Tomoko Ohshima

Bacterial Interactions in Dental Biofilm Development

Recent analyses with ribosomal RNA-based technologies have revealed the diversity of bacterial populations within dental biofilms, and have highlighted their important contributions to oral health and disease. Dental biofilms are exceedingly complex and multispecies ecosystems, where oral bacteria interact cooperatively or competitively with other members. Bacterial interactions that influence dental biofilm communities include various different mechanisms. During the early stage of biofilm formation, it is known that planktonic bacterial cells directly attach to surfaces of the oral cavity or indirectly bind to other bacterial cells that have already colonized. Adherence through co-aggregation may be critical for the temporary retention of bacteria on dental surfaces, and may facilitate eventual bacterial colonization. It is likely that metabolic communication, genetic exchange, production of inhibitory factors (e.g., bacteriocins, hydrogen peroxide, etc.), and quorum-sensing are pivotal regulatory factors that determine the bacterial composition and/or metabolism. Since each bacterium can easily access a neighboring bacterial cell and its metabolites, genetic exchanges and metabolic communication may occur frequently in dental biofilms. Quorum-sensing is defined as gene regulation in response to cell density, which influences various functions, e.g., virulence and bacteriocin production. In this review, we discuss these important interactions among oral bacteria within the dental biofilm communities.

Growth suppression of human carcinoma cells by reintroduction of the p300 coactivator

The p300 and closely related cAMP response element binding protein (CREB)-binding protein (CBP) acetyltransferases function as global transcriptional coactivators and play important roles in a broad spectrum of biological processes, including cell proliferation and differentiation. A role of p300/CBP in tumor suppression has been proposed from the fact that these coactivators are targeted by viral oncoproteins and that biallelic mutations of p300 have been identified in carcinomas. Here, we show that transcriptional response to the transforming growth factor β (TGF-β), an inhibitor of epithelial cell growth, was severely impaired in human carcinoma cell lines carrying p300 mutations accompanied by inactivation of the second allele, and that wild-type expression restored TGF-β-dependent transcriptional activity. Furthermore, reintroduction of wild-type p300 suppressed the growth of p300-deficient carcinoma cells, whereas p300 did not inhibit the growth of carcinoma cells examined, which have no detectable alterations in p300 protein and retain the TGF-β-dependent transcriptional response. In addition, tumor-derived mutants missing the bromodomain or glutamine-rich region, which are respectively important for chromatin interaction and coactivator activities, lost the suppressive activity. In contrast, CBP exhibited no or reduced ability to suppress the growth of p300-deficient carcinoma cells. These results provide experimental evidence to show that p300 acts as a suppressor of tumor cell growth and suggest a distinct role of p300 in suppression of epithelial tumors.

Distribution of Salivary Lactobacillus and Bifidobacterium Species in Periodontal Health and Disease

We surveyed the distribution of salivary Lactobacillus and Bifidobacterium species in periodontitis patients and healthy subjects. Approximately 700 lactobacilli and 300 bifidobacterial isolates were obtained from 16 young, orally healthy subjects (mean age ± standard deviation: 21.0±2.0 y), 16 periodontitis patients (51.6±13.8 y), and 14 well-maintained former periodontitis patients (60.2±9.6 y). Among eleven Lactobacillus species detected in saliva, L. salivarius, L. gasseri, and L. fermentum were prevalent, but no species was specifically associated with periodontal health. In contrast, of four Bifidobacterium species, B. adolescentis was specifically (P<0.05) prevalent in young healthy subjects compared with the other two groups. Furthermore, the bifidobacterial count of the well-maintained subjects was the highest (P<0.05) among the groups. These results suggest that bifidobacterial count and species might be associated with periodontal health status and/or age.

Interactions between salivary Bifidobacterium adolescentis and other oral bacteria : in vitro coaggregation and coadhesion assays

Coaggregation assays were performed to investigate interactions between oral Bifidobacterium adolescentis and other oral bacterial species. Bifidobacterium adolescentis OLB6410 isolated from the saliva of healthy humans did not coaggregate with Actinomyces naeslundii JCM8350, Streptococcus mitis OLS3293, Streptococcus sanguinis JCM5708, Veillonella parvula ATCC17745 or Porphyromonas gingivalis OB7124, but it did coaggregate with Fusobacterium nucleatum JCM8532. Subsequent examination of biofilm formation on saliva-coated hydroxyapatite discs using FISH revealed that B. adolescentis OLB6410 could not directly adhere to the coated discs. It did, however, adhere to biofilms of A. naeslundii, V. parvula, and F. nucleatum, although it did not coaggregate with A. naeslundii nor with V. parvula. These results suggest that the adhesion of B. adolescentis to tooth surfaces is mediated by other oral bacteria. Heat- or proteinase K-treated F. nucleatum could not coaggregate with B. adolescentis. Similarly, the coaggregation and coadhesion of proteinase K-treated B. adolescentis were strongly inhibited. It is therefore probable that proteinaceous factors on the cellular surface of B. adolescentis and F. nucleatum are involved in their interaction. The data presented in this study add to our understanding of bifidobacterial colonization in the human oral cavity.

Reduction of vitamin K concentration by salivary Bifidobacterium strains and their possible nutritional competition with Porphyromonas gingivalis

Aims:  To assess the possibility that bifidobacteria compete with Porphyromonas gingivalis for their mutual growth factor vitamin K. This study also examined whether salivary Bifidobacterium species decrease vitamin K concentration in the growth medium.

Therapeutic Application of Synbiotics, a Fusion of Probiotics and Prebiotics, and Biogenics as a New Concept for Oral Candida Infections: A Mini Review

Candida is a major human fungal pathogen causing infectious conditions predominantly in the elderly and immunocompromised hosts. Although Candida resides as a member of the oral indigenous microbiota in symbiosis, some circumstances may cause microbial imbalance leading to dysbiosis and resultant oral candidiasis. Therefore, oral microbial symbiosis that suppresses the overgrowth of Candida is important for a healthy oral ecosystem. In this regard, probiotics, prebiotics, and synbiotics can be considered a potential therapeutic and preventive strategy against oral candidiasis. Prebiotics have a direct effect on microbial growth as they stimulate the growth of beneficial bacteria and suppress the growth of pathogens. Probiotics render a local protective effect against pathogens and a systemic indirect effect on immunological amelioration. Synbiotics are fusion products of prebiotics and probiotics. This mini review discusses the potential use and associated limitations of probiotics, prebiotics, and synbiotics for the prevention and treatment of oral candidiasis. We will also introduce biogenics, a recent concept derived from the work on probiotics. Biogenics advocates the use of beneficial bioactive substances produced by probiotic bacteria, whose activities are independent from the viability of probiotic bacteria in human bodies.

Predominant Bacteria Recovered from a Periodontitis Site in a Hamster Model Raised by Silk-Ligature with Prophyromonas gingivalis Infection

We isolated oral bacteria that coexisted with Porphyromonas gingivalis in a hamster periodontitis model. As predominant bacteria in the periodontitis site, Collinsella-reltaed strains, Eubacterium-reltaed strains, Streptococcus suis-related strains, and Veillonella parvula-reltaed strains were detected. In addition, Actinomyces, Bacteroides, and P. gingivalis were also isolated predominantly. The results suggest that the bacterial composition of the periodontitis site in hamsters is complex, as in human periodontitis.

The Individual Cell Properties of Oral Squamous Cell Carcinoma and p53 Tumor Suppressor Gene Mutation

Abstract There is no consensus on the relationship between variations in TP53 mutations and tumor properties in oral squamous cell carcinoma (OSCC). To further the basic research required to eventually develop individualized (order-made) treatments and prognoses for OSCC, we established six human OSCC lines from patients within our department. Together with another nine cell lines derived from donations by other organizations, we determined the TP53 mutation and single nucleotide polymorphism (SNP) of codon 72 in a total of 15 cell lines, and examined in vitro cell invasion activity and anti-cancer drug sensitivity. The missense mutation at codon 248 was most abundant, and was noted in four cell lines, but other diverse mutation variations were also revealed. The cells which expressed the mutated p53 protein (the p53(+) group) showed slightly higher invasion activity than did the p53 (−) group. In p53(+) group, the 72R of SNP (72P/R) was higher than the 72P in invasion activity, although the difference was not significant. Surprisingly, an anti-cancer drug sensitivity test with four different types of drugs showed that the p53 (−) group was more resistant in other than CDDP, and that 72R was more sensitive than 72P in the p53(+) group. To clarify the characteristics of the R248Q mutation, which is the most abundant missense mutation, the gene was introduced with an expression plasmid vector into a TP53 null Saos-2 cell. The transformant of R248Q mutation gained higher activity of invasion, while its anti-cancer drug sensitivity also increased. Our findings suggest that it may be possible to estimate oral cancer cell characteristics and the malignancy level based on differences in the TP53 mutation.

Interleukin-4 released from human gingival fibroblasts reduces osteoclastogenesis

OBJECTIVE Human gingival epithelium is continuously exposed to bacteria and acts as the first line of defense in periodontal tissues. It is crucial to maintain healthy, non-inflamed gingival tissue to avoid gingivitis and periodontitis. The purpose of this study was to investigate the influence of IL-4 in human gingival fibroblasts (hGF) on the activation of osteoclasts. DESIGN Two hGF samples were obtained from two healthy patients, and one was collected from a commercially available resource. The hGFs were cultured, and conditioned medium of hGF (hGF-CM) was stocked at -80°C. The mRNA was isolated from the hGF cultures and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of suppressive osteoclastogenetic mediators, such as interleukin (IL)-4, osteoprotegerin (OPG), IL-10, IL-27, and IL-33. The hGF-CM was used to investigate the inhibitory function of mouse macrophages supplemented with either glutathione S-transferase-Receptor activator of NF-kB ligand (GST-RANKL), human recombinant (rh)IL-4, or rhOPG but not a combination. Differentiation of osteoclasts was examined by tartrate resistant acid phosphatase (TRAP) staining and TRAP assay. The suppressive role of IL-4 was assessed by neutralizing IL-4 antibody in the TRAP assay. RESULTS The hGF-CM reduced both TRAP positive staining and activity in a dose-dependent manner. IL-4 and OPG mRNA expressions were expressed in hGF-CM from three different donors but that of IL-10, IL-27, or IL-33 was not detected. In the RAW264 culture, rhIL-4 and rhOPG reduced TRAP positive staining as well as activity in a dose-dependent manner. Moreover, addition of neutralizing antibodies for IL-4 reduced the inhibitory effect of conditioned medium from gingival fibroblasts in the RAW264 culture. CONCLUSION We concluded that hGF potentially contained suppressive mediators, such as IL-4 and OPG, for osteoclastogenesis. Moreover, we confirmed that the differential inhibition of osteoclast is caused by OPG as well as IL-4 in hGF-CM.

Antibacterial Effects of Cocoa on Periodontal Pathogenic Bacteria

We examined the antibacterial effects of cocoa on periodontal pathogenic bacteria, including Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia, compared with its effects on indigenous oral streptococci. A colony-forming unit (CFU) assay in the presence and absence of 1.0% and 3.0% (w/v) cocoa revealed that the growth of periodontal pathogenic bacteria was significantly suppressed by cocoa in concentration- and incubation time-dependent manners, although cocoa had no effect on the growth of indigenous streptococci. Methanol- and ethanol-extractable fractions from cocoa were also subjected to the CFU assay to determine and characterize the component (s) responsible for these effects. Fractions containing mainly cocoa polyphenols showed antibacterial effects. After treatment with polyvinylpolypyrrolidone, an absorbent of polyphenols, the methanol-extractable fraction lost its effect. These results suggest that cocoa has significant antibacterial effects against periodontal pathogenic bacteria and that polyphenols are responsible.