The neutrophil-osteogenic cell axis promotes bone destruction in periodontitis.

The immune-stromal cell interactions play a key role in health and diseases. In periodontitis, the most prevalent infectious disease in humans, immune cells accumulate in the oral mucosa and promote bone destruction by inducing receptor activator of nuclear factor-κB ligand (RANKL) expression in osteogenic cells such as osteoblasts and periodontal ligament cells. However, the detailed mechanism underlying immune-bone cell interactions in periodontitis is not fully understood. Here, we performed single-cell RNA-sequencing analysis on mouse periodontal lesions and showed that neutrophil-osteogenic cell crosstalk is involved in periodontitis-induced bone loss. The periodontal lesions displayed marked infiltration of neutrophils, and in silico analyses suggested that the neutrophils interacted with osteogenic cells through cytokine production. Among the cytokines expressed in the periodontal neutrophils, oncostatin M (OSM) potently induced RANKL expression in the primary osteoblasts, and deletion of the OSM receptor in osteogenic cells significantly ameliorated periodontitis-induced bone loss. Epigenomic data analyses identified the OSM-regulated RANKL enhancer region in osteogenic cells, and mice lacking this enhancer showed decreased periodontal bone loss while maintaining physiological bone metabolism. These findings shed light on the role of neutrophils in bone regulation during bacterial infection, highlighting the novel mechanism underlying osteoimmune crosstalk.

Localization and pathogenic role of the cysteine protease dentipain in Treponema denticola.

The Msp protein complex and the serine protease dentilisin are the best-characterized virulence factors in Treponema denticola, the major etiological agent of chronic periodontitis. In addition to these outer sheath factors, the cysteine protease dentipain contributes to pathogenicity, but its secretion, processing, cellular localization, and role in T. denticola virulence are not fully understood. In this study, we found that full-sized dentipain (74-kDa) and the 52-kDa truncated form of the enzyme are located, respectively, in the outer sheath derived from T. denticola dentilisin- and the Msp-deficient mutants. Furthermore, dentipain was barely detected in the wild-type strain. These results suggest that dentilisin and Msp, the major outer sheath proteins, are involved in the secretion and maturation of dentipain. Inactivation of the dentipain gene slowed the growth of T. denticola, and the effect was more profound in serum-free medium than in serum-containing medium. Several genes, including those encoding transporters and methyl-accepting chemotaxis proteins, were differentially expressed in the dentipain-deficient mutant. Furthermore, the mutant strain was more hydrophobic than the wild-type strain. Finally, the mutant showed less autoaggregation activity and adhesion to IgG in a serum-free medium than the wild-type strain. These findings suggest that dentipain contributes to the virulence of T. denticola by facilitating adhesion and acquisition of nutrients essential for colonization and proliferation in the gingival crevice under serum-rich conditions.

Effect of Treponema Denticola Infection on Epithelial Cells.

Chronic periodontitis is an infectious disease caused by periodontopathic bacteria in subgingival plaque. One major pathogen of this disease, Treponema denticola, has several virulence factors, including a major surface protein (Msp) and the surface protease dentilisin. The cytopathic effects of periodontopathic bacteria on epithelial cells disrupt the integrity of the barrier junction, resulting in the inflammation of periodontal tissue. The aim of this study was to investigate the effect of T. denticola virulence factors dentilisin and Msp on epithelial cells. The effects of T. denticola wild-type, Msp-mutant, and dentilisin-mutant strains on the contact junction in Madin-Darby canine kidney epithelial cells was evaluated based on ohmic values. Cultured oral carcinoma epithelial cells were scratched and exposed to the selected T. denticola strains and cell migration determined. Subsequent degradation of adherence proteins and proteins in the contact junctions was evaluated. Dissociation of cell contact junctions was detected in cells infected with wild-type T. denticola approximately 30 min after infection, but not in those exposed to the mutants. Inhibition of migration was observed in the wild-type and Msp-deficient mutants. The adherent proteins focal adhesion kinase, ZO-1, and paxillin were hydrolyzed by infection with the wild-type and Msp mutants. These results indicate that T. denticola disrupts the function of epithelial cells by hydrolyzing proteins at the intercellular junction and inhibiting healing of epithelial cells via hydrolyzed proteins associated with focal adhesion; Msp was also associated with these effects.

Systemic administration of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-Ig abrogates alveolar bone resorption in induced periodontitis through inhibition of osteoclast differentiation and activation: An experimental investigation.

BACKGROUND/OBJECTIVES: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical immunoregulatory molecule expressed on T cells. CTLA-4 also binds to the surfaces of monocytes and macrophages, precursors of osteoclasts. Research on rheumatoid arthritis demonstrated that CTLA-4 suppresses inflammation and bone resorption. However, its effects on alveolar bone have yet to be understood. The purpose of this study was to investigate the role and potential mechanism of CTLA-4 in bone resorption in periodontitis. MATERIALS AND METHODS: In vivo, the effects of systemic administration of CTLA-4 immunoglobulin fusion protein (CTLA-4-Ig) on alveolar bone resorption were investigated using a periodontitis mouse model. A total of 20 C57BL/6J mice were randomly assigned to two groups according to the administration modes. Periodontitis was induced by placing a ligature around the left maxillary second molar. The contralateral tooth was left un-ligated. In the CTLA-4-Ig (+) group, CTLA-4-Ig was administered by intraperitoneal injection at 1 and 3 days after ligature placement. Animals in the CTLA-4-Ig (-) group were given only phosphate-buffered saline each time. At 5 days after ligature placement, bone resorption was assessed by micro-computed tomography and histological examination, and the prevalence of osteoclast-like cells was assessed by tartrate-resistant acid phosphatase (TRAP) staining. In vitro, the effects of CTLA-4-Ig on osteoclasts were evaluated. Viability of RAW 264.7 cells treated with receptor activator of nuclear factor-κB ligand (RANKL) and CTLA-4-Ig was tested by WST-1 assay. Osteoclast-like cells were enumerated by TRAP staining, and osteoclast activity was evaluated by resorption pit assay. Gene expression levels of osteoclast differentiation markers (macrophage-colony stimulating factor receptor, carbonic anhydrase II, cathepsin K, and Trap) and protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, were assessed by quantitative real-time polymerase chain reaction. The effect of CTLA-4-Ig on the nuclear factor-κB (NF-κB) activation was assessed by enzyme-linked immunosorbent assay. RESULTS: In vivo, ligature-induced bone resorption and the numbers of osteoclast-like cells were significantly decreased by the administration of CTLA-4-Ig. In vitro, treatment with RANKL and CTLA-4-Ig had no significant effect on cell viability. CTLA-4-Ig significantly reduced the prevalence and activation of osteoclast-like cells and decreased the expressions of osteoclast differentiation markers, compared with the RANKL-treated control. CTLA-4-Ig significantly suppressed RANKL-induced phosphorylation of NF-κB p65 but increased PP2A expression. CONCLUSION: These results suggest that CTLA-4-Ig abrogates bone resorption in induced periodontitis, possibly via inhibition of osteoclast differentiation and activation. The regulation of the NF-κB pathway and PP2A expression may be one mechanism by which CTLA-4-Ig suppresses osteoclast behavior.

Patient-specific establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering.

BACKGROUND AND OBJECTIVE: Dysbiosis, a loss of balance in the microbiota, is a potential factor of peri-implantitis. However, compositional change of the peri-implant microbiota soon after implant uncovering is still unknown. In this study, bacterial composition in the peri-implant sulcus was examined to understand the establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering. METHODS: Microbiota samples were collected at weeks 1, 2, 4, and 6 after stage-two surgery. Bacterial DNA was isolated from the samples, and a 16S rRNA gene library was constructed. Sequence reads were obtained using a high-throughput sequencing platform and were taxonomically assigned at the phylum and genus levels. RESULTS: Alpha diversity indices, which did not include taxonomic information, were at similar levels throughout the four time points. At 1 and 2 weeks, the bacterial composition was similar among patients with the predominance of Firmicutes and Proteobacteria. However, the composition was diverse at 4 and 6 weeks and significantly dissimilar to the composition at 1 week. CONCLUSIONS: At 1 week, the peri-implant microbiota was already formed with alpha diversity as high as that at the later time points. However, the bacterial composition was not highly dissimilar among patients at 1 week. The composition changed over the passage of several weeks and was specific for each patient.

Inhibitory Effect of Resveratrol on Candida albicans Biofilm Formation.

Candida albicans is the primary candidiasis-causing fungal pathogen in humans, and one of its most important virulence factors is the ability to form biofilms. Moreover, these biofilms are often resistant to antifungal agents, so there is a need to develop alternative elimination strategies and therapeutic agents for such infections. The antifungal activity of resveratrol, a phytoalexin polyphenolic compound, impairs the morphological transition of C. albicans under various hypha-inducing conditions and inhibits growth of the yeast-form and mycelia. The purpose of this study was to investigate the effect of resveratrol against C. albicans biofilm formation. The developmental, sustained, and mature stages of biofilm formation were affected or inhibited by resveratrol. Exposure to resveratrol at the developmental stage inhibited growth of C. albicans in a dose-dependent manner. A >30% reduction was observed in sustained biofilm growth in the presence of 200 µg/ml resveratrol in comparison with in its absence. In terms of disruption of matured biofilm, 6.25-100 µg/ml resveratrol significantly reduced cell viability of C. albicans compared with in a control sample (p<0.05). The present results indicate that resveratrol has the potential to serve as an anti-Candida treatment and preventive tool which functions by inhibiting existing or under-forming C. albicans biofilms.

Inhibitory Effects of Lingonberry Extract on Oral Streptococcal Biofilm Formation and Bioactivity.

Phenolic compounds in fruits such as cranberries have been shown to promote a number of biological activities. The purpose of this study was to investigate the effects of polyphenolic compound-containing lingonberry extract on oral streptococci and compare them with the known anti-cariogenic activity of cranberries. Water-soluble and polyphenol-rich fractions (Fractions I and II, respectively) were isolated from cranberries and lingonberries. The effects of those fractions on the biofilm formation ability and bioactivity of Streptococcus mutans MT8148R, Streptococcus sobrinus 6715, and Streptococcus sanguinis ATCC 10556 were then evaluated. Cranberry or lingonberry Fraction II (at 0.5-1 mg/ml) significantly reduced biofilm formation by S. mutans, S. sobrinus, and S. sanguinis. In contrast, cranberry or lingonberry Fraction I (at 0.5-2 mg/ml) increased biofilm formation by S. mutans and S. sobrinus, but not by S. sanguinis. Fractions I and II (at 1-2 mg/ml) also reduced the bioactivity of S. mutans, while Fraction II (at 0.5 mg/ml) enhanced the bioactivity of all tested strains. The results revealed that lingonberries contained a larger amount of polyphenol than cranberries and that they showed almost the same level of activity against the biofilm formation ability and bioactivity of oral streptococci. This indicates that polyphenol-rich lingonberry fraction offers a promising natural food derivative for prevention of dental caries.

Treponema denticola Induces Epithelial Barrier Dysfunction in Polarized Epithelial Cells.

Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with periodontal disease and has a variety of virulence factors. Although in vitro studies have shown that T. denticola is able to penetrate epithelial cell monolayers, its effect on the epithelial barrier junction is not known. Human gingival epithelial cells are closely associated with adjacent membranes, forming barriers in the presence of tight junction proteins, including zonula occludens-1 (ZO-1), claudin-1, and occludin. Tight junction proteins are also expressed by Madin-Darby canine kidney (MDCK) cells in culture. In this study, the MDCK cell profile was investigated following infection with T. denticola (ATCC 35405) wild-type, as well as with its dentilisin-deficient mutant, K1. Basolateral exposure of MDCK cell monolayers to T. denticola at a multiplicity of infection (MOI) of 104 resulted in a decrease in transepithelial electrical resistance (TER). Transepithelial electrical resistance in MDCK cell monolayers also decreased following apical exposure to T. denticola (MOI=104), although this took longer with basolateral exposure. The effect on the TER was time-dependent and required the presence of live bacteria. Meanwhile, MDCK cell viability showed a decrease with either basolateral or apical exposure. Immunofluorescence analysis demonstrated decreases in the amounts of immunoreactive ZO-1 and claudin-1 in association with disruption of cell-cell junctions in MDCK cells exposed apically or basolaterally to T. denticola. Western blot analysis demonstrated degradation of ZO-1 and claudin-1 in culture lysates derived from T. denticola-exposed MDCK cells, suggesting a bacteria-induced protease capable of cleaving these tight junction proteins.

Characterization of a novel potential peptide import system in Treponema denticola.

Treponema denticola is a major etiologic agent of chronic periodontitis. On the outer sheath of T. denticola, several proteins, such as the major outer sheath protein and dentilisin were detected, and among them, a 95 kDa protein which has not yet been characterized. The aim of this study was to characterize the function of this 95 kDa protein containing gene cluster. A gene encoding this 95 kDa protein (TDE_1072) of T. denticola was inactivated by homologous recombination. We compared growth curves between the TDE_1072 mutant and wild-type strains as well as differences in gene expression by DNA microarray analysis. Differential expression of genes identified by microarray analysis was confirmed by quantitative reverse transcription-polymerase chain reaction. The proteins encoded by TDE_1072, TDE_1073, TDE_1074, TDE_1075, and TDE_1076 shared respective similarities to the substrate-binding domain (DppA) of an ABC-type dipeptide/oligopeptide/nickel transport system, and to the permease components (DppB and DppC) and ATPase components (DppD and DppF) of an ABC-type dipeptide/oligopeptide/nickel transport system. Inactivation of dppA attenuated the growth of T. denticola and dppA-dppF were co-transcribed. In contrast, expression of oppB-oppF was up-regulated in the mutant. Our findings indicate that TDE_1072 may be a potential periplasmic solute binding protein encoded by dppA that is involved in the organization of a peptide uptake system with dppB-dppF.

Effect of Porphyromonas gingivalis infection in the placenta and umbilical cord in pregnant mice with low birth weight.

OBJECTIVE: Growing evidence indicates an association between periodontitis and delivery outcome; however, the mechanism is unclear. This study aimed to investigate the influence of Porphyromonas gingivalis (Pg) infection on delivery outcome in mice. MATERIALS AND METHODS: Bacteremia was induced in pregnant Slc:ICR mice (8 weeks old) by intravenous injection of Pg. Mice were randomly divided into a control group (CO), and those receiving Pg injection at gestational day 1 (GD1), gestational day 15 (GD15) or every day (ED). Delivery outcome, Pg infection, and gene expression in the placenta and umbilical cord were evaluated. RESULTS: Birth weight was lower in the ED and GD15 groups than in the CO group. A remarkable increase in anti-Pg IgG antibody was observed in the ED and GD1 groups, although Pg was not detected in the placenta or umbilical cord. mRNA expression of Tnfα and Il6 in the placenta, and Hif1α in the umbilical cord, was significantly increased in the ED group. Microarray analysis of the umbilical cord revealed increased expression of several genes including Orm1, Mgl2, Rps6ka3 and Trim15 in the ED group. CONCLUSIONS: Pg infection during the third trimester caused low birth weight and inflammation in the placenta and umbilical cord.

Elevation of pro-inflammatory cytokine levels following anti-resorptive drug treatment is required for osteonecrosis development in infectious osteomyelitis.

Various conditions, including bacterial infection, can promote osteonecrosis. For example, following invasive dental therapy with anti-bone resorptive agents, some patients develop osteonecrosis in the jaw; however, pathological mechanisms underlying these outcomes remain unknown. Here, we show that administration of anti-resorptive agents such as the bisphosphonate alendronate accelerates osteonecrosis promoted by infectious osteomyelitis. Potent suppression of bone turnover by these types of agents is considered critical for osteonecrosis development; however, using mouse models we found that acceleration of bone turnover by teriparatide injection did not prevent osteonecrosis but rather converted osteoclast progenitors to macrophages expressing inflammatory cytokines, which were required for osteonecrosis development. In fact, we demonstrate that TNFα-, IL-1α/ß- or IL-6-deficient mice as well as wild-type mice administered a TNFα-inhibitor were significantly resistant to development of osteonecrosis accompanying infectious myelitis, even under bisphosphonate treatment. Our data provide new insight into mechanisms underlying osteonecrosis and suggest new ways to prevent it.

Effect of water containing organic acids on aspiration pneumonia-causative bacteria in the biofilm on the tooth surface.

BACKGROUND/PURPOSE: The tooth surface is a source of oral microbes in dentulous individuals, it is difficult for elderly people requiring nursing care to perform mechanical tooth cleaning by themselves. The objective of this study was to investigate the antimicrobial effect of water containing organic acids (WOA) made by some organic acids as food additives on chemical cleaning for elderly people on aspiration pneumonia-causative bacteria in the biofilm on the tooth surface. MATERIALS AND METHODS: Ninety-six specimens made from bovine incisors were divided into four groups and incubated with one of four aspiration pneumonia-causative bacteria. Each group was further divided into six subgroups according to treatment as follows: control group (DW), chlorhexidine gluconate solution group (CHX), WOA group (WOA), ultrasonic treatment in distilled water group (DW-U), ultrasonic treatment in chlorhexidine gluconate solution group (CHX-U) or ultrasonic treatment in WOA group (WOA-U). After treatment, the levels of viable microbes in the biofilm were evaluated by quantitative adenosine triphosphate analysis and compared among the six groups. RESULTS: For every evaluated microbe, there were significant differences between DW and WOA, and DW and WOA-U. However, there was no significant difference among the WOA, DW-U, CHX-U and WOA-U groups. These results suggested that the antimicrobial effect of WOA on microbes attached to the tooth surface was similar to that of ultrasonic cleaning. CONCLUSION: WOA has an antimicrobial effect on microbes in the biofilm on the tooth surface.

Treponema denticola invasion into human gingival epithelial cells.

Host cell invasion is important for periodontal pathogens in evading host defenses and spreading into deeper areas of the periodontal tissue. Treponema denticola has been implicated in a number of potentially pathogenic processes, including periodontal tissue penetration. Here we tested the ability of T. denticola strains to invade human gingival epithelial cells (HGEC). After 2 h infection, intracellular location of T. denticola cells was confirmed by confocal laser scanning microscopy (CLSM). Results from an antibiotic protection assay following [(3)H]uridine labeling indicated that invasion efficiency reached a maximum at 2 h after infection. Internalized T. denticola cells were still observed in HGEC at 24 h by CLSM. A dentilisin deficient mutant exhibited significantly decreased invasion (p < 0.05) compared with the wild-type strain. In inhibition assays, phenylmethylsulfonyl fluoride and metabolic inhibitors such as methyl-ß-cyclodextrin and staurosporine significantly reduced T. denticola invasion. Under CLSM, T. denticola colocalized with GM-1 ganglioside-containing membrane microdomains in a cholesterol-dependent manner. These results indicated that T. denticola has the ability to invade into and survive within HGECs. Dentilisin activity of T. denticola and lipid rafts on HGEC appear to play important roles in this process.

Inhibitory effects of a novel cationic dodecapeptide [CL(14-25)] derived from cyanate lyase of rice on endotoxic activities of LPSs from Escherichia coli and periodontopathic Aggregatibacter actinomycetemcomitans.

OBJECTIVE: CL(14-25), a dodecapeptide of cyanate lyase from rice, is a novel cationic α-helical antimicrobial peptide. In this study, we examined inhibitory ability of CL(14-25) against endotoxic activities of lipopolysaccharides (LPSs) from Escherichia coli and periodontal pathogenic Aggregatibacter actinomycetemcomitans. METHODS: Endotoxin-neutralizing activity of CL(14-25) was evaluated by inhibition to induction of cytokine and nitric oxide in human aortic endothelial cells (HAECs) and RAW264 mouse macrophage cells, respectively. Protective effect of CL(14-25) was determined in mice against lethal toxicity of LPS. RESULTS: IL-6 in HAECs was induced by stimulation with LPS preparations of A. actinomycetemcomitans and E. coli tested in this study, and addition of CL(14-25) to the medium caused inhibition of their induction in a dose-dependent manner. CL(14-25) inhibited NO induction in RAW264 cells by a smooth type LPS of E. coli O55:B5 and an Rc type LPS of E. coli J5 as well as lipid A of E. coli R515 in a dose-dependent manner. Simultaneous injection of E. coli O55:B5 LPS and CL(14-25) in BALB/c mice resulted in prevention of lethal toxicity of the former. The results of a Limulus amebocyte lysate assay and surface plasmon resonance analysis of interaction between CL(14-25) and E. coli LPS or lipid A showed that CL(14-25) specifically binds to a lipid A moiety of LPS. CONCLUSION: The results of present study suggest that CL(14-25) has a potential to be used as a nutraceutical agent for periodontal therapy.

Evaluation of application possibility of water containing organic acids for chemical denture cleaning for older adults.

AIM: The purpose of the present study was to evaluate the application possibility of water containing organic acids (WOA), made by some organic acids used as food additives, for chemical denture cleaning for older adults by microbial investigation. METHODS: Using an in vitro biofilm study, we determined the effects of WOA on Streptococcus sanguinis, S. pneumoniae and Candida albicans attached to heat-cured acrylic resins. Specimens were divided into three groups as follows: control group (TW), commercial denture cleaner group (DC) and WOA group (WOA). Specimens were treated with each for 5 min, 30 min or 8 h, and the numbers of attached microbes were determined by counting colony-forming units or adenosine triphosphate analysis. Using an in vivo biofilm study, we studied the effects of these same solutions on 60 complete dentures. The dentures were divided randomly and blindness into three groups as described above, and treated for 10 min. The numbers of microbes attached to dentures before and after treatment were determined by counting colony-forming units. RESULTS: For the in vitro biofilm study, there were significant differences in the numbers of microbes between WOA and TW, although there were no significant differences between WOA and DC except for C. albicans. For the in vivo biofilm study, there were significant differences between WOA, DC and TW, although there was no significant difference between WOA and DC. CONCLUSION: We conclude that water containing organic acids exerts antimicrobial effects as strong as commercial denture cleaner, and it has an application possibility of use for safe chemical denture cleaning for older adults.

Role of extracytoplasmic function sigma factors in biofilm formation of Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis has been implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis biofilm formation in the subgingival crevice plays an important role in the ability of the bacteria to tolerate stress signals outside the cytoplasmic membrane. Some bacteria use a distinct subfamily of sigma factors to regulate their extracytoplasmic functions (the ECF subfamily). The objective of this study was to determine if P. gingivalis ECF sigma factors affect P. gingivalis biofilm formation. METHODS: To elucidate the role of ECF sigma factors in P. gingivalis, chromosomal mutants carrying a disruption of each ECF sigma factor-encoding gene were constructed. Bacterial growth curves were measured by determining the turbidity of bacterial cultures. The quantity of biofilm growing on plates was evaluated by crystal violet staining. RESULTS: Comparison of the growth curves of wild-type P. gingivalis strain 33277 and the ECF mutants indicated that the growth rate of the mutants was slightly lower than that of the wild-type strain. The PGN_0274- and PGN_1740-defective mutants had increased biofilm formation compared with the wild-type (p < 0.001); however, the other ECF sigma factor mutants or the complemented strains did not enhance biofilm formation. CONCLUSION: These results suggest that PGN_0274 and PGN_1740 play a key role in biofilm formation by P. gingivalis.

Infection with Porphyromonas gingivalis exacerbates endothelial injury in obese mice.

BACKGROUND: A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity. METHODS: In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression. RESULTS: Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate. CONCLUSIONS: Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.

Csa2, a member of the Rbt5 protein family, is involved in the utilization of iron from human hemoglobin during Candida albicans hyphal growth.

Csa2 is a member of both the Candida albicans Rbt5 protein family and the Common in Fungal Extracellular Membranes (CFEM) protein superfamily. CFEM proteins are characterized by an internal domain containing eight equally spaced cysteine residues. Csa2 is involved in iron uptake from hemoglobin and heme proteins; however, its precise role is unclear. Here, we provide quantitative evidence of the involvement of Csa2 in the utilization of iron from human hemoglobin during C. albicans hyphal growth. The ability of the hyphal form of the wild-type (wt), a homozygote csa2Δ mutant, and a complemented strain of C. albicans to utilize hemoglobin as an iron source under iron-restricted conditions was examined through growth studies and a crystal violet-staining assay. Hemoglobin-binding activity was assessed indirectly using a hemoglobin-sensitized tube method. Although hyphal growth of the wt and csa2Δ/Δ::CSA2 strains was completely recovered when a high concentration of human hemoglobin was added to the iron-restricted culture medium, the recovery of the csa2Δ/Δ mutant was significantly diminished. Furthermore, hemoglobin binding was impaired in the csa2Δ/Δ mutant compared with the wt and csa2Δ/Δ::CSA2 strains, revealing that Csa2 is involved in the utilization of hemoglobin as an iron source by the hyphal form of C. albicans.

Vinculin and Rab5 complex is required [correction of requited]for uptake of Staphylococcus aureus and interleukin-6 expression.

Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.

Effect of clinical factors on bacterial contamination of bone chips collected during implant surgery.

PURPOSE: The objective of this study was to investigate the relationship between various clinical factors and bacterial contamination of bone chips (BC) collected during dental implant surgery and to elucidate how bacterial contamination might be minimized. MATERIALS AND METHODS: Implants were installed in 55 partially edentulous patients (36 men and 19 women), among whom the relationship between various clinical factors and bacterial contamination of BC collected by bone trap was investigated in 37. The effect of rinsing with a saline on BC was determined in 18 patients. Number of contaminating microorganisms was expressed as colony-forming units (CFUs). RESULTS: CFUs in the maxilla were lower than those in the mandible (P < 0.01). CFUs at the incisors or canines were lower than those at the premolars or molars (P < 0.01). Logistic regression analysis revealed a relationship between average bacterial count and duration of surgery (odds ratio, 1.046; 95% CI, 1.012-1.081). Rinsing of BC reduced bacterial contamination. CONCLUSION: Duration of surgery is a major clinical factor affecting contamination risk in BC, and rinsing of BC with a sterile saline solution reduces bacterial number.