Search

This in vitro study assessed the efficacy of functionalized graphene oxide (f-GO) nanocomposites on the decalcification of dentin, because dental caries of the root surface is becoming one of the new problems in aged society. Hydroxyapatite plates (HAP) and dentin slices were coated with f-GO nanocomposites by comparing them to silver diamine fluoride as a positive control, then treated with decalcification solutions such as ethylenediaminetetraacetic acid and citrate at 37°C for 24 h. Scanning electron microscopy (SEM) revealed significant protection of the surface morphology of HAP and dentin. On the other hand, a cariogenic Streptococcus mutans growth was inhibited by f-GO nanocomposites. In addition, cytotoxicity of them to epithelial cells was much less than that of povidone-iodine, which is commonly used for oral disinfectant. We synthesized 5 different f-GO nanocomposites such as GO–silver (Ag), GO-Ag–calcium fluoride (CaF2), GO-CaF2, GO-zinc, and GO–tricalcium phosphate (Ca3(PO4)2). They were standardized by evaluating under SEM, transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetry analysis (TGA), and Raman spectra after being synthesized in an aseptic technique. The abilities of GO-Ag, GO-Ag-CaF2, and GO-CaF2 nanocomposites were most preventive for decalcification. In addition, GO-Ag and GO-Ag-CaF2 almost completely inhibited S. mutans growth. However, they did not exhibit cytotoxicity to epithelial cells except at the highest concentration (0.1 w/v%) of GO-Ag and GO-Ag-CaF2. Furthermore, these f-GO nanocomposites exhibited less or no discoloration of dentin, although commonly used silver diamine fluoride causes discoloration of dentin to black. Thus, these f-GO nanocomposites are useful to protect dental caries on the tooth root that becomes a social problem in aged society.

Background Basic fibroblast growth factor (bFGF) has been applied for periodontal regeneration. However, the application depends on bone defect morphology because bFGF diffuses rapidly from defect sites. In a previous study, collagen‐binding bFGF (CB‐bFGF) has been shown to enhance bone formation by collagen‐anchoring in the orthopedic field. The aim of this study is to demonstrate the efficacy of CB‐bFGF with collagen scaffolds in bone regeneration of horizontal bone defect. Methods Cell proliferation activity and collagen binding activity of CB‐bFGF was confirmed by WST‐8 assay and collagen binding assay, respectively. The retention of CB‐bFGF in the collagen sheet (CS) was measured by fluorescence imaging. The rat horizontal alveolar bone defect model was employed to investigate the efficacy of CB‐bFGF with collagen powder (CP). After 4 and 8 weeks, the regenerative efficacy was evaluated by microcomputed tomography, histological, and immunohistochemical analyses. Results CB‐bFGF had a comparable proliferation activity to bFGF and a collagen binding activity. CB‐bFGF was retained in CS longer than bFGF. At 8 weeks postoperation, bone volume, bone mineral content, and new bone area in CB‐bFGF/CP group were significantly increased compared with those in other groups. Furthermore, epithelial downgrowth was significantly suppressed in CB‐bFGF/CP group. At 4 weeks, the numbers of osteocalcin, proliferating cell nuclear antigen, and osteopontin‐positive cells at the regeneration site in CB‐bFGF/CP group were greater than those in other groups. Conclusions CB‐bFGF/CP effectively promoted bone regeneration of horizontal bone defect possibly by sustained release of bFGF. The potential of CB‐bFGF composite material for improved periodontal regeneration in vertical axis was shown.

Osteoporosis is a common disease characterized by a systemic impairment of bone mass and microarchitecture that results in fragility fractures. Severe bone loss due to osteoporosis triggers pathological fractures and consequently decreases the daily life activity and quality of life. Therefore, prevention of osteoporosis has become an important issue to be addressed. We have reported that the fungal secondary metabolite (+)-terrein (TER), a natural compound derived from Aspergillus terreus, has shown receptor activator of nuclear factor-κB ligand (RANKL)–induced osteoclast differentiation by suppressing nuclear factor of activated T-cell 1 (NFATc1) expression, a master regulator of osteoclastogenesis. TER has been shown to possess extensive biological and pharmacological benefits; however, its effects on bone metabolism remain unclear. In this study, we investigated the effects of TER on the femoral bone metabolism using a mouse-ovariectomized osteoporosis model (OVX mice) and then on RANKL signal transduction using mouse bone marrow macrophages (mBMMs). In vivo administration of TER significantly improved bone density, bone mass, and trabecular number in OVX mice (p < 0.01). In addition, TER suppressed TRAP and cathepsin-K expression in the tissue sections of OVX mice (p < 0.01). In an in vitro study, TER suppressed RANKL-induced phosphorylation of PKCα/βII, which is involved in the expression of NFATc1 (p < 0.05). The PKC inhibitor, GF109203X, also inhibited RANKL-induced osteoclastogenesis in mBMMs as well as TER. In addition, TER suppressed the expression of osteoclastogenesis-related genes, such as Ocstamp, Dcstamp, Calcr, Atp6v0d2, Oscar, and Itgb3 (p < 0.01). These results provide promising evidence for the potential therapeutic application of TER as a novel treatment compound against osteoporosis.

Periodontal disease is a chronic inflammatory disease of the periodontal tissue. The periodontal inflamed surface area (PISA) is a proposed index for quantifying the inflammatory burden resulting from periodontitis lesions. This study aimed to investigate longitudinal changes in the periodontal status as evaluated by the PISA following the active periodontal treatment. To elucidate the prognostic factors of PISA, mixed-effect modeling was performed for clinical parameters, tooth-type, and levels of periodontal pathogens as independent variables. One-hundred-twenty-five patients with chronic periodontitis who completed the active periodontal treatment were followed-up for 24 months, with evaluations conducted at 6-month intervals. Five-times repeated measures of mean PISA values were 130+/−173, 161+/−276, 184+/−320, 175+/−417, and 209+/−469 mm2. Changes in clinical parameters and salivary and subgingival periodontal pathogens were analyzed by mixed-effect modeling. Plaque index, clinical attachment level, and salivary levels of Porphyromonas gingivalis were associated with changes in PISA at the patient- and tooth-level. Subgingival levels of P. gingivalis and Prevotella intermedia were associated with changes in PISA at the sample site. For most patients, changes in PISA were within 10% of baseline during the 24-month follow-up. However, an increase in the number of bleeding sites in a tooth with a deep periodontal pocket increased the PISA value exponentially.

Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis-specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis.

Background We report the first protocol for a multicenter, randomized comparison study to compare the efficacies of periodontal scaling and root-planing treatment against that of tooth-brushing treatment for nonalcoholic fatty liver disease (NAFLD) (PERION: PERIOdontal treatment for NAFLD). Nonalcoholic steatohepatitis (NASH) is an advanced form of NAFLD, which can progress to cirrhosis and hepatocellular carcinoma. Increased endotoxemia is associated with the progression of NAFLD. Periodontal bacteria possess endotoxins; Porphyromonas gingivalis is well-known as a major pathogenic bacterium in periodontitis, and serum antibody levels for P. gingivalis are high in patients with periodontitis. Several reports have indicated that P. gingivalis is related to NAFLD. This study aims to investigate the effect of periodontal treatment for liver damage, P. gingivalis infection, and endotoxemia on patients with NAFLD. Methods We will include adult patients (20–85 years old) with NAFLD, alanine aminotransferase (ALT) ≥ 40 IU/L, and equivalent steatosis grade ≥ 1 (target sample size, n = 40 patients; planned number of patients with outcome data, n = 32). Participants will be randomly assigned to one of two groups: a scaling and root-planing group or tooth-brushing as the usual group. The primary outcome will be the change in ALT levels from baseline to 12 weeks; the key secondary outcome will be the change in the serum immunoglobulin G (IgG) antibody titer for P. gingivalis at 12 weeks. Discussion This study should determine whether periodontal treatment decreases liver damage, P. gingivalis infection, and endotoxemia in patients with NAFLD. Trial registration University Hospital Medical Information Network (UMIN) Clinical Trials Registry, ID: UMIN000022079.

The recruitment of tissue‐resident stem cells is important for wound regeneration. Periodontal ligament cells (PDL cells) are heterogeneous cell populations with stemness features that migrate into wound sites to regenerate periodontal fibres and neighbouring hard tissues. Cell migration is regulated by the local microenvironment, coordinated by growth factors and the extracellular matrix (ECM). Integrin‐mediated cell adhesion to the ECM provides essential signals for migration. We hypothesized that PDL cell migration could be enhanced by selective expression of integrins. The migration of primary cultured PDL cells was induced by platelet‐derived growth factor‐BB (PDGF‐BB). The effects of blocking specific integrins on migration and ECM adhesion were investigated based on the integrin expression profiles observed during migration. Up‐regulation of integrins α3, α5, and fibronectin was identified at distinct localizations in migrating PDL cells. Treatment with anti‐integrin α5 antibodies inhibited PDL cell migration. Treatment with anti‐integrin α3, α3‐blocking peptide, and α3 siRNA significantly enhanced cell migration, comparable to treatment with PDGF‐BB. Furthermore, integrin α3 inhibition preferentially enhanced adhesion to fibronectin via integrin α5. These findings indicate that PDL cell migration is reciprocally regulated by integrin α3‐mediated inhibition and α5‐mediated promotion. Thus, targeting integrin expression is a possible therapeutic strategy for periodontal regeneration.

Atherosclerosis, a chronic inflammatory disease in arterial blood vessels, is one of the major causes of death in worldwide. Meanwhile, periodontal disease is a chronic inflammatory disease caused by infection with periodontal pathogens such as P. gingivalis (Porphyromonas gingivalis). Several studies have reported association between periodontal infection and atherosclerosis, but direct investigation about the effects of periodontal treatment on atherosclerosis has not been reported. We have planned Japanese local clinics to determine the relationship between periodontal disease and atherosclerosis under collaborative with medical and dental care. A prospective, multicentre, observational study was conducted including 38 medical patients with lifestyle-related diseases in the stable period under consultation at participating medical clinics and 92 periodontal patients not undergoing medical treatment but who were consulting at participating dental clinics. Systemic and periodontal examinations were performed before and after periodontal treatment. At baseline, LDL-C (low-density lipoprotein cholesterol) levels and percentage (%) of mobile teeth were positively related to plasma IgG (immunoglobulin) antibody titer against P. gingivalis with multivariate analysis. Corresponding to improvements in periodontal clinical parameters after treatment, right and left max IMT (maximum intima-media thickness) levels were decreased significantly after treatment (SPT-S: start of supportive periodontal therapy, SPT-1y: at 1 year under SPT, and SPT-3y: at 3 years under SPT). The present study has clarified our previous univariate analysis results, wherein P. gingivalis infection was positively associated with progression of atherosclerosis. Thus, routine screening using plasma IgG antibody titer against P. gingivalis and periodontal treatment under collaborative with medical and dental care may prevent cardiovascular accidents caused by atherosclerosis.

Malnutrition causes prolonged inflammation, resulting in delayed wound healing. High mobility group box-1 (HMGB1) is a damage-associated molecular pattern that is present in the nuclei of macrophages and is secreted into the extracellular milieu in response to stimuli. It stimulates the production of interleukin-1β (IL-1β) through the receptors for advanced glycation end products (RAGE), inducing an inflammatory response, which is an essential response to initiate wound healing. We hypothesized that malnutrition may interfere with this cascade, causing abnormal inflammation and ultimately delaying wound healing. We used tooth-extracted mice with malnutrition fed with low-casein diet for two weeks. On days 3 and 7 after tooth extraction, the wound tissue was histologically observed and analyzed for several factors in the inflammation-regeneration lineage, including IL-1β, mesenchymal stem cells, myeloperoxidase activity, HMGB1, macrophage polarization, and adenosine 5-triphosphate (ATP). On day 7, delayed wound healing was observed with the following findings under malnutrition conditions: decreased mRNA expression of genes for regeneration and mesenchymal stem cell (MSC) accumulation, an obvious increase in myeloperoxidase and IL-1β mRNA expression, an increase in HMGB1 levels, and an increase in ATP concentration in tissues with elevated proportion of M2 macrophages. These results suggest that the significantly increased secretion of HMGB1 associated with the upregulated production of ATP and IL-1β secretion via the RAGE pathway may interfere with the resolution of inflammation and wound healing under the state of malnutrition.

Cells behave in a variety of ways when they perceive changes in their microenvironment; the behavior of cells is guided by their coordinated interactions with growth factors, niche cells, and extracellular matrix (ECM). Modulation of the microenvironment affects the cell morphology and multiple gene expressions. Rho/Rho-associated coiled-coil-containing protein kinase (ROCK) signaling is one of the key regulators of cytoskeletal dynamics and actively and/or passively determines the cell fate, such as proliferation, migration, differentiation, and apoptosis, by reciprocal communication with the microenvironment. During periodontal wound healing, it is important to recruit the residential stem cells into the defect site for regeneration and homeostasis of the periodontal tissue. Periodontal ligament (PDL) cells contain a heterogeneous fibroblast population, including mesenchymal stem cells, and contribute to the reconstruction of tooth-supporting tissues. Therefore, bio-regeneration of PDL cells has been the ultimate goal of periodontal therapy for decades. Recent stem cell researches have shed light on intrinsic ECM properties, providing paradigm shifts in cell fate determination. This review focuses on the role of ROCK activity and the effects of Y-27632, a specific inhibitor of ROCK, in the modulation of ECM-microenvironment. Further, it presents the current understanding of how Rho/ROCK signaling affects the fate determination of stem cells, especially PDL cells. In addition, we have also discussed in detail the underlying mechanisms behind the reciprocal response to the microenvironment.

Key Clinical Message Oral bacteria cause infective endocarditis (IE), so severe periodontitis is thought to be high risk for IE. We suggest the identification of high‐risk patients by an IgG antibody titer test against periodontal bacteria might become common screening test.

Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, β4, and β6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, β4, and β6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.

The periodontal ligament (PDL) cells contain heterogeneous mesenchymal cell populations, which have the ability to differentiate into cells that produce adjacent mineralized tissues and abundant extracellular matrix (ECM). ECM is essential not only for the homeostasis of the periodontal tissue, but also for controlling the differentiation of the PDL cells. The process of differentiation involves mechanotransduction, which links the ECM to the cytoskeleton. The present study investigated the roles of Rho-associated coiled-coil containing protein kinase (ROCK) signaling, a crucial regulator of the cytoskeleton, during ECM-mediated osteogenic differentiation of PDL cells in vitro. The PDL cells were isolated from human periodontal ligaments of extracted teeth and cultured in osteogenic medium with or without Y-27632, a pharmacological inhibitor of ROCK. ECM-coated plates were used for ECM-mediated differentiation. The osteogenic phenotype was evaluated at different time points by real-time RT-PCR for the gene encoding alkaline phosphatase (ALP) and an ALP activity assay. The effects of ROCK on cytoskeletal changes and ECM synthesis were examined by immunofluorescence analysis. Y-27632 significantly inhibited ALP at the mRNA and protein activity levels in the late stage of differentiation; concomitantly, the actin filament content and the extracellular levels of collagen-I and fibronectin were markedly decreased by Y-27632. Exogenous collagen-I and fibronectin temporally increased ALP activity, with fibronectin showing a more pronounced effect. Importantly, ECM-mediated differentiation was almost completely inhibited by Y-27632. These findings indicated that ECM-mediated differentiation is dependent on ROCK signaling, and ROCK signaling contributes to the establishment of the ECM microenvironment for PDL cell differentiation.

Contamination of dental unit waterlines (DUWL) with heterotrophic bacteria can cause problems in immune compromised patients (aged, tumor and organ transplantation-patients). We focused on the use of low-concentrated ozonized water (OZW) as the biofilm formation restraint system for DUWL. Here, we examined the effects of low-concentrated OZW on the growth of bacteria and related biofilm formation and harmfulness to dental unit components (DUCs) in vitro. Objectives To evaluate the bactericidal effects of OZW on biofilms in DUWL and DUC in vitro. Methods Low-concentrated OZW (0.4 mg/L) was generated using an OZS-PTDX generator. Heterotrophic bacterial biofilms in old DUWL tubes and Candia albicans solution (control microbe) were treated with OZW for 1 h with gentle agitation before static culturing for 96 h in Reasoner's 2A liquid media. The control solutions were 0.1% cetylpyridinium chloride (CPC), chlorinated tap water (TW), and phosphate-buffered saline (PBS). Adenosine triphosphate (ATP) amounts of the microbes were measured and the biofilms of these microbes were observed using scanning electron microscopy (SEM). Moreover, surfaces of DUC soaked in OZW and TW were observed by SEM. Results The OZW reduced ATP levels in microbes to 50% compared to TW and PBS treatment, although CPC reduced it below detection limits. SEM observation revealed deformation of microbes cultured with OZW, whereas no changes were seen on DUC surfaces. Conclusions Low-concentrated OZW is bactericidal against heterotrophic bacteria biofilms and it is not harmful to DUC, suggesting that it might be useful in preventing DUWL contamination.

Objective Gingival epithelial cells play an important role in preventing the initiation of periodontitis, by their hemidesmosomal adhesion to the tooth root surface. Adhesion requires integrin-extracellular matrix (ECM) interactions that are intricately regulated by transforming growth factor-β (TGF-β) signaling. However, the mechanisms underlying the interplay between adhesion molecules and TGF-β, especially the respective roles of Smad2 and Smad3, remain elusive. In this study, we examined the effects of Smad overexpression on gingival epithelial cell adhesion and expression profiles of integrin and ECM-related genes. Methods Human gingival epithelial cells immortalized by the SV40 T-antigen were transfected with Smad2- and Smad3-overexpression vectors. A cell adhesion assay involving fluorescence detection of attached cells was performed using the ArrayScan imaging system. Real-time PCR was performed to examine the kinetics of integrin and ECM gene expression. In vitro and in vivo localization of adhesion molecules was examined by immunofluorescence analysis. Results By using SB431542, a specific inhibitor of the TGF-β type I receptor, Smad2/3 signaling was confirmed to be dominant in TGF-β1-induced cell adhesion. The Smad2-transfectant demonstrated higher potency for cell adhesion and integrin expression (α2, α5, β4, and β6) than the Smad3-transfectant, whereas little or no change in ECM expression was observed in either transfectant. Moreover, the gingival epithelium of transgenic mice that overexpressed Smad2 driven by the keratin 14 promoter showed increased integrin α2 expression. Conclusion These findings indicate the crucial role of Smad2 in increased adhesion of gingival epithelial cells via upregulation of integrin α2.

The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence.

Pathophysiological bone resorption is commonly associated with periodontal disease and involves the excessive resorption of bone matrix by activated osteoclasts. Receptor activator of nuclear factor (NF)-κB ligand (RANKL) signaling pathways have been proposed as targets for inhibiting osteoclast differentiation and bone resorption. The fungal secondary metabolite (+)-terrein is a natural compound derived from Aspergillus terreus that has previously shown anti-interleukin-6 properties related to inflammatory bone resorption. However, its effects and molecular mechanism of action on osteoclastogenesis and bone resorption remain unclear. In the present study, we showed that 10 µM synthetic (+)-terrein inhibited RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner and without cytotoxicity. RANKL-induced messenger RNA expression of osteoclast-specific markers including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), the master regulator of osteoclastogenesis, cathepsin K, tartrate-resistant acid phosphatase (Trap) was completely inhibited by synthetic (+)-terrein treatment. Furthermore, synthetic (+)-terrein decreased RANKL-induced NFATc1 protein expression. This study revealed that synthetic (+)-terrein attenuated osteoclast formation and bone resorption by mediating RANKL signaling pathways, especially NFATc1, and indicated the potential effect of (+)-terrein on inflammatory bone resorption including periodontal disease.

High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that is secreted into the extracellular milieu in response to inflammatory stimuli. The secreted HMGB1 mediates various inflammatory diseases, including periodontitis; however, the underlying mechanisms of HMGB1-induced periodontal inflammation are not completely understood. Here, we examined whether anti-HMGB1 neutralizing antibody inhibits periodontal progression and investigated the molecular pathology of HMGB1 in vitro and in vivo. In vitro analysis indicated that HMGB1, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-1β (IL-1β) were secreted in response to tumor necrosis factor-α (TNF-α) stimuli in human gingival epithelial cells (HGECs) and human monocytic leukemia cells (THP-1) treated with phorbol myristate acetate. Increased levels of GM-CSF and IL-1β were observed in the conditioned media from TNF-α-stimulated HGECs and THP-1 in vitro . Simultaneous stimulation with TNF-α and anti-HMGB1 antibody significantly decreased TNF-α-induced inflammatory cytokine secretion. Experimental periodontitis was induced in mice using Porphyromonas gingivalis -soaked ligatures. The extracellular translocation was confirmed in gingival epithelia in the periodontitis model mice by immunofluorescence analysis. Systemic administration of anti-HMGB1 neutralizing antibody significantly inhibited translocation of HMGB1. The anti-HMGB1 antibody inhibited periodontal inflammation, expression of IL-1β and C-X-C motif chemokine ligand 1 (CXCL1), migration of neutrophils, and bone resorption, shown by bioluminescence imaging of myeloperoxidase activity, quantitative reverse transcription-PCR (RT-PCR), and micro-computed tomography analysis. These findings indicate that HMGB1 is secreted in response to inflammatory stimuli caused by periodontal infection, which is crucial for the initiation of periodontitis, and the anti-HMGB1 antibody attenuates the secretion of a series of inflammatory cytokines, consequently suppressing the progression of periodontitis.

We aimed to evaluate molecular imaging as a novel diagnostic tool for mice periodontitis model induced by ligature and Porphyromonas gingivalis (Pg) inoculation. Twelve female mice were assigned to the following groups: no treatment as control group (n = 4); periodontitis group induced by ligature and Pg as Pg group (n = 4); and Pg group treated with glycyrrhizinic acid (GA) as Pg + GA group (n = 4). All mice were administered a myeloperoxidase (MPO) activity-specific luminescent probe and observed using a charge-coupled device camera on day 14. Image analysis on all mice was conducted using software to determine the signal intensity of inflammation. Additionally, histological and radiographic evaluation for periodontal inflammation and bone resorption at the site of periodontitis, and quantitative enzyme-linked immunosorbent assay (ELISA) were conducted on three mice for each group. Each experiment was performed three times. Levels of serum IgG antibody against P. gingivalis were significantly higher in the Pg than in the Pg + GA group. Histological analyses indicated that the number of osteoclasts and neutrophils were significantly lower in the Pg + GA than in the Pg group. Micro-CT image analysis indicated no difference in bone resorption between the Pg and Pg + GA groups. The signal intensity of MPO activity was detected on the complete craniofacial image; moreover, strong signal intensity was localized specifically at the periodontitis site in the ex vivo palate, with group-wise differences. Molecular imaging analysis based on MPO activity showed high sensitivity of detection of periodontal inflammation in mice. Molecular imaging analysis based on MPO activity has potential as a diagnostic tool for periodontitis.

High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that is secreted into the extracellular milieu in response to inflammatory stimuli. The secreted HMGB1 has been suggested to mediate various inflammatory diseases. However, it is still unknown whether HMGB1 is involved in a healing process in the tooth extraction socket, the tissue containing gingival epithelium, and alveolar bone that is exposed to oral bacteria. In this study, we constructed a murine tooth extraction model with anti-HMGB1 neutralization antibody administration and observed the inflammatory response and bone healing process in tooth extraction sockets by molecular imaging of myeloperoxidase (MPO) activity, histological analysis, and quantitative RT-PCR. The translocation of HMGB1 from the nucleus to the cytoplasm in gingival epithelial cells and inflammatory cells was inhibited by anti-HMGB1 antibody administration. The MPO activity around the tooth extraction socket was significantly reduced, and the numbers of CD31- and CD68-positive cells were significantly lower in the anti-HMGB1 antibody treatment samples than in the control samples. The TRAP-positive cells, osteocalcin positive cells, and the neoplastic bone area were significantly lower in anti-HMGB1 antibody treatment samples than in control samples. The expression levels of IL-1β and VEGF-A were also decreased in anti-HMGB1 antibody treatment samples compared to that in control samples. Secreted HMGB1 induced initial acute inflammation and inflammatory cells recruitment after tooth extraction. HMGB1 was associated with angiogenesis and bone remodeling by osteoclast and osteoblast activation and promoted bone healing in the tooth extraction socket.

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.

Serotonin (5-hydroxytryptamine: 5-HT) is recognized as a neurotransmitter in the central nerve system and as a regulator of systemic blood pressure in the peripheral tissues. Recently, it was reported that 5-HT2 receptors (5-HT2Rs) were expressed in cartilage tissues lacking both vessels and neurons, suggesting possible novel functions of 5-HT during cartilage development and regeneration. Our previous data indicated that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) plays a central role in cartilage development and regeneration. Therefore, the aim of this study was to investigate the effect of 5-HT on the production of CCN2 in chondrocytes. Firstly, we showed that the mRNAs of 5-HT2R subtypes 5-HT2AR and 5-HT2BR, were expressed in a human chondrocytic cell line, HCS-2/8; however, 5-HT2CR mRNA was not detected. In addition, exogenously added 5-HT did not affect the 5-HT2AR and 5-HT2BR expressions. Next, we demonstrated that CCN2 production was increased by treatment with a 5-HT2AR agonist and the combination of 5-HT and 5-HT2BR antagonist. In contrast, treatment with a 5-HT2BR agonist and the combination of 5-HT and 5-HT2AR antagonist decreased CCN2 production. Furthermore, we showed that phosphorylation of Akt and p38 MAPK were increased by treatment with 5-HT2AR agonist, and that phosphorylation of PKCε, PKCζ, ERK1/2 and JNK were increased by treatment with 5-HT2BR agonist. Finally, we found that 5-HT2AR was localized in the growth plate, whereas 5-HT2BR was localized in the articular cartilage. These findings suggest that 5-HT promotes CCN2 production through the 5-HT2AR in growth plates, and that it represses CCN2 production through the 5-HT2BR in articular cartilage for harmonized development of long bones.

Interleukin (IL)-6 is a proinflammatory cytokine that performs a wide variety of biological functions, including important roles in the progression of chronic inflammatory diseases such as periodontal disease. (+)-Terrein, a secondary bioactive fungal metabolite isolated from Aspergillus terreus, has various biological activities; however, its anti-inflammatory effects are still unknown. The purpose of this study was to examine the effect of synthetic (+)-terrein on IL-6 signaling and related protein production in human gingival fibroblasts. To our knowledge, this study is the first to report that synthetic (+)-terrein is not cytotoxic at concentrations less than 20 μM and suppresses IL-6/soluble IL-6 receptor (sIL-6R)-induced phosphorylation of signal transducer and activator of transcription-3, extracellular signal-regulated kinase 1/2, and c-jun N-terminal kinase 1/2-signaling proteins that are downstream of IL-6 signaling. In addition, synthetic (+)-terrein suppresses IL-6/sIL-6R-induced vascular endothelial growth factor (VEGF) secretion in a concentration-dependent manner (p0.01). These data suggest that synthetic (+)-terrein has potential anti-IL-6 signaling activity and suppresses VEGF-associated inflammatory disease progression.

It has been revealed that atherosclerosis and periodontal disease may have a common mechanism of “chronic inflammation”. Several reports have indicated that periodontal infection is related to atherosclerosis, but none have yet reported such an investigation through the cooperation of local clinics. This study was performed in local Japanese clinics to examine the relationship between periodontal disease and atherosclerosis under collaborative medical and dental care. A pilot multicenter cross-sectional study was conducted on 37 medical patients with lifestyle-related diseases under consultation in participating medical clinics, and 79 periodontal patients not undergoing medical treatment but who were seen by participating dental clinics. Systemic examination and periodontal examination were performed at baseline, and the relationships between periodontal and atherosclerosis-related clinical markers were analyzed. There was a positive correlation between LDL-C level and plasma IgG antibody titer to Porphyromonas gingivalis. According to the analysis under adjusted age, at a cut-off value of 5.04 for plasma IgG titer to Porphyromonas gingivalis, the IgG titer was significantly correlated with the level of low-density lipoprotein cholesterol (LDL-C). This study suggested that infection with periodontal bacteria (Porphyromonas gingivalis) is associated with the progression of atherosclerosis. Plasma IgG titer to Porphyromonas gingivalis may be useful as the clinical risk marker for atherosclerosis related to periodontal disease. Moreover, the application of the blood examination as a medical check may lead to the development of collaborative medical and dental care within the local medical clinical system for the purpose of preventing the lifestyle-related disease.

Porphyromonas gingivalis is a major etiological agent of periodontal diseases and the outer membrane vesicles (OMVs) contain virulence factors such as LPS and gingipains. In this study, we investigated the potential role of the OMVs in host immune response and tissue destruction during P. gingivalis infection. Firstly, we found that sera from periodontitis patients had significantly stronger reactivity against an OMV-producing wild type strain than the isogenic OMV-depleted strain. OMVs were found to be highly antigenic, as absorption of patient sera with OMVs greatly reduced reactivity with whole cells of P. gingivalis. LC-MS/MS analysis of OMVs revealed multiple forms of gingipains and several gingipain-related proteins. Western blots of OMVs using patient sera revealed a conserved immunoreactive antigen profile resembling the profile of OMV antigens that were recognized by gingipain antiserum, suggesting a potential role of OMV-associated gingipains in triggering antibody-mediated immune responses to P. gingivalis infection. When OMVs were added to a monolayer of an oral squamous epithelial cell line, OMVs caused cell detachment, which was inhibited by preincubating OMVs with anti-gingipain antiserum. These data suggest that gingipain-laden OMVs may contribute to tissue destruction in periodontal diseases by serving as a vehicle for the antigens and active proteases.

[Background] : Eosinophil cationic protein (ECP) was reported previously to be involved in allergic inflammation with cytotoxic activity. On the other hand, recent studies showed that ECP did not induce cell death but inhibited the growth of cancer-derived cells. Our previous study indicated that human ECP enhanced differentiation of rat neonatal cardiomyocytes and stress fiber formation in Balb/c 3T3 mouse fibroblasts, while the effects of human ECP on human fibroblasts are unknown. [Objective] : The present study was performed to determine the effects of human ECP on cytokine expression in human fibroblasts by protein array. [Methods] : The effects of recombinant human ECP (rhECP) on normal human dermal fibroblasts (NHDF) were examined by assaying cell growth. Furthermore, cytokine expression of NHDF stimulated by ECP, which could influence cell growth, was evaluated by protein array. [Results] : ECP was not cytotoxic but enhanced the growth of NHDF. The peak rhECP concentration that enhanced the cell counts by 1.56-fold was 100 ng/mL, which was significantly different from cultures without ECP stimulation (ANOVA/Scheffe’s test, P < 0.05). Array analyses indicated that ciliary neurotrophic factor (CNTF), neutrophilactivating peptide (NAP)-2, and neurotrophin (NT)-3 were significantly upregulated in NHDF stimulated with 100 ng/mL ECP compared to those without stimulation. [Conclusion] : ECP is not cytotoxic but enhances the growth of NHDF. CNTF, NAP-2, and NT-3 were suggested to be involved in enhancing the growth of NHDF. These findings will contribute to determination of the role of ECP in allergic inflammation. (Asian Pac J Allergy Immunol 2013;31:271-6)

Background and Objective Spatiotemporal inhibition of apical migration and proliferation of gingival epithelium are significant factors involved in periodontal regeneration. Transforming growth factor β (TGF-β) is important in multiple aspects of wound healing, and Smad2, a downstream transcription factor of TGF-β, has an inhibitory effect on re-epithelialization during gingival wound healing. Therefore, we investigated the effects on migration and proliferation status, and intra/extracellular signaling regulated by Smad2 overexpression in gingival epithelial cells. Material and Methods Gingival epithelial cells were isolated from the palatal gingival tissue of transgenic mice overexpressing Smad2 driven by the Keratin14 promoter. Smad2 expression was identified by western blotting and immunofluorescence analysis. Scratch assay and 5-bromo-2′-deoxyuridine staining were performed to assess cell migration and proliferation. To inactivate TGF-β type I receptor, the cultures were supplemented with SB431542. Secreted TGF-β was quantified by ELISA. Smad2 target gene expression was examined by real-time RT-PCR and in vivo immunofluorescence analysis of gingival junctional epithelium. Results Smad2-overexpressing cells were confirmed to have significant phosphorylated Smad2 in the nucleus. Scratch assay and 5-bromo-2′-deoxyuridine staining indicated that Smad2-overexpressing cells showed no significant differences in migration, but had reduced proliferation rates compared to wild-type controls. SB431542 significantly inhibited Smad2 phosphorylation, which coincided with restoration of the proliferation rate in Smad2-overexpressing cells. ELISA of TGF-β release did not show any differences between genotypes. The cell cycle inhibitors, p15 and p21, showed significant upregulation in Smad2-overexpressing cells compared to wild-type controls. Moreover, junctional epithelium of the transgenic mice showed increased expression of P-Smad2, p15 and p21. Conclusion The signaling activation triggered by overexpression of Smad2 was dependent on TGF-β type I receptor, and the activated Smad2 increased p15 and p21 expression, responsible for inhibiting cell cycle entry, resulting in antiproliferative effects on gingival epithelial cells. Understanding of Smad2-induced signaling would be useful for possible clinical application to regulate gingival epithelial downgrowth.

Both group I (HSP60) and group II (CCT) chaperonins are targets of autoantibodies. Autoimmune reactions to HSP60 have been well characterized, while immune reactions to group II chaperonin have not been clarified. Methanobrevibacter oralis is a suspected periodontal pathogen with group II chaperonin. In this study, serum responses to M. oralis chaperonin, human HSP60, and CCT subunits were examined using sera from patients with periodontitis and autoimmune diseases. In comparison with healthy controls, periodontitis patients showed significantly higher responses to CCT4 and CCT8 on dot blot analysis. Signals for CCT3 and CCT8 in autoimmune disease patients were significantly higher than in controls. Significant differences were also demonstrated by Western blotting in anti-CCT4 response in both patient groups. All subjects showed strong reactivity to M. oralis chaperonin and faint signals to human HSP60. Autoantibodies were raised against CCT rather than HSP60; and CCT3, CCT4, and CCT8 were shown to be the main targets. Host immune systems may be frequently exposed to chaperonins of Archaea in various habitats. Although further studies of the cross-reactivity between M. oralis chaperonin and human CCT are required, anti-CCT autoantibodies may be involved in the pathogenesis of periodontitis and autoimmune diseases.

Summary Recent advances in molecular biological techniques have yielded large amounts of information regarding the oral microflora. The microbiological communities were shown to be more diverse than previously thought and to include a number of previously uncharacterized microorganisms. The range of research targets of microorganisms associated with oral diseases has been expanded to include these unknown or uncharacterized organisms. These organisms include the Archaea. A series of recent reports suggested these microorganisms to be potential pathogens involved in periodontitis and apical periodontitis mainly based on the detection frequency or their increased numbers in diseased sites in association with the severity or symptoms of disease. However, it cannot be concluded that Archaea are oral pathogens based on such circumstantial evidence. Further studies are required to investigate the potential pathogenic mechanisms of action of these organisms. This will require investigation of the antigenic properties of the Archaea and synergism with other established oral pathogens. Especially, studies of the host immune response will provide insight into the medical impact of Archaea as suspected pathogens.

We previously isolated rat 14.7K-interacting protein-2 (rFIP-2) from the rat-wounded pulp. The protein, homologous to human FIP-2, is known as optineurin and was initially identified as a novel tumor necrosis factor-α (TNF-α)-inducible protein, and more recently, as an autophagy receptor. However, the biological role of optineurin in dental pulp remains elusive. We hypothesized that optineurin has a crucial role in regulating molecular processes during pulp inflammatory responses induced by TNF-α. We examined the kinetics of optineurin expression in pulp inflammation. Optineurin localization and expression were examined using rat pulp fibroblasts. The cells were treated with pharmacological inhibitors for TNF-α-induced inflammatory signals or with hydrogen peroxide as apoptotic stimuli. Stable optineurin-knockdown cells (OPTN-KD cells) were established by transfecting normal rat kidney cells with a vector expressing optineurin-specific small interfering RNA. Cell proliferation and the profiles of cytokines and intracellular signaling molecules were examined using OPTN-KD cells stimulated by TNF-α. Optineurin was localized in the cytoplasm and then translocated into the nucleus upon apoptotic stimuli. Optineurin expression was increased by TNF-α and decreased by a specific inhibitor of c-Jun N-terminal kinase. The OPTN-KD cells secreted smaller amounts of monocyte chemotactic protein-1 (MCP-1) and intracellular MCP-1 mRNA, and cell proliferation was significantly increased. Apoptosis-related signaling molecules were downregulated in OPTN-KD cells. These results demonstrated that optineurin is a crucial molecule mediated by TNF-α, which induces the production of inflammatory factors and apoptosis signaling, suggesting the presence of signaling interactions between optineurin and a transcription factor for MCP-1.

Background The diagnosis of the progression of periodontitis presently depends on the use of clinical symptoms (such as attachment loss) and radiographic imaging. The aim of the multicenter study described here was to evaluate the diagnostic use of the bacterial content of subgingival plaque recovered from the deepest pockets in assessing disease progression in chronic periodontitis patients. Methods This study consisted of a 24-month investigation of a total of 163 patients with chronic periodontitis who received trimonthly follow-up care. Subgingival plaque from the deepest pockets was recovered and assessed for bacterial content of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans using the modified Invader PLUS assay. The corresponding serum IgG titers were measured using ELISA. Changes in clinical parameters were evaluated over the course of 24 months. The sensitivity, specificity, and prediction values were calculated and used to determine cutoff points for prediction of the progression of chronic periodontitis. Results Of the 124 individuals who completed the 24-month monitoring phase, 62 exhibited progression of periodontitis, whereas 62 demonstrated stable disease. The P. gingivalis counts of subgingival plaque from the deepest pockets was significantly associated with the progression of periodontitis (p

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients (ClinicalTrials.gov number NCT01658475).

During periodontal regeneration, inhibition of gingival downgrowth is necessary to promote migration of mesenchymal cells into the defects. Transforming growth factor (TGF)-β is a pleiotropic cytokine that has numerous cell functions, including regulation of epithelial growth. Recent studies have shown that Smad2, a downstream transcription factor of TGF-β, plays crucial roles in wound healing in the epithelia. Therefore, we investigated the effects of Smad2 overexpression on re-epithelialization of gingival wounds. Transgenic mice overexpressing smad2 driven by the keratin 14 promoter ( k14-smad2) were confirmed to have significant Smad2 phosphorylation in gingival basal epithelia. Punch wounds were made in the palatal gingiva, and wound healing was assessed histologically for 7 days. Re-epithelialization was significantly retarded on day 2, while collagen deposition was enhanced on day 7 in k14-smad2 compared with wild-type mice. Moreover, expression of keratin 16 (K16), an indicator of keratinocyte migration, was significantly inhibited in wound-edge keratinocytes in k14-smad2. The inhibition of K16 coincided with the induction of Smad2 in the corresponding epithelia, while BrdU incorporation was unaffected. These results indicated that Smad2 has inhibitory effects in regulating keratinocyte migration during gingival wound healing. TGF-β/Smad2 signaling mediating alteration of K16 expression must be tightly regulated during periodontal regeneration.

Chronic periodontitis is associated with systemic diseases such as atherosclerosis. In this study, we evaluated the efficacy of serum IgG antibody titer to periodontal bacteria for prognosis of periodontitis recurrence during supportive periodontal therapy (SPT) phase. The 139 patients during SPT phase were selected and divided to two groups as follows: “Stable” and “Recurrence” group at SPT phase for case–control study: “High IgG titer” and “Normal IgG titer” group before transition to SPT phase for cohort study. We examined whether clinical findings or serum IgG antibody titers to periodontal bacteria are risk factors for the development of periodontitis recurrence. Case–control study showed thatthere were significant differences between the stable and recurrence groups in age and number of teeth. The serum IgG antibody titer to Eikenella corrodens FDC1073, Porphyromonas gingivalis SU63, and Campylobacter rectus ATCC33238 was significantly higher in the recurrence group. Next, we found, that the recurrence ratio in the high IgG titer group to Gram‐negative obligate anaerobe, Prevotella intermedia, Treponema denticola, and C. rectus was significantly higher than that of the normal IgG titer group. Taken together, serum IgG antibody titer test is useful in the prognosis of periodontitis recurrence during the SPT phase. J. Clin. Lab. Anal. 25:25–32, 2011. © 2011 Wiley‐Liss, Inc.

Control of bacterial infection-induced inflammatory responses is one of the effective therapeutic approaches of periodontal diseases. Natural products such as lipid mediators and metabolites from microorganisms have been used for decreasing inflammation. We previously reported that (+)-terrein inhibited activation of STAT3 and ERK1/2 in interleukin-6 (IL-6) signaling cascade, leading to prevent vascular endothelial growth factor (VEGF) secretion in human gingival fibroblasts (HGFs). However, little is still known about the role of (+)-terrein on inflammatory responses. In this study, we provided the possibility of novel action that (+)-terrein inhibits activation of Janus-activated kinase 1 (JAK1), which has a central function in IL-6 signaling cascade, and alters expression of mRNAs and proteins induced by IL-6/soluble IL-6 receptor (sIL-6R) stimulation in HGFs. First, we performed PCR array to examine IL-6/sIL-6R-induced mRNA expression, and then expression of mRNA and protein of colony stimulating factor-1 (CSF1) and VEGF were clearly determined by quantitative RT-PCR and ELISA, respectively. Treatment with (+)-terrein suppressed expression of mRNA and protein of CSF1 and VEGF by IL-6/sIL-6R stimulation. Next, to test the effect of (+)-terrein on IL-6/sIL-6R signaling cascade, we demonstrated whether (+)-terrein affects phosphorylation of JAK1 and its downstream proteins, Akt and SHP-2. Western blotting revealed that (+)-terrein inhibited IL-6/sIL-6R-induced phosphorylation of JAK1, Akt, and SHP-2. Therefore, (+)-terrein suppresses IL-6/sIL-6R-induced expression of CSF1 and VEGF via inhibition of JAK1, Akt, and SHP-2. Based on our results, we suggest that (+)-terrein is a candidate compound for anti-inflammatory effect associated with IL-6 signaling.

In this study we attempted to clarify the mechanism of the inhibitory effects of PGE 2 on DNA synthesis in Gin-1 (fibroblasts derived from healthy human gingiva) from the aspect of the cyclic AMP-dependent protein kinase signal transduction pathway. PGE 2 upregulated intracellular cyclic AMP accumulation and inhibited DNA synthesis in Gin-1 in a dose-dependent manner. When the PGE 2 -induced intracellular cyclic AMP accumulation was further enhanced by treatment with the cyclic AMP-phosphodiesterase inhibitor, IBMX, the inhibitory effect of PGE 2 on DNA synthesis was also enhanced. Furthermore, when we examined the effects of forskolin, an activator of cyclic AMP production, on intracellular cyclic AMP accumulation and DNA synthesis, similar results were obtained. However, inhibitors of cyclic AMP-dependent protein kinase (protein kinase A) such as HA1004 did not diminish the inhibitory effect of PGE 2 on DNA synthesis in Gin-l. These results suggest that in Gin-l, PGE 2 -induced cyclic AMP accumulation may not lead to the activation of protein kinase A or protein kinase A activity may not relate directly to the growth inhibitory effect of PGE 2 , and that PGE 2 does not inhibit DNA synthesis through the cyclic AMP-protein kinase A signal transduction pathway in Gin-1.

Methanobrevibacter oralis is an archaeal species frequently isolated from sites of severe periodontitis. However, its pathogenic roles remain unclear. Here, we aimed to isolate group II chaperonin from M. oralis and examine its antigenicity. The genes encoding two chaperonin subunits (Cpn-1 and Cpn-2) were cloned from M. oralis using polymerase chain reaction and genome walking procedures. Recombinant proteins Cpn-1 and Cpn-2 were generated, and the reactivities of sera from patients with periodontitis were examined by Western immunoblotting. The open reading frames of Cpn-1 and Cpn-2 genes consisted of 1641 and 1614 base pairs, respectively. Putative ATP-binding domains conserved among the chaperonin family were observed in both genes. The deduced amino acid sequences of the two genes showed 28.8-40.0% identity to each of the subunits of human CCT (CCT1-8). Thirty and 29 of 36 patients' sera reacted with the recombinant Cpn-1 and recombinant Cpn-2, respectively. Western immunoblotting using antiserum against human CCT subunits indicated that anti-CCT3 and anti-CCT8 antibodies recognized recombinant Cpn-1. In addition, anti-CCT1, CCT3, CCT6, and CCT8 antibodies recognized an antigen of approximately 60 kDa in M. oralis. The results suggested that the chaperonin subunits of M. oralis were antigenic molecules that were recognized by periodontitis patients and that may cross-react with human chaperonin CCT.

Aggregatibacter actinomycetemcomitans, a potent pathogen of periodontitis, typically grows as a rough and adherent colony on primary isolated cultures. The colony transforms into a smooth phenotype during repeated subculture. In this study, we aimed to identify highly expressed genes in the rough-colony-forming phenotype for isolation of host-induced genes. Using a cDNA-subtractive hybridization technique, three genes, homologous to a macrophage infectivity potentiator gene (mip), peroxiredoxin gene (prx) and outer membrane protein gene (ompA), were identified. The expression levels of these genes in the rough-colony-forming phenotype were 4-10-fold higher as compared with the smooth-colony-forming phenotype. Attention was focused on the mip-like gene, and a recombinant protein and a deficient mutant were constructed. The recombinant protein reacted with sera from patients with periodontitis, suggesting the production of the Mip-like protein in periodontal lesions. Viable quantitative invasion assay demonstrated that the viable cell counts of the wild-type strain that invaded HeLa cells were more than fourfold as compared with the mip-deficient mutant. The expression of the mip-like gene, prx-like gene and ompA-like gene may be enhanced in the host, and the mip-like gene may play an important role in the infection of A. actinomycetemcomitans, especially in its invasion of the epithelium.

A genome-association study is a powerful tool for analyzing small gene effects in complex diseases such as chronic periodontitis (CP), although the cost of analysis is prohibitive. We designed a study using the DNA pooling method, which could be a breakthrough in lowering such costs. This study was conducted to assess the genetic association in severe CP in a Japanese population.We adopted a DNA pooling method by genotyping 454 densely spaced microsatellite (MS) markers in chromosome 19 as a pilot study, with the possibility of future use in a whole-genome study. This can reduce the high cost and technical burden, which is generally unavoidable in a genomic association study. Pooled DNA samples from 300 case subjects, 300 control subjects, and 200 systemically healthy subjects were screened by genotyping MS markers. The case-control association in the candidate region was analyzed by individual typing of MS and single nucleotide polymorphisms (SNPs).The single MS marker allele 17 of 1902G31 was isolated in association with severe CP (P = 0.0012 for 2 x 2; P0.046 for 2 x m, where m refers to the number of polymorphic alleles observed in a population). No other SNP or MS polymorphism hypothesized to affect biologic functions in the critical region was found in the linkage disequilibrium block analysis.We efficiently isolated the susceptible locus for severe CP in chromosome 19 and identified a useful marker to evaluate the risk for disease. This approach can be applied to a whole-genome study in severe CP.

Objective Biomaterials with anti-microbial properties are highly desirable in the oral cavity. Ideally, bactericidal molecules should be immobilized within the biomaterial to avoid unwanted side-effects against surrounding tissues. They may then however loose much of their antibacterial efficiency. The aim of this study was to investigate how much antibacterial effect an immobilized bactericidal molecule still has against oral bacteria. Methods Experimental resins containing 0, 1 and 3% cetylpyridinium chloride (CPC) were polymerized, and the bacteriostatic and bactericidal effects against Streptococcus mutans were determined. Adherent S. mutans on HAp was quantitatively determined using FE-SEM and living cells of S. mutans were quantified using real-time RT-PCR. The amount of CPC released from the 0%-, 1%- and 3%-CPC resin sample into water was spectrometrically quantified using a UV–vis recording spectrophotometer. Results UV spectrometry revealed that less than 0.11 ppm of CPC was released from the resin into water for all specimens, which is lower than the minimal concentration generally needed to inhibit biofilm formation. Growth of S. mutans was significantly inhibited on the surface of the 3%-CPC-containing resin coating, although no inhibitory effect was observed on bacteria that were not in contact with its surface. When immersed in water, the antibacterial capability of 3%-CPC resin lasted for 7 days, as compared to resin that did not contain CPC. Significance These results demonstrated that the bactericidal molecule still possessed significant contact bacteriostatic activity when it was immobilized in the resin matrix.

OBJECTIVE: To understand the effects of aging on cellular motility of human periodontal ligament (PDL) cells, and to determine the possible link between cell proliferation and migration in relation to cellular aging. MATERIALS AND METHODS: The chemotactic response of PDL cells from three juvenile and four older donors were compared. Then, migrated or unmigrated cells were examined for their cell cycle by morphological and immunocytochemical procedureS. Finally, migrated or unmigrated cells were examined for the expression of c-fos and c-myc by in situ hybridization. RESULTS: PDL cells from older donors showed lower chemotaxis compared with the cells from juvenile donors (P < 0.05).Cells undergoing migration were found not to be in the S- or M-phase of the cell cycle. However, all migrated cells were found to express c-fos, while many of the cells which did not migrate were found not to express c-fos. CONCLUSIONS: Cellular motility of PDL cells decreases with donor age as well as cell proliferation. Since the cells reaching senescence fail to express c-fos, the mechanisms linked to cellular senescence may be a possible underlying mechanism for low migration seen in the older cells.

OBJECTIVE: The aim of this study is to compare the in vitro proliferative capacity of periodontal ligament (PDL) cells from aged and juvenile donors. MATERIALS AND METHODS: Flow-cytometric analysis of the cell cycle was used to compare the length of each cell cycle, and the ratio of the cells progressing through the cycles between four PDL cells from juvenile donors and four cells from aged donors. Then, replicative capacity of the PDL cells from three juvenile and three aged donors was compared by serial cultureS. Finally, expression of c-fos was compared between cells proliferating and cells which had reached senescent. RESULTS: Flow-cytometric analysis of the cell cycle had revealed that although there were no differences in the length of each phase of the cell cycle, significant differences were found in the ratio of the cells entering from Gap 1 to DNA synthesis phase of the cell cycle (P< 0.025).Replicative capacity was much longer in two cells from juvenile donors (about 20 population doublings), while all cells from aged donors showed short dividing abilities (less than eight population doublings), hence entered senescent phases shortly. Additionally, no c-fos was detected in cells which had reached senescence upon stimulation with serum. CONCLUSIONS: It is generally believed that aged humans have an impaired wound healing ability. We believe that more fibrotic PDL tissues seen in aged humans might be the reason for this, and suggest that this phenomena might be due to the progressive accumulation of senescent cell populations.

Goals The commercially available saliva substitute Oralbalance (R) has been reported to alleviate symptoms of post-radiotherapy xerostomia in head and neck cancer patients. Oralbalance (R) may also be effective for xerostomia in patients undergoing hematopoietic cell transplantation (HCT) with high-dose chemotherapy and total-body irradiation. However, HCT patients are severely compromised, and saliva substitute must therefore not promote infection. This study was performed to determine the effects of Oralbalance (R) on microbial species identified during HCT. Patients and methods Microbial identification of oral mucosa was performed in 28 patients undergoing HCT. The antimicrobial effects of Oralbalance (R) against bacteria and fungi detected in the HCT period were examined in vitro. Briefly, bacteria and fungi were spread on agar plates, and 0.1g of Oralbalance (R) gel was applied (about phi 1cm). After incubation at 37 degrees C for 24h, the presence of a transparent zone of inhibition around Oralbalance (R) was observed. Main results Not only bacterial species constituting normal flora of the oral mucosa but also those not usually constituting normal flora, e.g., coagulase-negative Staphylococcus, were detected. A transparent zone was observed around Oralbalance (R) in all bacterial species examined. No transparent zone was observed for Candida albicans, but growth was inhibited in the area where Oralbalance (R) was applied. Conclusions Oralbalance (R) does not facilitate increases in microorganisms in the HCT period. Oral care with Oralbalance (R) does not promote infection in patients undergoing HCT.

Background: Dentists generally recognize the importance of periodontal treatment inpatients with leukemia, with the most attention paid to preventing the development of odontogenic infection. For physicians, the worst type of infection is one caused by multidrug-resistant bacteria. Here, we report a patient with an abnormal increase in multidrug-resistant opportunistic bacteria in the gingiva during hematopoietic cell transplantation (HCT). Methods: A 53-year-old woman receiving HCT for leukemia had an insufficient blood cell count for invasive periodontal treatment before HCT. Even brushing caused difficulties with hemostasis. Therefore, frequent pocket irrigation and local minocycline administration were performed. Results: The multidrug-resistant opportunistic bacterium Stenotrophomonas maltophilia was detected first in phlegm 2 days before HCT, and it was detected in a gingival smear and a blood sample 7 and I I days after HCT, respectively. The patient developed sepsis on day I I and died 14 days after HCT. Frequent irrigation and local antibiotic application were ineffective against S. maltophilia on the gingiva. Inflammatory gingiva without scaling and root planing showed bleeding tendency, and this interfered with the eradication of this bacterium. Conclusions: The gingiva in patients undergoing leukemia treatment acts as sites of proliferation and reservoirs for multidrug-resistant opportunistic bacteria. Severe systemic infection by multidrug-resistant bacteria in such patients with leukemia also may involve the gingiva. To prevent abnormal increases in such bacteria on the gingiva, scaling and/or root planing before chemotherapy, which reduces bleeding on brushing during the neutropenic period caused by chemotherapy, may contribute to infection control in such patients, although it was impossible in this case.

Objective: Recent studies suggested macrophages were integrated in adipose tissues, interacting with adipocytes, thereby exacerbating inflammatory responses. Persistent low-grade infection by gram-negative bacteria appears to promote atherogenesis. We hypothesized a ligand for toll-like receptor 4 (TLR4), bacterial lipopolysaccharide (LPS), would further exaggerate macrophage-adipocyte interaction. Research Methods and Procedures: RAW264.7 macrophage cell line and differentiated 3T3-L1 preadipocytes were co-cultured using transwell system. As a control, each cell was cultured independently. After incubation of the cells with or without Escherichia coli LPS, tumor necrosis factor (TNF)-α and interleukin (IL)-6 production was evaluated. Results: Co-culture of macrophages and adipocytes with low concentration of Escherichia coli LPS (1 ng/mL) markedly up-regulated IL-6 production (nearly 100-fold higher than that of adipocyte culture alone, p < 0.01), whereas TNF-α production was not significantly influenced. This increase was partially inhibited by anti-TNF-α neutralizing antibody. Recombinant TNF-α and LPS synergistically up-regulated IL-6 production in adipocytes. However, this increase did not reach the level of production observed in co-cultures stimulated with LPS. Discussion: A ligand for TLR-4 stimulates macrophages to produce TNF-α. TNF-α, thus produced, cooperatively up-regulates IL-6 production with other soluble factors secreted either from adipocytes or macrophages in these cells. Markedly up-regulated IL-6 would greatly influence the pathophysiology of diabetes and its vascular complications.

Cyclosporin A (CsA) is an immunosuppressant with severe side effects including gingival overgrowth. We have previously reported that CsA impairs the activity of the lysosomal enzymes cathepsin B and L in human gingival fibroblasts (HGFs). Here, we have examined the effects of CsA on the DNA-binding activity of the cyclic AMP response element-binding protein (CREB) and cell viability, and the effects of CREB on cathepsin B and L synthesis and activity in HGFs. We have confirmed that CsA down-regulates cathepsin B and L synthesis. Further, CsA has no effect on cell viability and dramatically impairs CREB-DNA binding activity. Importantly, the synthesis of cathepsin B and L is down-regulated, and their activity is also significantly impaired in HGFs transfected with plasmid expressing dominant-negative CREB. These results suggest that CREB is essential for the CsA-mediated down-regulation of cathepsin B and L synthesis in HGFs.

The purpose of this study was to profile gene expression in periapical lesions during root canal treatment (RCT). Periapical lesions were induced experimentally by exposing the pulp in Sprague-Dawley rats. After 3 wk, the animals received root canal filling (RCF) and were sacrificed 1 or 4 wk later. From the periapical tissues, total RNA was extracted and processed for cDNA-microarray analysis. The lesions were histologically and radiographically confirmed to expand 4 wk after pulp exposure (inflammation phase) and to stabilize 4 wk after RCF (healing phase). In approximately 30,000 genes on the microarray, 203 genes were up-regulated to more than 5-fold (e.g., IL-1beta), and 864 genes were down-regulated to less than 20% of baseline level (e.g., caspase 8) in inflammation phase. Compared with inflammation phase, we found that 133 genes were up-regulated (e.g., IL-1alpha) and 50 genes were down-regulated (e.g., defensin alpha5) in healing phase. Corresponding to the gene expression profiles, accumulation of IL-1alpha and IL-1beta was observed in the periapical lesions by immunohistochemistry. These gene profiles might be useful in diagnosing the healing process of periapical lesions.

Summary Several reports have demonstrated a possible association of periodontal infections with coronary heart disease (CHD) by elevated antibody titre to periodontopathic bacteria in CHD patients compared with non-diseased controls. Although each periodontopathic bacterium may vary in virulence for periodontitis and atherosclerosis, antibody response to multiple bacteria in CHD patients has not been understood fully. Therefore, serum levels of antibody to 12 periodontopathic bacteria together with other atherosclerotic risk markers were compared among 51 patients with CHD, 55 patients with moderate to severe chronic periodontitis and 37 healthy individuals. The antibody response was the most prevalent for Porphyromonas gingivalis, a major causative organism, in CHD as well as periodontitis patients. However, antibody positivity was different between CHD and periodontitis if the response was analysed for two different strains of P. gingivalis, namely FDC381 and Su63. While periodontitis patients were positive for both P. gingivalis FDC381 and Su63, a high frequency of antibody positivity for P. gingivalis Su63 but not for FDC381 was observed in CHD patients. The results indicate that the presence of particular periodontopathic bacteria with high virulence may affect atherogenesis. Identifying the virulence factors of P. gingivalis Su63 may gain insight into the new therapeutic modality for infection-induced deterioration of atherosclerosis.