Effect of bovine milk fermented with Lactobacillus rhamnosus L8020 on periodontal disease in individuals with intellectual disability: a randomized clinical trial.

OBJECTIVE: Studies on the oral health of individuals with intellectual disability (ID) have identified problems that include a high prevalence of periodontal disease. The use of probiotics to treat periodontal disease has been the focus of considerable research, and bovine milk fermented with Lactobacillus rhamnosus L8020 (L8020 yogurt) has been shown to reduce the oral prevalence of four periodontal pathogens. The aim of this randomized, double-blind, placebo-controlled trial was to compare the effects of L8020 yogurt (test group) with those of placebo yogurt (placebo group) on the papillary-marginal-attached (PMA) index, gingival index (GI), and probing depth (PD) in 23 individuals with ID. METHODOLOGY: All patients were required to consume the allocated yogurt after breakfast for 90 days. PMA index and GI scores as well as PDs were assessed before the start of yogurt consumption (baseline), after 45 and 90 days of consumption, and 30 days after the cessation of consumption. Student's t-test, Mann-Whitney U test or Fisher's exact test was used for inter-group comparisons, and the mixed effect model of repeated measurements was used for data analysis. RESULTS: The decrease in PMA index score was significantly greater in the test group than in the placebo group (p<0.001). The GI score also decreased during the study, with a tendency for greater decrease in the test group. Furthermore, decreases in PD between baseline, 45 and 90 days tended to be greater in the test group than in the placebo group. CONCLUSION: These results suggest that regular consumption of bovine milk fermented with L. rhamnosus L8020 can lower the risk of periodontal disease in individuals with ID.

Effect of bovine milk fermented with Lactobacillus rhamnosus L8020 on periodontal disease in individuals with intellectual disability: a randomized clinical trial

Abstract Studies on the oral health of individuals with intellectual disability (ID) have identified problems that include a high prevalence of periodontal disease. The use of probiotics to treat periodontal disease has been the focus of considerable research, and bovine milk fermented with Lactobacillus rhamnosus L8020 (L8020 yogurt) has been shown to reduce the oral prevalence of four periodontal pathogens. Objective The aim of this randomized, double-blind, placebo-controlled trial was to compare the effects of L8020 yogurt (test group) with those of placebo yogurt (placebo group) on the papillary-marginal-attached (PMA) index, gingival index (GI), and probing depth (PD) in 23 individuals with ID. Methodology All patients were required to consume the allocated yogurt after breakfast for 90 days. PMA index and GI scores as well as PDs were assessed before the start of yogurt consumption (baseline), after 45 and 90 days of consumption, and 30 days after the cessation of consumption. Student's t-test, Mann-Whitney U test or Fisher's exact test was used for inter-group comparisons, and the mixed effect model of repeated measurements was used for data analysis. Results The decrease in PMA index score was significantly greater in the test group than in the placebo group (p<0.001). The GI score also decreased during the study, with a tendency for greater decrease in the test group. Furthermore, decreases in PD between baseline, 45 and 90 days tended to be greater in the test group than in the placebo group. Conclusion These results suggest that regular consumption of bovine milk fermented with L. rhamnosus L8020 can lower the risk of periodontal disease in individuals with ID.

CdtC-Induced Processing of Membrane-Bound CdtA Is a Crucial Step in Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Holotoxin Formation.

Aggregatibacter actinomycetemcomitans is an oral pathogen causing periodontal disease and bacterial endocarditis. It produces cytolethal distending toxin (CDT) that could damage mammalian cells and tissues. CDT is a tripartite protein toxin composed of CdtA, CdtB, and CdtC. We have previously indicated that CdtA is a lipoprotein and that the proteolytic processing of CdtA is important for biogenesis and secretion of CDT holotoxin. Here, we established an in vitro processing assay of CdtA and investigated the interactions of CdtA with other Cdt subunits. This assay demonstrated that incubation of membrane-bound CdtA (MCdtA), CdtB, and CdtC immediately generated a processed form of CdtA (CdtA'), which is recovered from the soluble fraction. In contrast, incubation of soluble membrane-unbound CdtA with CdtB and CdtC did not yield any CdtA'. Furthermore, incubation of CdtC with MCdtA was enough to induce rapid processing of MCdtA, whereas CdtB alone was unable to induce the processing. Coimmunoprecipitation demonstrated that CdtA' and CdtC formed a complex. Furthermore, subsequent addition of CdtB to this reaction mixture resulted in complete CDT holotoxin complex. The cytolethal distending activity assay demonstrated that CDT complex containing CdtA' showed far stronger cytotoxicity than that containing CdtA. Collectively, our data suggest that CDT holotoxin formation in vivo is a sequential event: interaction of MCdtA and CdtC induces proteolytic processing of MCdtA, and the released CdtA' forms a complex with CdtC. Subsequent binding of CdtB to the CdtA'/CdtC complex results in CDT holotoxin formation.

Inhibition of RANKL-dependent cellular fusion in pre-osteoclasts by amiloride and a NHE10-specific monoclonal antibody.

The functions of Na(+) /H(+) exchangers (NHEs) during osteoclastic differentiation were investigated using the NHE inhibitor amiloride and a monoclonal antibody (MAb). Compared with sRANKL-stimulated control cells, amiloride decreased the number of large TRAP-positive osteoclast cells (OCs) with ≥10 nuclei and increased the number of small TRAP-positive OCs with ≤10 nuclei during sRANKL-dependent osteoclastic differentiation of RAW264.7 cells. NHE10 mRNA expression and OC differentiation markers were increased by sRANKL stimulation in dose- and time-dependent manners. NHEs 1-9 mRNA expression was not increased by sRANKL stimulation. Similar to amiloride, a rat anti-mouse NHE10 MAb (clone 6B11) decreased the number of large TRAP-positive OCs, but increased the number of small TRAP-positive OCs. These findings suggested that inhibition of NHEs by amiloride or an anti-NHE10 MAb prevented sRANKL-promoted cellular fusion. The anti-NHE10 MAb has the potential for use as an effective inhibitor of bone resorption for targeted bone disease therapy.

Delivery and application of dietary polyphenols to target organs, tissues and intracellular organelles.

At present, dietary polyphenols are popular with consumers because regular consumption of polyphenol-rich foods is likely to be beneficial for human health. However, administrated polyphenols are extensively metabolized in the digestive tract or some other parts before reaching the target organs. Additionally, some of the polyphenols are photosensitive, easily oxidized and are in unfavorable forms. Therefore, a lot of work has been performed to ensure delivery of intact polyphenols to the target organs. We here summarize recent progress in polyphenol-delivery to individual organs, tissues, and cells, in regard to relatively new delivery systems. Polyphenol-delivery systems can be divided into three categories: (i) before delivery into the blood stream (skin, mouth, gastrointestine), (ii) in the blood stream (plasma), and (iii) after the blood stream (brain, spleen, bone marrow, kidney). Polyphenols before the delivery into blood stream must overcome several obstacles to avoid converting into inactive forms by commensal microorganisms, environmental pH, and some others. In the blood, plasma-polyphenol interactions and modifications are very effective for the bioavailability of polyphenols with numerous enzymes. Native forms of polyphenols, successfully out of the blood stream, further go through obstacles such as the blood brain barrier to reach target organs. Recent progress in delivering polyphenols is here discussed on 3 main delivery systems, nanoparticle, liposome, and microemulsion. Moreover, we also focused on delivery systems to intracellular organelles (cell surface, lysosome, mitochondria, nucleus), which are the final targets of polyphenols to perform their beneficial reactions.

Prevalence of drug-resistant opportunistic microorganisms in oral cavity after treatment for oral cancer.

Drug-resistant opportunistic infections may cause health problems in immunocompromised hosts. Representative microorganisms in opportunistic infections of the oral cavity are Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. We investigated the prevalence of drug-resistant opportunistic microorganisms in elderly adults receiving follow-up examinations after primary treatment of oral cancer. Oral microorganisms were collected from patients satisfactorily treated for oral cancer (defined as good outcomes to date) and a group of healthy adults (controls). After identification of microorganisms, the prevalence of drug-resistant microorganisms was studied. Pulsed-field gel electrophoresis (PFGE) and staphylococcal cassette chromosome mec (SCCmec) typing were also performed for methicillin-resistant S aureus (MRSA). Statistical analysis revealed no significant differences in the prevalences of the three microorganisms between the groups. Surprisingly, 69.2% of S aureus isolates showed oxacillin resistance, suggesting that MRSA colonization is increasing among older Japanese. These MRSA isolates possessed SCCmec types II and IV but no representative toxin genes. Our results indicate that a basic infection control strategy, including standard precautions against MRSA, is important for elderly adults, particularly after treatment for oral cancer.

The green tea polyphenol (-)-epigallocatechin gallate precipitates salivary proteins including alpha-amylase: biochemical implications for oral health.

Green tea is a popular drink throughout the world, and it contains various components, including the green tea polyphenol (-)-epigallocatechin gallate (EGCG). Tea interacts with saliva upon entering the mouth, so the interaction between saliva and EGCG interested us, especially with respect to EGCG-protein binding. SDS-PAGE revealed that several salivary proteins were precipitated after adding EGCG to saliva. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting indicated that the major proteins precipitated by EGCG were alpha-amylase, S100, and cystatins. Surface plasmon resonance revealed that EGCG bound to alpha-amylase at dissociation constant (K(d)) = 2.74 × 10(-6) M, suggesting that EGCG interacts with salivary proteins with a relatively strong affinity. In addition, EGCG inhibited the activity of alpha-amylase by non-competitive inhibition, indicating that EGCG is effective at inhibiting the formation of fermentable carbohydrates involved in caries formation. Interestingly, alpha-amylase reduced the antimicrobial activity of EGCG against the periodontal bacterium Aggregatibacter actinomycetemcomitans. Therefore, we considered that EGCG-salivary protein interactions might have both protective and detrimental effects with respect to oral health.

Titanium immobilized with an antimicrobial peptide derived from histatin accelerates the differentiation of osteoblastic cell line, MC3T3-E1.

The objective of this study was to evaluate the effect of titanium immobilized with a cationic antimicrobial peptide (JH8194) derived from histatin on the biofilm formation of Porphyromonas gingivalis and differentiation of osteoblastic cells (MC3T3-E1). The titanium specimens (Ti) were immobilized with JH8194, according to the method previously described. The colonization of P. gingivalis on JH8194-Ti was significantly lower than that on control- and blocking-Ti. JH8194-Ti enhanced the mRNA expressions of Runx2 and OPN, and ALPase activity in the MC3T3-E1, as compared with those of control- and blocking-Ti. These results, taken together, suggested the possibility that JH8194-Ti may be a potential aid to shorten the period of acquiring osseointegration.

Cytolethal distending toxin from Aggregatibacter actinomycetemcomitans induces DNA damage, S/G2 cell cycle arrest, and caspase- independent death in a Saccharomyces cerevisiae model.

Cytolethal distending toxin (CDT) is a bacterial toxin that induces G(2)/M cell cycle arrest, cell distension, and/or apoptosis in mammalian cells. It is produced by several Gram-negative species and may contribute to their pathogenicity. The catalytic subunit CdtB has homology with DNase I and may act as a genotoxin. However, the mechanism by which CdtB leads to cell death is not yet clearly understood. Here, we used Saccharomyces cerevisiae as a model to study the molecular pathways involved in the function of CdtB from Aggregatibacter actinomycetemcomitans, a cause of aggressive periodontitis. We show that A. actinomycetemcomitans CdtB (AaCdtB) expression induces S/G(2) arrest and death in a DNase-catalytic residue and nuclear localization-dependent manner in haploid yeasts. Yeast strains defective in homologous recombination (HR) repair, but not other DNA repair pathways, are hypersensitive to AaCdtB, suggesting that HR is required for survival upon CdtB expression. In addition, yeast does not harbor the substrate for the other activity proposed for CdtB function, which is phosphatidylinositol-3,4,5-triphosphate phosphatase. Thus, these results suggest that direct DNA-damaging activity alone is sufficient for CdtB toxicity. To investigate how CdtB induces cell death, we examined the effect of CdtB in yeast strains with mutations in apoptotic regulators. Our results suggest that yeast death occurs independently of the yeast metacaspase gene YCA1 and the apoptosis-inducing factor AIF1 but is partially dependent on histone H2B serine 10 phosphorylation. Therefore, we report here the evidence that AaCdtB causes DNA damage that leads to nonapoptotic death in yeast and the first mutation that confers resistance to CdtB.

Increased prevalence and clonal dissemination of multidrug-resistant Pseudomonas aeruginosa with the blaIMP-1 gene cassette in Hiroshima.

OBJECTIVES: The aim of this study was to evaluate the dissemination of metallo-beta-lactamase (MBL)-encoding genes among multidrug-resistant (MDR) Pseudomonas aeruginosa isolates recovered from major hospitals in the Hiroshima region. METHODS: During July to December from 2004 to 2006, a surveillance of eight major hospitals in the Hiroshima region identified 387 non-duplicate isolates resistant to imipenem (MIC >or= 16 mg/L). They were screened for resistance to amikacin (MIC >or= 64 mg/L) and ciprofloxacin (MIC >or= 4 mg/L) and MBL-encoding genes. The structure of the variable regions of the integrons was determined using PCR mapping. Clonality was assessed using PFGE and multilocus sequence typing (MLST). RESULTS: The frequency of MBL-positive isolates in MDR P. aeruginosa isolates significantly increased from 42.3% in 2004 to 81.4% in 2006. Most of the MBL-positive isolates produced IMP-1 followed by VIM-2. The bla(IMP-1) and bla(VIM-2) genes were present in class 1 integrons. Characterization of the variable regions of the integron showed the presence of six different gene cassette arrays in bla(IMP-1) cassettes and a single array in bla(VIM-2) cassettes. The IMP-1 producers belonged to two clonal lineages using PFGE and MLST analyses and the integron variations correlated well with the clonal complexes. Among them, strains positive for a newly identified In113-derived bla(IMP-1) gene cassette array were most widely distributed in Hiroshima. CONCLUSIONS: This study shows a dramatic increase in MBL genes, primarily bla(IMP-1), in MDR P. aeruginosa isolates in Hiroshima during these 3 years. In addition, MDR P. aeruginosa with the newly discovered In113-derived bla(IMP-1) gene cassette array appears to be clonally expanding.

Cytolethal distending toxin induces caspase-dependent and -independent cell death in MOLT-4 cells.

Cytolethal distending toxin (CDT) induces apoptosis using the caspase-dependent classical pathway in the majority of human leukemic T cells (MOLT-4). However, we found the process to cell death is only partially inhibited by pretreatment of the cells with a general caspase inhibitor, z-VAD-fmk. Flow cytometric analysis using annexin V and propidium iodide showed that a 48-h CDT treatment decreased the living cell population by 35% even in the presence of z-VAD-fmk. z-VAD-fmk completely inhibited caspase activity in 24 h CDT-intoxicated cells. Further, CDT with z-VAD-fmk treatment clearly increased the cell population that had a low level of intracellular reactive oxygen. This is a characteristic opposite to that of caspase-dependent apoptosis. Overexpression of bcl2 almost completely inhibited cell death using CDT treatment in the presence of z-VAD-fmk. The data suggest there are at least two different pathways used in CDT-induced cell death: conventional caspase-dependent (early) apoptotic cell death and caspase-independent (late) death. Both occur via the mitochondrial membrane disruption pathway.

First isolation of blaIMP-7 in a Pseudomonas aeruginosa in Japan.

We report here the first isolation in Japan of a carbapenem-resistant Pseudomonas aeruginosa strain that carries the metallo-beta-lactamase gene bla(IMP-7). This isolate revealed high-level resistance to all of the tested antibiotics except for piperacillin, showing a multidrug-resistant phenotype.

Molecular characterization of imipenem-resistant Pseudomonas aeruginosa in Hiroshima, Japan.

Pseudomonas aeruginosa showing resistance to imipenem were found in 100 of 1,058 strains (9.5%) from six hospitals (a-f) in Hiroshima City, Japan. Of the 100 strains, 14 (14%) were double disk synergy test positive using sodium mercaptoacetic acid disks, and 18 (18%) were bla(IMP-1) or bla(VIM-2) allele positive by polymerase chain reaction (PCR). Among 100 imipenem-resistant strains, 32 were categorized into multi-drug resistant strains, in which 13 were positive for the metallo-beta-lactamase gene. Fifty-one strains (51%) among the 100 imipenem-resistant strains had elevated RND efflux pump activity against levofloxacin. But only 6 of 51 strains were classified as multi-drug resistant strains. The pulsed field gel electrophoresis analysis of the Spe I-digested DNA from the 100 isolates suggested not only clonal spread but spread of heterogeneous clones started to contribute to the prevalence of metallo-beta-lactamase producing P. aeruginosa strains in Japanese hospitals.

Single nucleotide polymorphism in the cytolethal distending toxin B gene confers heterogeneity in the cytotoxicity of Actinobacillus actinomycetemcomitans.

Clinical Actinobacillus actinomycetemcomitans produces cytolethal distending toxin (CDT) with titers ranging from 10(2) to 10(8) U/mg. Single nucleotide polymorphism analysis of the cdt gene in clinical isolates identified a variation of a single amino acid at residue 281 of CdtB, which significantly affected CDT toxicity by modulating the chromatin-degrading activity of CdtB.

Biogenesis of the Actinobacillus actinomycetemcomitans cytolethal distending toxin holotoxin.

The cell cycle G2/M specific inhibitor cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is composed of CdtA, CdtB, and CdtC coded on the cdtA, cdtB, and cdtC genes that are tandem on the chromosomal cdt locus. A. actinomycetemcomitans CdtA has the lipid binding consensus domain, the so-called "lipobox", at the N-terminal signal sequence. Using Escherichia coli carrying plasmid pTK3022, we show that the 16th residue, cysteine, of CdtA bound [3H]palmitate or [)H]glycerol. Further, posttranslational processing of the signal peptide, CdtA, was inhibited using globomycin, an inhibitor of lipoprotein-specific signal peptidase II. Fractionation and immunoblotting show the lipid-modified CdtA is present in the outer membrane. Immunoprecipitation and the pull-down assay of the CDT complex from E. coli carrying a plasmid containing cdtABC demonstrated that the CDT complex in the periplasm is composed of CdtA, CdtB, and CdtC and that the CDT complex in culture supernatant is an N-terminally truncated (36 to 43 amino acids) form of CdtA (CdtA'), CdtB, and CdtC. This suggests that CDT is present as a complex both in the periplasm and the supernatant where CdtA undergoes posttranslation processing to CdtA' in the process of biogenesis and secretion of CDT holotoxin into the culture supernatant. Site-directed mutagenesis of the 16th cysteine residue to glycine in CdtA altered localization of CdtA in the cell and reduced the amount of CDT activity in the culture supernatant. This suggests that CDT forms a complex inside the periplasm for lipid modification where posttranslational processing of CdtA plays an important role for the efficient production of CDT holotoxin into the culture supernatant.

Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial cells.

OBJECTIVES: Antimicrobial peptides are one of the factors involved in innate immunity. The susceptibility of periodontopathogenic and cariogenic bacteria to the major antimicrobial peptides produced by epithelia was investigated. METHODS: Synthetic antimicrobial peptides of human beta-defensin-1 (hBD1), hBD2, hBD3 and LL37 (CAP18) were evaluated for their antimicrobial activity against oral bacteria. They included Actinobacillus actinomycetemcomitans (20 strains), Porphyromonas gingivalis (6), Prevotella intermedia (7), Fusobacterium nucleatum (7), Streptococcus mutans (5), Streptococcus sobrinus (5), Streptococcus salivarius (5), Streptococcus sanguis (4), Streptococcus mitis (2) and Lactobacillus casei (1). RESULTS: Although the four peptides had bactericidal activity against all bacteria tested, the degree of antibacterial activity was variable against the different strains and species. The antibacterial activity of hBD1 was lower than that of the other peptides. Among the bacteria tested in this study, F. nucleatum was highly susceptible to hBD3 and LL37, and S. mutans was highly susceptible to hBD3. We measured the Zeta-potential, representing the net charge of whole bacteria, to study the relationship between susceptibility to cationic peptide and the net charge of the bacteria. Although we found some correlation in A. actinomycetemcomitans strains, we did not find a definite correlation with all the bacterial species. CONCLUSIONS: These results indicate that beta-defensins and LL37 have versatile antibacterial activity against oral bacteria.

Mutation analysis of the histidine residues in the glycylglycine endopeptidase ALE-1.

A novel staphylolytic enzyme, ALE-1, is a glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. ALE-1 possesses seven histidines. Chemical modification studies using diethylpyrocarbonate and iodoacetic acid suggested that a histidine or tyrosine residue(s) in the molecule is important for the organism's staphylolytic activity. All of the histidine residues, one tyrosine, and one aspartic acid residue in the N-terminally truncated ALE-1 (DeltaN-term ALE-1) were systematically altered by site-directed mutagenesis, and the enzyme activities and metal contents of the variants were measured. Our studies indicated that His-150, His-200, His-231, His-233, and Asp-154 are essential for the enzyme activity of DeltaN-term ALE-1. Except for His-150 and Asp-154, all of these amino acids were located within the 38-amino-acid region conserved among 11 proteins, including 5 staphylolytic endopeptidases. Inductively coupled plasma-mass spectrometric analysis of DeltaN-term ALE-1 revealed that it contains one atom of zinc per molecule. Measurement of the zinc content of the mutant DeltaN-term ALE-1 suggested that His-150 and -233 are important for zinc binding; their loss in these variant enzymes coincided with the loss of staphylolytic activity. These results strongly suggest that ALE-1 is a novel member of zinc metalloproteases.

Cytolethal distending toxin: a bacterial bullet targeted to nucleus.

Cytolethal distending toxin (Cdt) is a newly added member of bacterial protein toxins that hijack the control system of eukaryotic cells. Cdts are produced by several pathogenic bacteria causing chronic infectious diseases. They are composed of three subunits, CdtA, CdtB and CdtC, which together form a ternary complex. CdtB is the active component, and CdtA and CdtC are involved in delivering the CdtB into the cells. The sophisticated strategy of Cdt to control host cells is CdtB-mediated limited DNA damage of the host cell chromosome, which triggers the response of the cell cycle checkpoint and results in G2 arrest in the cells. Cdt also induces apoptotic cell death of lymphocytes, which may be relevant to onset or persistence of chronic infection by the producing bacteria. The study of this toxin is expected to provide us information on a novel strategy by which bacteria interact with host cells.

The gate controlling cell wall synthesis in Staphylococcus aureus.

Glucosamine-6-P occupies a central position between cell wall synthesis and glycolysis. In the initial steps leading to peptidoglycan precursor formation glucosamine-6-P is processed sequentially to UDP-N-acetylglucosamine, while to enter the glycolysis pathway, glucosamine-6-P is isomerized by NagB to fructose-6-P. Although we could not demonstrate NagB activity, nagB inactivation significantly reduced growth. Mutational analysis showed that NagA was involved in glucosamine-6-P formation from N-acetylglucosamine-6-P, and GlmS in that from fructose-6-P. Inactivation of glmS prevented growth on glucose as sole carbon source, which resumed after complementation with N-acetylglucosamine. Transcription of glmS as well as the amount of GlmS was reduced in the presence of N-acetylglucosamine. This and the preferential incorporation of N-acetylglucosamine over glucose into cell wall material showed that N-acetylglucosamine was used exclusively for cell wall synthesis, while glucose served both cell wall synthesis and glycolysis. These observations suggest furthermore GlmS to be the key and only enzyme leading from glucose to cell wall synthesis in Staphylococcus aureus, and show that there exists a tight regulation and hierarchy in sugar utilization. Inactivation of nagA, nagB or glmS affected the susceptibility of S. aureus to cell wall synthesis inhibitors, suggesting an interdependence between efficiency of cell wall precursor formation and resistance levels.