Reconsideration of Leeway Space Calculated Using Models from Same Patients at Different Growth Stages.

In this study, original leeway space was determined using plaster models prepared from the same individuals over successive years and compared with that reported in an earlier study. The plaster models used for measurement were prepared from a normal dentition group comprising 64 patients and a crowded dentition group comprising 29. The mesiodistal crown width of the deciduous canines, deciduous first molars, and deciduous second molars, as well as that of the canines, first premolars, and second premolars was measured. Leeway space was then calculated and the results compared with that previously reported. The average maxillary and mandibular leeway spaces calculated in the same individuals were 1.46 and 2.66 mm, respectively, whereas those calculated from data collected from different individuals were 0.89 and 2.83 mm, respectively. No direct statistical comparison of leeway space between this and the earlier report was possible. The present results did indicate, however, that it was larger in the maxilla than previously reported. A larger leeway space is advantageous for the normal arrangement of the permanent teeth. While the mesiodistal crown width of some of the lateral segment teeth in the present study was significantly larger in boys than in girls, no significant difference was observed in leeway space between sexes. The mesiodistal width was larger in multiple permanent teeth such as canines in the crowded dentition group, and this difference was significant in boys. On the other hand, no difference was observed in mesiodistal width in the deciduous teeth between the two groups. These results showed a tendency toward larger teeth and less leeway space in the permanent dentition in the crowded dentition group, which may have been a factor in why such crowding occurred.

Melatonin suppresses the antiviral immune response to EMCV infection through intracellular ATP deprivation caused by mitochondrial fragmentation.

Melatonin, a sleep hormone derived from the pineal gland, has an anti-inflammatory effect on the immune system in addition to modulating the brain nervous system. Previous studies have shown that melatonin suppresses signaling pathways downstream of multiple pattern recognition receptors on the innate immune cells during pathogen infection, but the specific mechanism of suppression has not been well understood. Using an encephalomyocarditis virus (EMCV) infection model in macrophages, we investigated the effects of melatonin on the antiviral response in innate immunity and found that melatonin attenuated the uptake of viral particles into macrophages. Furthermore, melatonin suppressed cytoskeletal regulation by decreasing ATP production by mitochondria. Finally, in an in vivo infection experiment, we also found that melatonin administration partially exacerbated the infection in the mouse brain. These results suggest that melatonin may have an inhibitory effect on excessive inflammation by suppressing cytoskeletal regulation in the innate immune system, but also suggest that suppression of inflammation may lead to insufficient protection against EMCV infection in vivo.

Caries and Its Association with Infant Feeding and Oral Health-related Behavior in 18-month and 3-year-old Japanese Children.

The primary aim of this study was to determine the prevalence of dental caries and its association with infant feeding and oral health-related behavior in Japanese children between the ages of 18 months and 3 years. A total of 387 infants were initially examined at the age of 18 months (or 19 months in some cases) and then again at 3 years (or at 3 years 1 month in some cases). The primary objective was to identify factors contributing to the prevalence of caries in children aged 18 months. The secondary objective was to follow up children with no caries at the age of 18 months to seek potential correlations between background factors and the increment of caries by the age of 3 years. In an adjusted multivariate logistic regression model, children who continued breastfeeding were approximately 7 times more likely to have dental caries at 18 months of age than those who did not. Infants brought in for an oral examination and consultation at around 12 months of age were less likely to develop dental caries at 18 months of age than those who were not. Multivariate logistic regression analysis revealed that infants who did not receive a dental check-up at 12 months of age showed a significantly higher incidence of dental caries at 3 years of age. The present results suggest that prolonged breastfeeding is a risk factor for early childhood caries at 18 months of age, and that infants in whom a regular oral care program is implemented from the age of 12 months are less likely to develop early childhood caries at 18 months or 3 years.

Microbiomes of colored dental biofilms in children with or without severe caries experience.

BACKGROUND: Biofilm coloration can compromise maturation and increase the risk of oral disease in adulthood, though children with colored biofilm do not always demonstrate a poor oral health status. AIM: The microbial compositions of colored and white biofilms in children were compared. DESIGN: Thirty-two dental biofilm samples from 16 children (age < 13 years) were analyzed using 16S rRNA pyrosequencing, then the subjects were divided into severe caries and healthy (caries-free) groups. Correlations between microbiomes and oral health status were also examined. RESULTS: Phylogenetic analysis revealed no distinctly different patterns between colored and white biofilms. In the severe caries group, genus Actinomyces, Cardiobacterium, Kingella, Lautropia, and Veillonella, and family Neisseriaceae were detected, though abundance was significantly different between colored and white biofilm specimens, in contrast to the healthy group. In addition, five colored biofilm samples from the severe caries group contained greater than 15% Actinomyces, which led us to consider that genus to be possibly associated with formation of colored biofilm in children. CONCLUSIONS: Our findings indicate that differences in bacterial composition between colored and white biofilms are higher in individuals with severe caries. Additional research may reveal the significance of colored dental biofilm in children.

Treatment of Child with Four Lost Maxillary Incisors due to Traffic Injury.

Tooth injury is frequently encountered in pediatric clinical practice. A clinician may be requested to not only respond at the time of injury, but also properly diagnose how such injury will affect the teeth and dentition. Here, we present a case of a child who lost 4 bilateral maxillary incisors due to a traffic accident, and in whom marked mesial inclination of the bilateral maxillary canines and bilateral maxillary first premolars would subsequently occur. Dental management was provided over an extended period. The boy, aged 9 years 11 months, visited our department with the chief complaint of premature loss of 4 bilateral maxillary incisors and eating disturbance associated with the loss of these teeth. Initially, a denture was fitted. The bilateral maxillary canines subsequently demonstrated marked mesial inclination, however, and erupted from an area equivalent to that of the bilateral central incisors. The bilateral maxillary first premolars also showed mesial inclination and rotation. Taking esthetics into consideration, a Nance holding arch with resin buttons that extended to the maxillary incisors and attached to artificial teeth was used. This appliance was also equipped with a retracting hook, and the bilateral maxillary canines were moved downward and distally. Presently, the removable partial denture is worn for the purposes of retention, esthetics, and mastication. Long-term oral management will be necessary. Therefore, final prosthetic treatment for the missing maxillary incisors is planned for when the patient reaches adulthood.

Influence of Lifestyle Factors on Risk of Dental Caries among Children Living in Urban China.

The prevalence of dental caries has been decreasing among kindergarten children in Shanghai, China, over recent years, although it still remains at an unacceptably high level. The purpose of this study was to identify which factors were important in providing oral health guidance and achieving further improvement in the oral health status of kindergarten children in urban China. A survey was conducted on dental caries in 128 Japanese and 368 Chinese kindergarten children and a questionnaire given to their parents/guardians on each child's lifestyle and dietary habits from birth to the present. Correlations between responses to each questionnaire item and the status of dental caries were statistically analyzed. The dft index score (p=0.0016), prevalence of dental caries (p=0.0002), and percentages of children with decayed (untreated caries-affected) teeth (p<0.0001) were significantly higher in the Chinese than in the Japanese children. Many differences were observed in lifestyle factors between the two groups. The percentage of parents failing to control the child's snacking habits between meals was higher in China, and weaning was significantly delayed in China compared with in Japan. These lifestyle factors were considered to be associated closely with the high risk of dental caries in Chinese kindergarten children. These findings indicate that oral health guidance for kindergarten children in Shanghai, China, should focus on control of dietary habits, including control of inter-meal snacking, and breastfeeding practices. The results of this study may help improve the status of dental caries among Chinese children.

Hydrogen peroxide contributes to the epithelial cell death induced by the oral mitis group of streptococci.

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H2O2) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H2O2 may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H2O2-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited S. oralis cytotoxicity, and H2O2 alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H2O2 production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H2O2 induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H2O2 is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts.

Identification, characterization, and expression of dentin matrix protein 1 gene in Xenopus laevis.

Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein expressed mainly in bone and dentin, and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. The DMP1 gene, however, appears to evolve rapidly in comparison with other SIBLING genes, even though such functionally important molecules usually evolve more slowly than less important ones. The purpose of this study was to identify and characterize an ortholog of the DMP1 gene in an amphibian (Xenopus laevis; X. laevis) to clarify molecular evolutionary alterations in DMP1 associated with calcified tissues in tetrapods. Furthermore, we analyzed the mRNA expression of this gene to elucidate its functional change in bone and developing tooth germ in comparison with amniote DMP1s. The similarities of the deduced amino acid sequence of X. laevis DMP1 to that of the corresponding amniote proteins were low, although they did share several unique features specific to DMP1 and have similar properties. Expression of X. laevis DMP1 mRNA was predominant in osteocytes and odontoblasts, but only transiently observed in ameloblasts, as in amniotes. These results suggest that DMP1 has conserved several functions during tetrapod evolution. This indicates that continuity of biochemical properties has been more important in maintaining DMP1 functionality than that of the sequence of amino acid residues, which has undergone change over the course of molecular evolution.

Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species.

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, is a common streptococcal species implicated in infective endocarditis. Herein, we investigated the effects of infection with S. sanguinis on foam cell formation and cell death of macrophages. Infection with S. sanguinis stimulated foam cell formation of THP-1, a human macrophage cell line. At a multiplicity of infection >100, S. sanguinis-induced cell death of the macrophages. Viable bacterial infection was required to trigger cell death because heat-inactivated S. sanguinis did not induce cell death. The production of cytokines interleukin-1ß and tumor necrosis factor-α from macrophages was also stimulated during bacterial infection. Inhibition of the production of reactive oxygen species (ROS) resulted in reduced cell death, suggesting an association of ROS with cell death. Furthermore, S. sanguinis-induced cell death appeared to be independent of activation of inflammasomes, because cleavage of procaspase-1 was not evident in infected macrophages.

Pili of oral Streptococcus sanguinis bind to salivary amylase and promote the biofilm formation.

Streptococcus sanguinis is a member of oral streptococci and one of the most abundant species found in oral biofilm called dental plaque. Colonization of the oral streptococci on the tooth surface depends on the adhesion of bacteria to salivary components adsorbed to the tooth surface. Recently, we identified unique cell surface long filamentous structures named pili in this species. Herein, we investigated the role of S. sanguinis pili in biofilm formation. We found that pili-deficient mutant, in which the genes encoding the three pilus proteins PilA, PilB and PilC have been deleted, showed an impaired bacterial accumulation on saliva-coated surfaces. Confocal microscopic observations suggested that the mutant was incapable of producing typical three-dimensional layer of biofilm. Ligand blot analysis showed that the ancillary pilus proteins PilB and PilC bound to human whole saliva. Additional analysis demonstrated that PilC bound to multiple salivary components, and one of which was found to be salivary α-amylase. These results indicate that pilus proteins are members of saliva-binding proteins of oral S. sanguinis, and suggest the involvement of pili in its colonization on saliva-coated tooth surfaces and in the human oral cavity.

Specific behavior of intracellular Streptococcus pyogenes that has undergone autophagic degradation is associated with bacterial streptolysin O and host small G proteins Rab5 and Rab7.

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion.

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.

Comparative genomic analyses of Streptococcus mutans provide insights into chromosomal shuffling and species-specific content.

BACKGROUND: Streptococcus mutans is the major pathogen of dental caries, and it occasionally causes infective endocarditis. While the pathogenicity of this species is distinct from other human pathogenic streptococci, the species-specific evolution of the genus Streptococcus and its genomic diversity are poorly understood. RESULTS: We have sequenced the complete genome of S. mutans serotype c strain NN2025, and compared it with the genome of UA159. The NN2025 genome is composed of 2,013,587 bp, and the two strains show highly conserved core-genome. However, comparison of the two S. mutans strains showed a large genomic inversion across the replication axis producing an X-shaped symmetrical DNA dot plot. This phenomenon was also observed between other streptococcal species, indicating that streptococcal genetic rearrangements across the replication axis play an important role in Streptococcus genetic shuffling. We further confirmed the genomic diversity among 95 clinical isolates using long-PCR analysis. Genomic diversity in S. mutans appears to occur frequently between insertion sequence (IS) elements and transposons, and these diversity regions consist of restriction/modification systems, antimicrobial peptide synthesis systems, and transporters. S. mutans may preferentially reject the phage infection by clustered regularly interspaced short palindromic repeats (CRISPRs). In particular, the CRISPR-2 region, which is highly divergent between strains, in NN2025 has long repeated spacer sequences corresponding to the streptococcal phage genome. CONCLUSION: These observations suggest that S. mutans strains evolve through chromosomal shuffling and that phage infection is not needed for gene acquisition. In contrast, S. pyogenes tolerates phage infection for acquisition of virulence determinants for niche adaptation.

Cost effective DNA sequencing and template preparation from bacterial colonies and plasmids.

Cost-effective DNA sequencing and template preparation were evaluated for high-throughput screening of bacterial colonies. The rolling-circle amplification to generate template for DNA sequencing was carried out using 10-fold smaller amount of phi29 DNA polymerase and excluding two other enzymes (yeast pyrophosphatase and calf intestine alkaline phosphatase) used previously. Then, a 1/40 volume of the fluorescent terminator mix recommended by the manufacturer gave a usable sequencing result although a 1/26.7 volume of the mix was used to compare the protocol more accurately with manufacturer's protocol.

Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13.

Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis.

Streptococcus pyogenes infection induces septic arthritis with increased production of the receptor activator of the NF-kappaB ligand.

Bacterial arthritis is a rapidly progressive and highly destructive joint disease in humans, with Staphylococcus aureus and Neisseria gonorrhoeae the major causative agents, although beta-hemolytic streptococci as well often induce the disease. We demonstrate here that intravenous inoculation of CD-1 mice with the group A streptococcus (GAS) species Streptococcus pyogenes resulted in a high incidence of septic arthritis. Signs of arthritis emerged within the first few days after injection, and bacterial examinations revealed that colonization of the inoculated GAS in the arthritic joints persisted for 21 days. Induction of persistent septic arthritis was dependent on the number of microorganisms inoculated. Immunohistochemical staining of GAS with anti-GAS antibodies revealed colonization in the joints of infected mice. Cytokine levels were quantified in the joints and sera of infected mice by using an enzyme-linked immunosorbent assay. High levels of interleukin-1beta (IL-1beta) and IL-6 were detected in the joints from 3 to 20 days after infection. We noted that an increase in the amount of receptor activator of NF-kappaB ligand (RANKL), which is a key cytokine in osteoclastogenesis, was also evident in the joints of the infected mice. RANKL was not detected in sera, indicating local production of RANKL in the infected joints. Blocking of RANKL by osteoprotegerin, a decoy receptor of RANKL, prevented bone destruction in the infected joints. These results suggest that GAS can colonize in the joints and induce bacterial arthritis. Local RANKL production in the infected joints may be involved in bone destruction.

Infection by Streptococcus pyogenes induces the receptor activator of NF-kappaB ligand expression in mouse osteoblastic cells.

Group A Streptococcus pyogenes is known to induce nongonococcal septic arthritis in addition to pharyngitis, scarlet fever, and poststreptococcal sequelae. However, little is known about the interaction between S. pyogenes and bone cells. We report here that S. pyogenes strain JRS4 (M6) attached to and invaded mouse primary osteoblasts. Reverse transcription-PCR demonstrated that S. pyogenes infection of osteoblasts stimulated expression of mRNA for the receptor activator of NF-kappaB ligand (RANKL). Western blot analysis followed by ligand precipitation with the receptor activator of NF-kappaB receptor showed that there was an increase in RANKL protein in infected osteoblasts. Production of interleukin-6 was also stimulated, but no production of interleukin-1beta or tumor necrosis factor alpha was observed. Stimulation of RANKL production was not observed in osteoblasts stimulated with heat-inactivated S. pyogenes, suggesting that an active interaction of S. pyogenes with osteoblasts is essential for this phenomenon. A Western blot analysis performed with antibodies specific for phosphorylated signal transduction proteins demonstrated that S. pyogenes infection induces phosphorylation of p38 mitogen-activated protein kinase. A specific inhibitor of this kinase, SB203580, inhibited RANKL production by infected osteoblasts. These results suggest that infection of osteoblasts by S. pyogenes stimulates RANKL production and may trigger bone destruction in infected bone tissue.