Outer membrane vesicles of Porphyromonas gingivalis: Novel communication tool and strategy.

Extracellular vesicles (EVs) have been recognized as a universal method of cellular communications and are reportedly produced in bacteria, archaea, and eukaryotes. Bacterial EVs are often called "Outer Membrane Vesicles" (OMVs) as they were the result of a controlled blebbing of the outer membrane of gram-negative bacteria such as Porphyromonas gingivalis (P. gingivalis). Bacterial EVs are natural messengers, implicated in intra- and inter-species cell-to-cell communication among microorganism populations present in microbiota. Bacteria can incorporate their pathogens into OMVs; the content of OMVs differs, depending on the type of bacteria. The production of distinct types of OMVs can be mediated by different factors and routes. A recent study highlighted OMVs ability to carry crucial molecules implicated in immune modulation, and, nowadays, they are considered as a way to communicate and transfer messages from the bacteria to the host and vice versa. This review article focuses on the current understanding of OMVs produced from major oral bacteria, P. gingivalis: generation, characteristics, and contents as well as the involvement in signal transduction of host cells and systemic diseases. Our recent study regarding the action of P. gingivalis OMVs in the living body is also summarized.

[Real-time Lens Exposure Dose Measurement Using a Scintillator with Optical Fiber Dosimeter during Cardiac Catheter Operation].

In recent years, the exposure dose of the operator's eye lens during interventional radiology operations has become a problem. We therefore evaluated the feasibility of real-time lens dose measurement using scintillator with optical fiber (SOF) dosimeter. Given that the SOF dosimeter is calibrated for direct X-rays, we performed a calibration for scattered X-rays to investigate energy dependence and the accuracy of lens dose measurements. The detection limit was calculated using the Kaiser method. The SOF dosimeter and the radiophotoluminescence glass (RPLG) dosimeter were attached to the protective glasses worn by the operator, and the lens exposure dose of the operator during cardiac catheterization was measured. In the phantom experiment, the SOF dosimeter had an error rate of 5.45% based on the measured value of the ionization chamber dosimeter. The sensitivity characteristics of the SOF dosimeter were slightly reduced on the higher side of the effective energy. The difference in sensitivity was related to variations in the additional filter and energy dependency. The sensitivity difference was 18.5% at maximum. Furthermore, when the additional dose was displayed, the influence of noise on long-term measurement was considerable. Using the Kaiser method to obtain the detection limit, the accuracy of the integrated dose had SOF dosimeter error rates of 4.3% to 15.5% with respect to the integrated value of the RPLG dosimeter when calibrated by the ionization chamber dosimeter. The use of the SOF dosimeter allowed for the real-time visualization of the exposure status of the eye lens and measurements with a relatively high accuracy.

Suppressive effects of 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer on the adherence of Candida species and MRSA to acrylic denture resin.

OBJECTIVES: The effects of 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer on the adherence of microorganisms such as non-Candida albicans Candida (NCAC) and methicillin-resistant Staphylococcus aureus (MRSA), frequently detected in oral infections in immunocompromised and/or elderly people, to denture resin material, are still unclear. Here, we report the effects of MPC-polymer on the adherence of C. albicans, NCAC, and MRSA to acrylic denture resin. METHODS: Sixteen strains of C. albicans, seven strains of C. glabrata, two strains of C. tropicalis, one strain of C. parapsilosis, and six strains of MRSA were used. We cultured the fungal/bacterial strains and examined the cell growth and adherence of fungi/bacteria to mucin-coated acrylic denture resin plates (ADRP) with or without MPC-polymer coating, by scanning electron microscopy. The cell surface hydrophobicity of the fungal/bacterial strains was measured by the adsorption to hydrocarbons. RESULTS: MPC-polymer did not affect the growth of all strains of Candida species and MRSA, but significantly suppressed adherence to ADRP in most strains of C. albicans and all strains of NCAC and MRSA. A significant positive correlation was found between cell hydrophobicity and the reduction rates of microbial adherence to ADRP treated with 5% of MPC-polymer. CONCLUSIONS: MPC-polymer treatment for acrylic resin material suppresses the adherence of C. albicans, NCAC and MRSA via their hydrophilicity interaction. CLINICAL SIGNIFICANCE: The application of MPC-polymer for denture hygiene is potent to prevent oral candidiasis, denture stomatitis and opportunistic infection, caused by Candida species and MRSA, via suppressing the adherence of those fungus/bacteria.

Anti-inflammatory effects of olanexidine gluconate on oral epithelial cells.

BACKGROUND: Periodontitis is a biofilm-induced chronic inflammatory condition of the periodontium. Chemokines produced by the innate and acquired immune responses play a significant role in disease progression. Reducing biofilm formation and inflammatory response caused by chemokines is vital for preventing and treating periodontitis. Previously, we observed that treatment with 0.1% olanexidine gluconate (OLG) inhibited biofilm formation on saliva-coated hydroxyapatite. This study aimed to evaluate the anti-inflammatory effect of OLG on oral epithelial cells. METHODS: We examined if OLG could inhibit the inflammatory responses caused by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and heat-killed P. gingivalis in immortalized human oral keratinocytes (RT7). RESULTS: Treatment of RT7 with non-cytotoxic OLG concentrations significantly inhibited the production of inflammatory chemokines such as interleukin 8 (IL-8), C-C motif ligand 20 (CCL20), and growth-related oncogene protein-α (GRO-α), which are stimulated by P. gingivalis LPS in a concentration-dependent manner. Moreover, the inhibitory effects were observed regardless of the treatment time with P. gingivalis LPS (6, 12, or 24 h). OLG also significantly inhibited chemokine production stimulated by heat-killed P. gingivalis. CONCLUSIONS: The findings of this study suggest that treatment with OLG inhibits chronic inflammatory reactions in oral mucosal cells, such as periodontitis, caused by oral bacteria.

The Pathogenic Factors from Oral Streptococci for Systemic Diseases.

The oral cavity is suggested as the reservoir of bacterial infection, and the oral and pharyngeal biofilms formed by oral bacterial flora, which is comprised of over 700 microbial species, have been found to be associated with systemic conditions. Almost all oral microorganisms are non-pathogenic opportunistic commensals to maintain oral health condition and defend against pathogenic microorganisms. However, oral Streptococci, the first microorganisms to colonize oral surfaces and the dominant microorganisms in the human mouth, has recently gained attention as the pathogens of various systemic diseases, such as infective endocarditis, purulent infections, brain hemorrhage, intestinal inflammation, and autoimmune diseases, as well as bacteremia. As pathogenic factors from oral Streptococci, extracellular polymeric substances, toxins, proteins and nucleic acids as well as vesicles, which secrete these components outside of bacterial cells in biofilm, have been reported. Therefore, it is necessary to consider that the relevance of these pathogenic factors to systemic diseases and also vaccine candidates to protect infectious diseases caused by Streptococci. This review article focuses on the mechanistic links among pathogenic factors from oral Streptococci, inflammation, and systemic diseases to provide the current understanding of oral biofilm infections based on biofilm and widespread systemic diseases.

2-Methacryloyloxyethyl phosphorylcholine (MPC)-polymer suppresses an increase of oral bacteria: a single-blind, crossover clinical trial.

OBJECTIVES: The biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymers, which mimic a biomembrane, reduce protein adsorption and bacterial adhesion and inhibit cell attachment. The aim of this study is to clarify whether MPC-polymer can suppress the bacterial adherence in oral cavity by a crossover design. We also investigated the number of Fusobacterium nucleatum, which is the key bacterium forming dental plaque, in clinical samples. MATERIALS AND METHODS: This study was a randomized, placebo-controlled, single-blind, crossover study, with two treatment periods separated by a 2-week washout period. We conducted clinical trial with 20 healthy subjects to evaluate the effect of 5% MPC-polymer mouthwash after 5 h on oral microflora. PBS was used as a control. The bacterial number in the gargling sample before and after intervention was counted by an electronic bacterial counter and a culture method. DNA amounts of total bacteria and F. nucleatum were examined by q-PCR. RESULTS: The numbers of total bacteria and oral streptcocci after 5 h of 5% MPC-polymer treatment significantly decreased, compared to the control group. Moreover, the DNA amounts of total bacteria and F. nucleatum significantly decreased by 5% MPC-polymer mouthwash. CONCLUSIONS: We suggest that MPC-polymer coating in the oral cavity may suppress the oral bacterial adherence. CLINICAL RELEVANCE: MPC-polymer can be a potent compound for the control of oral microflora to prevent oral infection.

Antifungal and Mechanical Properties of Tissue Conditioner Containing Plant-Derived Component: An In Vitro Study.

PURPOSE: To evaluate the antifungal activity and mechanical properties of a novel antifungal tissue conditioner containing Juncus powder. MATERIALS AND METHODS: Juncus powder was mixed with GC tissue conditioner at concentrations of 2.5%, 5.0%, and 10.0% by mass. The cylindrical specimens of Juncus-mixed tissue conditioner (dimensions: 10 mm in diameter and 2 and 6 mm in height for antimicrobial and mechanical tests, respectively) were prepared. The specimens placed on the bottom of the 24-well tissue culture plate were cultured with Candida albicans CAD1 for 2 and 4 days. The proliferation of the C. albicans in the wells was determined by measuring the optical density of fungal culture, and the surface of the specimens were also observed by scanning electron microscopy (SEM). To assess the mechanical properties of the specimens, the fluidity and hardness of Juncus-mixed tissue conditioner were measured using the methods certified according to ISO 10139-1. RESULTS: Juncus-mixed tissue conditioner significantly exhibited growth inhibitory effect in a Juncus concentration-dependent manner after both 2- and 4- day cultures. SEM observation showed that the amount of C. albicans on Juncus-mixed specimens drastically decreased, and biofilm formation was markedly inhibited. Moreover, both mechanical properties were found to be within the ranges regulated and specified by ISO. CONCLUSION: These findings demonstrated that the tissue conditioner including Juncus powder has a significant growth inhibitory effect against C. albicans, and it is suggested that the application of Juncus-mixed tissue conditioner may prevent denture stomatitis and oral candidiasis in denture wearers.

Antibiofilm and Anti-Inflammatory Activities of Houttuynia cordata Decoction for Oral Care.

Dental biofilms that form in the oral cavity play a critical role in the pathogenesis of several infectious oral diseases, including dental caries, periodontal disease, and oral candidiasis. Houttuynia cordata (HC, Saururaceae) is a widely used traditional medicine, for both internal and external application. A decoction of dried HC leaves (dHC) has long been consumed as a health-promoting herbal tea in Japan. We have recently reported that a water solution of HC poultice ethanol extract (wHCP) exerts antimicrobial and antibiofilm effects against several important oral pathogens. It also exhibits anti-inflammatory effects on human keratinocytes. In our current study, we examined the effects of dHC on infectious oral pathogens and inflammation. Our results demonstrated that dHC exerts moderate antimicrobial effects against methicillin-resistant Staphylococcus aureus (MRSA) and other oral microorganisms. dHC also exhibited antibiofilm effects against MRSA, Fusobacterium nucleatum (involved in dental plaque formation), and Candida albicans and inhibitory effects on interleukin-8, CCL20, IP-10, and GROα productions by human oral keratinocytes stimulated by Porphyromonas gingivalis lipopolysaccharide (a cause of periodontal disease), without cytotoxic effects. This suggests that dHC exhibits multiple activities in microorganisms and host cells. dHC can be easily prepared and may be effective in preventing infectious oral diseases.

Royal Jelly Inhibits Pseudomonas aeruginosa Adherence and Reduces Excessive Inflammatory Responses in Human Epithelial Cells.

Pseudomonas aeruginosa is a Gram-negative bacterium and causes respiratory infection especially in elderly patients. Royal jelly has been used worldwide as a traditional remedy and as a nutrient; however, the effect against P. aeruginosa is unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly against P. aeruginosa. Wild-type strain PAO1 and clinical isolates of P. aeruginosa were used for antibacterial assay and antiadherent assay to abiotic surface and epithelial cells, which are pharynx (Detroit 562) and lung (NCI-H292) epithelial cells. In anti-inflammatory assay, epithelial cells were pretreated with royal jelly before bacterial exposure to investigate its inhibitory effect on interleukin (IL-8) and macrophage inflammatory protein-3α/CCL20 overproduction. Although royal jelly did not have antibacterial activity at concentration of 50% w/v, antiadherent activity was confirmed on the abiotic surface and epithelial cells under concentration of 25%. Pretreatment with royal jelly significantly inhibited overproduction of IL-8 and CCL20 from both cells. These results demonstrated that royal jelly inhibits P. aeruginosa adherence and protects epithelial cells from excessive inflammatory responses against P. aeruginosa infection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused by P. aeruginosa.

Effects of an autoinducer analogue on antibiotic tolerance in Pseudomonas aeruginosa.

Objectives: Antibiotic tolerance causes chronic, refractory and persistent infections. In order to advance the development of a new type of drug for the treatment of infectious diseases, we herein investigated the effects of a newly synthesized analogue of the Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 ( a uto i nducer a nalogue) on antibiotic tolerance in P. aeruginosa . Methods: A P. aeruginosa luminescent strain derived from PAO1 was injected into neutropenic ICR mice and bioluminescence images were acquired for a period of time after treatments with antibiotics and AIA-1. In vitro susceptibility testing and killing assays for the planktonic and biofilm cells of PAO1 were performed using antibiotics and AIA-1. The expression of quorum-sensing-related genes was examined using real-time PCR. Results: In vivo and in vitro assays showed that AIA-1 alone did not exert any bactericidal effects and also did not affect the MICs of antibiotics. However, the combined use of AIA-1 and antibiotics exerted markedly stronger therapeutic effects against experimental infection than antibiotics alone. The presence of AIA-1 also enhanced the killing effects of antibiotics in planktonic and biofilm cells. Although AIA-1 did not inhibit the expression of lasB and rhlA genes, which are directly regulated by quorum sensing, it clearly suppressed expression of the rpoS gene. Conclusions: The new compound, AIA-1, did not alter the antibiotic susceptibility of P. aeruginosa by itself; however, its addition enhanced the antibacterial activity of antibiotics. AIA-1 did not inhibit quorum sensing, but reduced the antibiotic tolerance of P. aeruginosa by suppressing rpoS gene expression.

Role of psl Genes in Antibiotic Tolerance of Adherent Pseudomonas aeruginosa.

Bacteria attached to a surface are generally more tolerant to antibiotics than their planktonic counterparts, even without the formation of a biofilm. The mechanism of antibiotic tolerance in biofilm communities is multifactorial, and the genetic background underlying this antibiotic tolerance has not yet been fully elucidated. Using transposon mutagenesis, we isolated a mutant with reduced tolerance to biapenem (relative to that of the wild type) from adherent cells. Sequencing analysis revealed a mutation in the pslL gene, which is part of the polysaccharide biosynthesis operon. The Pseudomonas aeruginosa PAO1ΔpslBCD mutant demonstrated a 100-fold-lower survival rate during the exposure of planktonic and biofilm cells to biapenem; a similar phenotype was observed in a mouse infection model and in clinical strains. Transcriptional analysis of adherent cells revealed increased expression of both pslA and pelA, which are directly regulated by bis-(3',5')-cyclic dimeric GMP (c-di-GMP). Inactivation of wspF resulted in significantly increased tolerance to biapenem due to increased production of c-di-GMP. The loss of pslBCD in the ΔwspF mutant background abolished the biapenem-tolerant phenotype of the ΔwspF mutant, underscoring the importance of psl in biapenem tolerance. Overexpression of PA2133, which can catalyze the degradation of c-di-GMP, led to a significant reduction in biapenem tolerance in adherent cells, indicating that c-di-GMP is essential in mediating the tolerance effect. The effect of pslBCD on antibiotic tolerance was evident, with 50- and 200-fold-lower survival in the presence of ofloxacin and tobramycin, respectively. We speculate that the psl genes, which are activated by surface adherence through elevated intracellular c-di-GMP levels, confer tolerance to antimicrobials.

A Pseudomonas aeruginosa Quorum-Sensing autoinducer analog enhances the activity of antibiotics against resistant strains.

In this study, we have investigated the effects of the newly synthesized analog of Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 (autoinducer analog) against antibiotic-resistant bacteria. In vitro susceptibility and killing assays for P. aeruginosa PAO1ΔoprD mutant and clinical isolates were performed by using antibiotics and AIA-1. In an in vivo assay, a luminescent carbapenem-resistant strain derived from PAO1ΔoprD was injected into neutropenic ICR mice and bioluminescence images were acquired after the treatment with antibiotics and AIA-1. Additionally, we investigated the effects of the combination use against carbapenem-resistant Enterobacteriaceae (CRE). Using killing assays in P. aeruginosa, the survival rates in the presence of antibiotics and AIA-1 significantly decreased in comparison with those with antibiotics alone. Furthermore, dual treatment of biapenem and AIA-1 was more effective than biapenem alone in a mouse infection model. AIA-1 did not change the MICs in P. aeruginosa, suggesting that AIA-1 acts on the mechanism of antibiotic tolerance. Conversely, the MICs of antibiotics decreased in the presence of AIA-1 in some CRE strains, indicating that AIA-1 may require additional mechanism to act on CRE. In conclusion, AIA-1 may be a potent drug for clinical treatment of infections caused by antibiotic-resistant bacteria. J. Med. Invest. 64: 101-109, February, 2017.

Explorative gene analysis of antibiotic tolerance-related genes in adherent and biofilm cells of Pseudomonas aeruginosa.

BACKGROUND: Antibiotic tolerance has attracted worldwide attention, as it leads to chronic, refractory, and persistent infections that are difficult to control. Bacterial biofilms are well known to be more tolerant to antibiotics compared to planktonic bacteria. We previously revealed that adherent bacteria on a solid surface also exhibited tolerance to antibiotics before forming a biofilm. However, little is known about the mechanisms of antibiotic tolerance for adherent or biofilm cells. OBJECTIVES: We investigated the mechanisms of antibiotic tolerance in the biofilm life cycle using adherent and biofilm cells, and evaluated the possibility that common mechanisms operate at each stage. METHODS: We constructed transposon mutants of Pseudomonas aeruginosa PAO1 and screened for low-tolerant mutants with two different methods, using adherent cells and biofilm cells. RESULTS: Fourteen and nine mutants exhibiting low antibiotic tolerance were detected in the adherent cells and biofilm cells, and 14 and 7 candidate genes linked to this tolerance were identified by sequencing, respectively. Eight of the 14 genes related to the antibiotic tolerance of the adherent cells were involved in biofilm formation. Two of the seven genes related to the antibiotic tolerance of biofilm cells participated in the antibiotic tolerance of adherent cells. CONCLUSIONS: The antibiotic tolerance of adherent cells and biofilm formation appear to be under the same regulation mechanism to promote survival in the presence of antibiotics. Antibiotic tolerance shows a complex regulation mechanism at each stage of biofilm formation.

DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis.

Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human ß-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.

Anti-inflammatory and protective effects of 2-methacryloyloxyethyl phosphorylcholine polymer on oral epithelial cells.

Periodontitis is a chronic inflammatory disease initiated by a microbial biofilm formed in the periodontal pocket. Gingival epithelium plays important roles as the first physical barrier to bacterial invasion and in orchestrating the innate immune reaction via toll-like receptors (TLRs), which recognize various bacterial products, and maintaining its function. Newly developed oral care products to inhibit bacterial adherence, subsequent inflammatory reaction and protect the gingival epithelium are expected. We previously reported that 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer coating decreased bacterial adhesion to human oral keratinocytes, RT-7, and mouth-rinsing with MPC-polymer inhibited the increase of oral bacteria. In this study, regarding the possibility of MPC-polymer application for preventing the adherence of periodontal pathogen, subsequent inflammatory reaction and protection of gingival epithelium, we examined the effects of MPC-polymer on the adherence of Porphyromonas gingivalis, major periodontitis-related pathogen, and TLR2 ligand to RT-7 and subsequent interleukin (IL)-8 production. MPC-polymer treatment significantly reduced P. gingivalis adherence by 44% and TLR2-mediated IL-8 production by blocking the binding of its specific-ligand in a concentration-dependent manner. Furthermore, MPC-polymer pretreatment protected RT-7 from injury by chemical irritants, cetylpyridinium chloride. These findings suggest that MPC-polymer is potentially useful for oral care to prevent oral infection and to maintain oral epithelial function.

Evaluation of a rapid oral bacteria quantification system using dielectrophoresis and the impedance measurement.

To clarify the availability of the dielectrophoretic impedance measurement (DEPIM) system as the evaluator for oral care, we evaluated the usefulness of DEPIM system by comparison with the standard plate counting (SPC) method. First, the relationship between the DEPIM results and bacterial concentration measured by SPC was clarified. Next, the measurement of the microorganism number in a mixed suspension was evaluated with DEPIM and SPC. The bacterial counts with DEPIM strongly correlated with those with SPC (r(2)=0.633-0.997) and this correlation was also shown in the measurement of a mixed bacterial suspension (ranging from 10(5) to 10(8) cfu/ml) of two bacterial species. Moreover, the experiments using dissociating enzymes to eliminate the influence of the size of the bacterial aggregates demonstrated that the microbial measurement results with DEPIM are unaffected by bacterial aggregates. This study demonstrated that bacterial counts with DEPIM strongly correlated with those with SPC and were unaffected by bacterial aggregates.

Involvement of commensal bacteria may lead to dysregulated inflammatory and autoimmune responses in a mouse model for chronic nonsuppurative destructive cholangitis.

BACKGROUND: We previously reported a mouse model of primary biliary cirrhosis (PBC)-like chronic nonsuppurative destructive cholangitis (CNSDC), in which frequent injections of Streptococcus intermedius induced CNSDC and autoantibody production. The present study was performed to verify the model by examining 1) the reappearance of the PBC-like CNSDC after lymphocyte transfer from model to naïve mice, 2) the involvement of autophagy, and 3) the influence of the strain difference. METHODS: Mice were inoculated with S. intermedius weekly for 8 weeks, then sacrificed to obtain samples. Spleen cells obtained from S. intermedius-inoculated mice were transferred to RAG2(-/-) mice. RESULTS: CNSDC and elevated serum level of anti-gp210 titers were observed in S. intermedius-inoculated C57BL/6 mice, similar to the results of our previous report using BALB/c mice. Portal inflammation was induced in the livers of RAG2(-/-) mice by the transfer of spleen cells from S. intermedius-inoculated C57BL/6 mice. Among the inflammatory cells in the RAG2(-/-) mice, CD3-positive cells were predominant. Autophagosome-like structures were detected histologically, in the cytoplasm of infiltrated cells around the bile ducts in the livers of S. intermedius-inoculated both C57BL/6 and BALB/c mice. In S. intermedius-inoculated C3H/HeJ mice, inflammation in the portal area was less extensive than that in the hepatic parenchyma. CONCLUSION: Bacterial component(s) and sequentially upregulated innate and acquired immune responses, accompanied by autophagy, might trigger CNSDC, via autoimmune mechanisms. Throughout the generation of bacteria-triggered PBC-like CNSDC, strain difference may influence the response to S. intermedius-inoculation in the liver.

Biofilm formation of Candida albicans on implant overdenture materials and its removal.

OBJECTIVES: The purposes of this study were to clarify the surface characteristics of various implant overdenture materials and the capabilities of Candida albicans adherence and biofilm formation on these surfaces, and to investigate the role of salivary mucin in biofilm formation. METHODS: Seven commonly used implant and restorative materials were assessed. The surface roughness averages of all materials were limited to 0.07-0.10 µm. Contact angles and salivary mucin absorption were measured. After 90-min initial adhesion and 2-day biofilm formation, the amounts of C. albicans were determined by counting colony-forming units and the morphological characteristics were observed by scanning electron microscopy (SEM). The effects of saliva coating and the influences of material surface property on initial adhesion, biofilm formation and its removability were analysed by univariate two-way analysis of variance and multiple linear regression analysis. RESULTS: Surface contact angle of materials, the index of hydrophobicity, was found to be correlated positively with initial adhesion and biofilm formation of C. albicans. A negative correlation between mucin absorption and removability of Candida biofilm indicates that mucin plays an important role in biofilm formation and its rigidity. SEM observation also revealed fewer Candida cells on saliva-coated Ti than on saliva-coated hydroxyapatite or acrylic resin. CONCLUSIONS: The materials with different hydrophobic property and compositions display diverse manners of salivary mucin absorption, initial adhesion and biofilm formation. The hydrophobic materials encourage enhanced initial adhesion, subsequently resulting in the active biofilm formation. Mucin has decisive effects on Candida immobilization and biofilm development on the materials. CLINICAL SIGNIFICANCE: Surface hydrophilic property and composition of materials and salivary proteins, especially mucin, affect the process of Candida biofilm formation and influence the amount and rigidity of formed biofilm. The present data may be applied as a reference for selecting materials in implant overdenture treatment from a microbiological point of view.

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells.

Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-κB translocation in human hepatic HepG2 cells, ILY did not affect NF-κB localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.