Nobuhiro Hanada

Systemic Diseases in Association with Microbial Species in Oral Biofilm from Elderly Requiring Care

Background: The oral cavity is a reservoir for colonization and infection of systemic organs by pathogenic bacteria. It is understood that aging, tooth eruption, hormonal changes, active disease, oral hygiene, and other factors have an influence on biofilm formation and bacterial accumulation in the oral cavity. Objective: To understand the influence of systemic health care on microfloral changes, we conducted epidemiological studies of nursing home residents in an attempt to elucidate the relationship between underlying systemic diseases and the isolation frequency of oral opportunistic pathogens. Methods: The prevalence of bacteria and fungi causing pneumonia in association with oral biofilm bacteria were determined using detection culture plates. The influences of gender, age, denture-wearing status, number of teeth, and bedridden status in the patients residing in nursing homes were then analyzed. Results: The isolation frequency rates of Candida albicans, Pseudomonadaceae, Staphylococcus spp., and some strains of Enterobacteriaceae in plaque samples, as well as C. albicans and Xanthomonas maltophilia in samples from the pharynx, were significantly higher in those requiring systemic care (mean age 83.9 years) than in those who did not require such care (mean 71.0 years). In particular, the frequencies of Pseudomonas spp., C. albicans, and Serratia marcescens in plaque were significantly higher in those who were bedridden. Furthermore, the isolation of Pseudomonas spp. and Klebsiella pneumoniae, and/or C. albicans in plaque was significantly associated with heart disease. Conclusion: The coexistence of Pseudomonas spp. and C. albicans in elderly with 10–19 teeth is a potential indicator of high risk for pneumonia and heart disease. Therefore, attention to oral hygiene and professional care for removing the indicators may diminish the occurrence of systemic disease in the elderly requiring systemic care.

Salivary biomarkers for predicting the progression of chronic periodontitis

OBJECTIVE Predicting the progression of periodontitis would allow for targeted supportive periodontal therapy. The purpose of this study was to determine the usefulness of salivary biomarkers for predicting the progression of periodontitis. DESIGN Eighty-five chronic periodontitis patients were enrolled in an 18-month longitudinal study. Amongst them, 57 experienced progression of periodontitis, indicated at the end of the 18 months by at least one site with >3mm loss of attachment compared with baseline. We determined the levels of aspartate aminotransferase, alanine aminotransferase (ALT), lactate dehydrogenase, alkaline phosphatase and free haemoglobin as biomarkers, as well as the counts of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia, which represented the periodontal bacteria, in the stimulated saliva. The Mann-Whitney U test was used to compare patients with and without progression. After categorising the diagnostic values, the chi-square test was applied. RESULTS Counts and ratios (ratio to total bacteria) of P. gingivalis and P. intermedia were found to be significant predictors of the progression of periodontitis. To increase prediction accuracy, combination analyses were performed. The combination of ALT level and the P. gingivalis ratio showed the highest likelihood (p<0.001, sensitivity 0.40, specificity 0.96, likelihood 11.30). CONCLUSION Our findings suggest that salivary ALT level and the P. gingivalis ratio may be potential indicators for the progression of periodontitis. Such a salivary test could be a useful diagnostic tool for predicting periodontal disease progression.

Tooth loss and intakes of nutrients and foods: a nationwide survey of Japanese dentists.

OBJECTIVES To clarify the association of tooth loss with dietary intakes among dentists, for whom sufficient dental care is available. METHODS We analyzed the data from 20 366 Japanese dentists (mean age +/- SD, 52.2 +/- 12.1 years; women 8.0%) who participated in a nationwide cohort study from 2001 to 2006. The baseline questionnaire included a validated food-frequency questionnaire to estimate intakes of foods and nutrients. We computed the geometric means of daily intakes by the number of teeth, adjusting for age, sex, smoking, physical activity, and history of diabetes. RESULTS The mean intakes of some key nutrients and food groups, such as carotene, vitamins A and C, milk and dairy products, and vegetables including green-yellow vegetables, decreased with the increasing number of teeth lost (P for trend <0.05). On the contrary, mean intakes of carbohydrate, rice, and confectioneries were increased among those with fewer teeth (P for trend <0.05). The difference in the geometric mean (%) between totally edentulous subjects and those with > or =25 teeth, that is [(Geometric mean for > or =25 teeth) - (Geometric mean for 0 teeth)]/(Geometric mean for > or =25 teeth) x 100, was 14.3%, 8.6%, 6.1%, and -6.1% for carotene, vitamin C, vitamin A, and carbohydrate, respectively. For food groups, it was 26.3%, 11.9%, 5.6%, -9.5%, and -29.6% for milk and dairy products, green-yellow vegetables, total vegetables, rice, and confectioneries, respectively. CONCLUSIONS Tooth loss was linked with poorer nutrition even among dentists.

Assessment of Genes Associated with Streptococcus mutans Biofilm Morphology

ABSTRACT Streptococcus mutans, the major pathogen responsible for dental caries in humans, is a biofilm-forming bacterium. In the present study, 17 different pulsed-field gel electrophoresis patterns of genomic DNA were identified in S. mutans organisms isolated clinically from whole saliva. The S. mutans isolates showed different abilities to form biofilms on polystyrene surfaces in semidefined minimal medium cultures. Following cultivation in a flow cell system in tryptic soy broth with 0.25% sucrose and staining using a BacLight LIVE/DEAD system, two strains, designated FSC-3 and FSC-4, showed the greatest and least, respectively, levels of biofilm formation when examined with confocal laser scanning microscopy. Further, image analyses of spatial distribution and architecture were performed to quantify the merged green (live cells) and red (dead cells) light. The light intensity of the FSC-3 biofilm was greater than that of the FSC-4 biofilm in the bottom area but not in the top area. S. mutans whole-genome array results showed that approximately 3.8% of the genes were differentially expressed in the two strains, of which approximately 2.2%, including bacitracin transport ATP-binding protein gene glrA and a BLpL-like putative immunity protein gene, were activated in FSC-3. In addition, about 1.6% of the genes, including those associated with phosphotransferase system genes, were repressed. Analyses of the glrA-deficient strains and reverse transcription-PCR confirmed the role of the gene in biofilm formation. Differential assessment of biofilm-associated genes in clinical strains may provide useful information for understanding the morphological development of streptococcal biofilm, as well as for colonization of S. mutans.

Relationship between the number of remaining teeth and physical activity in community-dwelling elderly.

The relationship between oral health and general health in the elderly has been much studied. However, further studies focussing on the influence of oral health on the quality of life (QOL) among the elderly are required. The goal of this study was to evaluate the relationship between oral health and physical or cultural activities. Subjects were 101 community-dwelling elderly persons who were functionally independent (mean age 70.3). Oral health status was evaluated according to the number of remaining teeth and the number of functional teeth. Physical and cultural activities were evaluated from self-reported information. The relationship between oral health and physical or cultural activities was examined by logistic regression analysis. About 60% of subjects took part in cultural activities, and less than half actively exercised (leisure sports, 33.6%; travel, 42.6%). Persons with 20 or more remaining teeth were more active in leisure sports (Odds ratio (OR)=4.86, 95% confidence interval (CI)=1.34, 17.38) and travel (OR=5.42, 95% CI=1.63, 18.08) than those with fewer than 20 remaining teeth. These results suggest that the number of remaining teeth is associated with physical activity in elderly persons.

Oral Streptococci Exhibit Diverse Susceptibility to Human β-Defensin-2: Antimicrobial Effects of hBD-2 on Oral Streptococci

We examined the antimicrobial effects of human β-defensin-2 (hBD-2) on 17 species of oral streptococci to investigate the involvement of antimicrobial peptide activity in oral microflora development and the clinical use of the antimicrobial peptide for oral microflora control. Oral streptococci exhibit diverse levels of susceptibility to human β-defensin-2 (hBD-2). Two major cariogenic bacterial species, Streptococcus mutans (S. mutans) and S. sobrinus, were found to be susceptible to the peptide, indicating that it is a potential therapeutic agent for preventing dental caries. S. mitis exhibited the lowest susceptibility to the peptide. S. mitis is a major indigenous bacterium in the oral microflora, and our results suggest that it might possess a certain resistance mechanism against hBD-2.

Different frequencies of Streptococcus anginosus infection in oral cancer and esophageal cancer

Multiple cancers frequently occur in the upper aerodigestive tract. The high incidence rate of multiple carcinomas in this region is often explained in terms of involvement of the same underlying risk factors. It has been reported that the oral bacterium Streptococcus anginosus (S. anginosus) is associated with esophageal, gastric, and pharyngeal cancer tissues. In this study, a highly specific quantification method for S. anginosus DNA using real‐time PCR was established. We employed this assay to determine whether S. anginosus is also associated with oral cancer tissues. This precise quantification method revealed different degrees of infection with S. anginosus in esophageal cancer and oral cancer. We assayed 10 ng of genomic DNA from cancer tissues, and found that eight of 18 samples (44%) from the esophagus contained a detectable level (>10 fg) of S. anginosus DNA, whereas this was the case for only five of 38 samples (13%) of oral cancer. The quantity of S. anginosus DNA in the esophageal cancer tissues was significantly higher than in oral cancer. The maximum amount of S. anginosus DNA was approximately ten times higher in esophageal than in oral cancer tissues. In addition, none of the five different oral cancer sites (floor of the mouth, mandibular gingival, maxillary gingival, buccal mucosal, and tongue) showed significant signs of S. anginosus infection. On the other hand, most non‐cancerous tissues of the esophagus and tongue showed an undetectable level of S. anginosus. These results suggest that S. anginosus is associated with esophageal cancer, but is not closely related with oral cancer.

Role of stromal-cell derived factor-1 in the development of autoimmune diseases in non-obese diabetic mice

The chemokine stromal‐cell derived factor‐1 (SDF‐1) controls maturation, trafficking, and homing of certain subsets, lymphoid cells including immunogenic B and T cells, as a ligand of the CXCR4 chemokine receptor. Insulin‐dependent diabetes mellitus (IDDM) and Sjögrens syndrome (SS), both highly regulated autoimmune diseases, develop spontaneously in non‐obese diabetic (NOD) mice. To investigate the role of SDF‐1 in the development of autoimmune diseases, we injected groups of NOD female mice with antibodies to SDF‐1 (anti‐SDF‐1), which resulted in a 30% reduction of diabetes up to 30 weeks of age, delayed average diabetes onset by 10 weeks, and suppressed insulitis. Autoimmune sialoadenitis was evident in anti‐SDF‐1‐injected mice (SDF‐1‐Ig group) at the same level as in all groups of mice, whether injected with non‐specific antibodies or not. In addition, in the SDF‐1‐Ig group, a greater number of immunoglobulin M (IgM)− IgD− B220low CD38+ CD43+ CD23− progenitor B cells and IgM+ IgD+ B220high CD43− CD38+ CD24+ CD23+ mature B cells remained in the bone marrow, whereas infiltration of mature IgM+ B cells was less extensive in peripheral tissues. Our results suggested that anti‐SDF‐1 antibodies injection was effective in inhibiting diabetes and insulitis without affecting autoimmune sialoadenitis or SS in NOD mice. SDF‐1 may be an essential chemokine for trafficking and migration of autoreactive B cells in the development of diabetes.

Human periodontal ligament fibroblasts are the optimal cell source for induced pluripotent stem cells.

Among the various kinds of fibroblasts existing in the human body, the periodontal ligament (PDL) fibroblasts have been suggested as multipotent cells. Periodontal ligament fibroblasts are characterized by rapid turnover, a high remodeling capacity and remarkable capacity for renewal and repair. They also differentiate into osteoblasts and cementoblasts. We established iPS cells from human PDL fibroblasts by introducing the ES cell markers Oct3/4, Sox2, Nanog, Klf4 and Lin28 by retrovirus transduction, even without the oncogene c-Myc. The iPS cells established in this study expressed the ES cell markers and formed teratomas in SCID mice. The c-Myc expression level in the PDL fibroblasts was higher than that in the iPS cells by quantitative RT-PCR. Therefore, we have concluded that PDL fibroblasts could be an optimal cell source for iPS cells.

Longitudinal study on the relationship between serum albumin and periodontal disease

AIM The purpose of this study was to evaluate the relationship between periodontal disease and the general health status in community-dwelling elderly using the serum albumin concentration as a criterion index of the severity of underlying disease and nutrition. MATERIAL AND METHODS Six hundred subjects aged 70 years underwent a baseline examination. Dental examinations were carried out at baseline and once a year for 4 years. Periodontal conditions were estimated for subjects with at least one remaining tooth. Clinical attachment levels at six sites of all teeth present were measured. A change in loss of attachment of 3 mm or greater in 1 year at each site was defined as periodontal disease progression. Data were analysed in subjects for whom data were available for 4 years. RESULTS Serum albumin concentration at baseline ranged from 3.4 to 5.0 g/dl with a mean of 4.3+/-0.2. When the analysis was stratified by smoking status, we found that serum albumin concentration had a significant effect on periodontal disease progression among non-smokers (standardized regression coefficient=-0.16; p=0.017), using multiple regression analysis. CONCLUSIONS The findings of the present study suggest that serum albumin concentration is a significant risk predictor of periodontal disease progression among elderly non-smokers.