found to be involved in tooth development, but only a few of these genes, such as MSX1, PAX9 and AXIN2, are related to the condition of non-syndromic oligodontia. The objective of the present work was to investigate the disease-causing gene of non-syndromic oligodontia in a Han Chinese family and analyse the pathogenesis of mutations that result in oligodontia.

Design

We examined all individuals of the oligodontia family by clinical and radiographic examinations. Based on the clinical manifestations, the candidate genes MSX, PAX9 and AXIN2 were selected to analyse and screen for mutations.

Results

The clinical evaluation suggested that the family might show non-syndromic oligodontia. DNA sequencing of the MSX1 gene revealed two mutations in the two patients with oligodontia: a heterozygotic silent mutation, c.348C > T (P.Gly116=), in exon 1 and a homozygotic deletion of 11 nucleotides (c.469 + 56delins GCCGGGTGGGG) in the intron. However, the silent mutation and the deletion mutation were thought to be known polymorphisms (rs34165410 and rs34341187) by bioinformatics analysis. We did not detect any mutations in the PAX9 and AXIN2 genes of oligodontia patients.

Conclusion

Our finding suggests that identified polymorphisms (c.348C > T and c.469 + 56delins GCCGGGTGGGG) may be responsible for the oligodontia phenotype in this Chinese family, but the association requires further study.     

Association of DLX3 gene polymorphism and dental caries susceptibility in Japanese children

Objective

In this study, we investigated whether single nucleotide polymorphisms (SNPs) in DLX3 are associated with dental caries susceptibility in Japanese children.

Design

Genomic DNA of 201 Japanese children was extracted from buccal epithelial cells. The subjects were divided into two groups: ‘low level’ group with <10,000 colony forming units (CFU) of Streptococcus mutans/mL saliva (level 0) and ‘high level’ group with ≥10,000 CFU/mL (more than level 1). Each group was further divided according to decayed, missing, filled teeth (dmft) into low caries experience (dmft ≤ 2) and high caries experience (dmft ≥ 3). Seven SNPs in DLX3 were genotyped using TaqMan^® SNP Genotyping Assay.

Results

Statistical significant association was observed between DLX3 (rs2278163) and caries experience in ‘high level Mutans streptococci’ group.

Conclusion

These findings suggest that rs2278163 SNP of DLX3 might be associated with dental caries susceptibility in Japanese children. T and C alleles of rs2278163 SNP may potentially be involved in caries susceptibility and caries protection respectively.     

Antibacterial effect of ozone on cariogenic bacterial species

Objective

The aim was to evaluate the antibacterial effect of ozone on cariogenic bacterial species with and without the presence of saliva and a possible effect on the salivary proteins.

Methods

Suspensions of Actinomyces naeslundii (ACTCC 12104^T), Lactobacilli casei (N CTC 151) and Streptococcus mutans (NCTC 10449), in salt buffer or in saliva, were exposed to ozone gas delivered by the ozone generator Healozone™ 2130C. Aliquots of the suspensions were taken after 10, 30 and 60 s ozone exposures and cultivated on agar plates. Initial number of bacteria per ml was 8.0 × 10⁷ (SD 2.2 × 10⁷) (A. naeslundii), 1.0 × 10⁸ (SD 3.1 × 10⁶) (L. casei) and 1.0 × 10⁸ (SD 7.0 × 10⁵) (S. mutans), respectively. The proteins were separated by SDS electrophoresis and visualized by silver staining.

Results

In salt buffer 92%, 73% and 64% of the initial numbers of A. naeslundii, S. mutans and L. casei, respectively, were killed already after 10 s ozone exposure, while approximately 99.9% of the bacteria were dead after a 60 s exposure. After 10 and 30 s, but not after 60 s exposure to ozone, S. mutans and L. casei were less efficiently killed in saliva compared to the salt buffer. Various saliva proteins were degraded by ozone after a 60 s exposure.

Conclusions

The cariogenic species S. mutans, L. casei and A. naeslundii were almost eliminated following 60 s of ozone treatment. This killing was reduced in the presence of saliva although increasing the ozone application time to 60 s overcame these reductants in saliva. Detection of altered salivary proteins indicates that saliva components constitute additional targets for ozone.     

Isolation of Candida dubliniensis in denture stomatitis

Objective

To describe the isolation of Candida dubliniensis from a patient with denture stomatitis and to compare with the presence of yeasts in the oral cavities of denture wearers.

Design

One hundred and fifty-two Candida isolates were recovered through oral swabs from denture as well as the underlying mucosa from 100 patients wearing denture. For detection and identification of fungal isolates, standard phenotypic and genotypic methods were used.

Results

Forty-five of 100 denture wearers suffered from denture stomatitis. Seventy-three Candida isolates were recovered from 38 denture wearers without denture stomatitis. In this group, Candida albicans was the predominant species (58.9%), followed by Candida tropicalis (15.1%), Candida guilliermondii (13.7%), Candida glabrata (9.6%), and Candida parapsilosis (2.7%).Seventy-nine isolates were yielded from 40 patients suffering from denture stomatitis. C. albicans was also the most frequently isolated species (58 isolates, 73.4%), followed by C. glabrata and C. tropicalis (7 isolates each, 8.9%), and Saccharomyces cerevisiae (2 isolates, 2.5%). One isolate was yielded of the following species: Candida famata, Candida krusei, C. parapsilosis and C. guilliermondii. Moreover 1 isolate was phenotypic and genotypic identified as C. dubliniensis genotype 1.

Conclusions

C. albicans is the predominant fungal species isolated from denture wearers. C. dubliniensis could be isolated from adults with denture stomatitis.     

In Vitro Evaluation of the Antibacterial Activities of MTAD in Combination with Nisin against Enterococcus faecalis

Introduction

MTAD is a common intracanal irrigant. Although MTAD inhibits Enterococcus faecalis in root canals, its bactericidal effect against E. faecalis remains to be improved. Nisin, an antibacterial peptide, possesses a strong bactericidal effect. The study evaluated the synergetic action between MTAD and nisin against E. faecalis.

Methods

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were used to measure the antibacterial activities of MTAD, MTAN (substitution of doxycycline with nisin), and MTADN (nisin in combination with doxycycline), respectively. The synergetic effect between nisin and doxycycline was evaluated by fractional inhibitory concentration (FIC) and time-killing curves. Furthermore, morphologic changes in E. faecalis were observed by scanning electron microscopy after E. faecalis was treated with MTAD, MTAN, or MTADN for 24 hours.

Results

The MBC of MTADN against E. faecalis was lower than that of MTAD and MTAN. The combination of nisin and doxycycline had a significantly synergetic antibacterial effect on E. faecalis. Among the 3 antimicrobial treatments, MTADN caused the most severe damage to E. faecalis.

Conclusions

The combination of nisin and doxycycline has a synergetic antibacterial effect on E. faecalis, and MTAD in conjunction with nisin inhibits E. faecalis better than MTAD alone.     

Identification of SNP markers on 1p36 and association analysis of EPB41 with mandibular prognathism in a Chinese population

Objective

The results of a genome-wide scan suggested that chromosome locus 1p36 might be linked to the etiology of mandibular prognathism (MP) amongst Asian ethnicities. In this study, we performed a two-stage case-control association study to determine whether one or more genes that confer susceptibility to MP are located within this genomic region.

Design

In the first stage of the study, we examined 103 single nucleotide polymorphisms (SNPs) on 1p36 across an 8.6 Mb critical region and within four candidate genes in 158 cases and 147 controls to identify genes associated with MP. In the second stage of the study, we examined an additional 23 SNPs within the erythrocyte membrane protein band 4.1 (EPB41) gene in 211 cases and 224 controls.

Results

Four SNPs located in the EPB41 gene showed possible allelic and genotypic associations with MP (P < 0.03 and P < 0.05, respectively) in the first stage. In the analysis of single SNPs in the second stage, the allele of rs4654388 showed the strongest significant association with MP (P = 0.008) and the rs4654388 G-allele was associated with a significantly increased risk of MP (OR: 1.78, 95% CI: 1.16-2.74). Haplotype analysis revealed that MP was associated significantly with haplotype GTTCAGGT (P_corrected = 0.031), which included the rs4654388 G-allele.

Conclusions

An association between genetic polymorphisms in the EPB41 gene and MP has been observed. Although the polymorphisms which may contribute to MP have not been determined, the results of our study suggest that the EPB41 gene could confer susceptibility to MP.     

Blockade of sympathetic β-receptors inhibits Porphyromonas gingivalis-induced alveolar bone loss in an experimental rat periodontitis model

Introduction

Periodontal disease is characterised by alveolar bone loss. Some studies have suggested the involvement of sympathetic nervous system in the deterioration of periodontal disease. Noradrenaline, released from sympathetic nerve terminals due to various stimuli, binds to specific adrenergic receptors on immune cells. Recently, we reported that restraint stress augmented the alveolar bone loss induced by Porphyromonas gingivalis infection. In this study, we investigated the effects of the β-blocker (propranolol) on alveolar bone loss induced by P. gingivalis infection to examine the involvement of sympathetic nerves in periodontal breakdown.

Methods

Sprague-Dawley rats were treated as follows: saline injection (Group A), propranolol injection (Group B), saline injection and oral challenge with P. gingivalis (Group C), and propranolol injection and oral challenge with P. gingivalis (Group D). Horizontal alveolar bone loss was evaluated by measuring the distance between the cemento-enamel junction and the alveolar bone crest. Specimens from periodontal tissue were evaluated by staining with hematoxylin-eosin and tartrate-resistant acid phosphatase.

Results

Blockade of β-receptors in periodontal tissue by propranolol inhibited osteoclast differentiation and prevented alveolar bone loss induced by P. gingivalis infection. Histological study revealed that the number of osteoclasts detected was proportional to the level of bone loss.

Conclusions

These results indicate that the sympathetic nervous system is involved in the development of periodontitis and suggest that sympathetic signal modulation with β-blockers enables the control of alveolar bone mass metabolism.     

Vitamin C attenuates the cytotoxic effects of Porphyromonas gingivalis on human gingival fibroblasts

Objective

Periodontitis is induced by an imbalance between bacterial virulence and host defense ability involving increased levels of oxidative stress. The aim of this study was to investigate the influence of vitamin C on the cytotoxic effects of Porphyromonas gingivalis on human gingival fibroblasts (HGF).

Methods

This in vitro study observed the interaction between HGF and P. gingivalis. HGF were cultured with medium containing vitamin C and exposed to P. gingivalis ATCC 33277 for a maximum of 180 min. The assessment of cell viability was followed by a 3-(4,5-dimethylthiazol-2-ly)-2,5-diphenyltetrazolium-bromide (MTT) assay. The apoptosis rate was detected by flow cytometry using Annexin-V-FITC and propidium iodide. Superoxide as an oxidative stress factor was measured photometrically by the reduction of ferricytochrome C.

Results

Vitamin C reduced the cytotoxic effects of P. gingivalis on HGF. Vitamin C-treated HGF showed significantly higher cell viability rates (89.0 ± 5.7%) in comparison to untreated HGF (77.0 ± 5.0%; p < 0.05). In vitamin C-treated HGF, lower apoptosis rates (40.0 ± 2.2%) were observed after P. gingivalis exposure than in untreated HGF (66.1 ± 1.6%; p < 0.05). The exposure of HGF to P. gingivalis led to a significant increase of superoxide concentration, but this effect was not influenced by vitamin C.

Conclusion

Vitamin C reduces the cytotoxic and apoptotic effects of P. gingivalis on HGF in vitro. These results suggest that the benefit of vitamin C should be further investigated clinically.     

Molecular characterization of Streptococcus mutans strains containing the cnm gene encoding a collagen-binding adhesin

Objective

Streptococcus mutans, known to be a major pathogen of dental caries, is also considered to cause infective endocarditis. Its 120-kDa Cnm protein binds to type I collagen, which may be a potential virulence factor. In this study, we characterized S. mutans clinical strains focusing on the cnm gene encoding Cnm.

Design

A total of 528 S. mutans strains isolated from Japanese, Finnish, and Thai subjects were investigated. Using molecular techniques, the distribution frequency of cnm-positive strains and location of the inserted cnm were analyzed. Furthermore, isogenic mutant strains were constructed by inactivation of the cnm gene, then their biological properties of collagen-binding and glucan-binding were evaluated. Southern hybridization of the genes encoding glucan-binding proteins was also performed.

Results

The distribution frequency of cnm-positive strains from Thai subjects was 12%, similar to that previously reported for Japanese and Finnish subjects. Furthermore, the location of insertion of cnm was the same in all cnm-positive clinical isolates. As for the cnm-inactivated mutant strains constructed from 28 clinical isolates, their collagen-binding activity was negligible. In addition, glucan-binding activity in the cnm-positive clinical isolates was significantly reduced and corresponded to a lack of gbpA encoding glucan-binding protein A.

Conclusions

Our results indicate that strains with cnm genes, the most crucial factor for the collagen-binding property of S. mutans, are detectable at similar frequencies over several different geographic locations. In addition, the common properties of these strains are a high level of collagen-binding activity and tendency for a low level of glucan-binding activity.