Serum γ-glutamyltransferase level is associated with periodontal disease independent of drinking habits in Japanese adults.

Background Non-alcoholic fatty liver disease is considered a hepatic manifestation of metabolic syndrome. Periodontal disease is a mild chronic inflammatory disease with systemic effects, and many studies have indicated an association between metabolic syndrome and periodontitis. In the present study, we investigated the relationship between periodontitis and liver biochemical parameters according to alcohol drinking habits through a cross-sectional study based on data from Japanese people in occupational settings. Material and Methods The subjects were 1510 employees (1218 males, 292 females, mean age 50.4 years) who underwent dental and medical checkups in 2012. Associations between the presence of periodontal pockets and serum levels of liver biochemical parameters were assessed. Results Alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) levels were higher in subjects with than without periodontal pockets. Multiple logistic regression analysis (adjusting for age, gender, cigarette smoking, and alcohol drinking habits, and components of metabolic syndrome) with GGT or ALT as the dependent variable revealed that there was a significant association between periodontal pockets and GGT (odds ratio, OR=1.48), but not ALT. Similar associations were observed when an analysis was performed according to the presence or absence of alcohol drinking habits; the OR was higher in subjects without (OR=1.84) than with drinking habits (OR=1.41). Conclusions The presence of periodontal pockets was associated with serum levels of GGT, a liver biochemical parameter, in Japanese adults with no drinking habit, suggesting that periodontal disease is associated with liver function, independent of alcohol ingestion.

Interleukin-17F affects cartilage matrix turnover by increasing the expression of collagenases and stromelysin-1 and by decreasing the expression of their inhibitors and extracellular matrix components in chondrocytes.

Interleukin (IL)-17, a proinflammatory cytokine, is produced primarily by activated Th17 cells. IL-17 consists of six ligands that signal through five receptors (IL-17Rs); IL-17A and IL-17F share the highest homology in the family. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during cartilage remodeling whereas tissue inhibitor of metalloproteinases (TIMPs) inhibit the action of MMPs. In the present study, we examined the effect of IL-17F on the degradation and synthesis of the extracellular matrix in cartilage using human articular chondrocytes. We examined the effect of IL-17F on the expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and cyclooxygenases (COXs), as well as on prostaglandin E2 (PGE2) production. We also examined the indirect effect of PGE2 on the above IL-17F-induced/reduced components using NS-398, a specific inhibitor of COX-2. Cells were cultured with or without IL-17F in the presence or absence of either an IL-17R antibody or NS-398 for up to 28 days. Expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and COXs at mRNA and protein levels was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. PGE2 production was determined by ELISA. The expression of all types of IL-17Rs was detected in chondrocytes. However, IL-17RE expression was extremely low, compared with other IL-17Rs. The expression of MMP-1, MMP-3, MMP-13, and COX-2 as well as PGE2 production were increased by addition of IL-17F, whereas the expression of IL-17RD, TIMP-2, TIMP-4, type II collagen, aggrecan, link protein, and COX-1 was decreased. The expression of IL-17RA, IL-17RB, IL-17RC, MMP-2, MMP-14, TIMP-1, and TIMP-3 was unaffected by addition of IL-17F. The IL-17R antibody blocked the stimulating/reducing effect of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, aggrecan, and link protein. NS-398 blocked the reducing effect of IL-17F on aggrecan expression, whereas it did not completely block the stimulating/reducing effects of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, and link protein. Our results suggest that IL-17F stimulates cartilage degradation by increasing the expression of collagenases (MMP-1 and -13) and stromelysin-1 (MMP-3) and by decreasing expression of their inhibitors (TIMP-2 and -4), type II collagen, aggrecan, and link protein in chondrocytes. Furthermore, our results suggest that the expression of aggrecan, link protein, and TIMP-4 decrease through the autocrine action of PGE2 in chondrocytes.

Association between periodontal disease and metabolic syndrome.

OBJECTIVES: Metabolic syndrome is a complex medical disorder characterized by visceral fat-type obesity involving hypertension, and abnormal glucose and lipid metabolism. The objective of this study was to investigate the relationship between periodontal disease and components of metabolic syndrome (obesity, lipid abnormality, hypertension, and hyperglycemia) in industrial workers of a single company in Tokyo, Japan. METHODS: The study subjects consisted of 2478 adult employees (2028 men and 450 women; mean age: 43.3 years). The association between the presence of periodontal pockets and components of metabolic syndrome was investigated cross-sectionally using multiple logistic regression analysis, odds ratios (ORs), and 95 percent confidence intervals (CIs). RESULTS: Body mass index, blood pressure, triglycerides, fasting blood glucose, and hemoglobin A1c (HbA1c) were significantly elevated (P < 0.05) in patients with periodontal pockets of 4 mm or more. We found that the OR of the presence of periodontal pockets adjusted for age, gender, and smoking habit was 1.8 (96 percent CI = 1.4-2.3) when the subjects with two positive components and without positive component were compared. And it was 2.4 (96 percent CI = 1.7-2.7) when the subjects with three or four positive components and without positive component were compared. CONCLUSIONS: Our findings suggest an association between periodontal disease and metabolic syndrome in Japanese workers between the ages of 20 and 60 years.

The oral health of children in a rural area of the Lao People's Democratic Republic.

The lack of information on oral health in Laos makes it difficult to estimate the need and methods for preventing oral disease. This study identified problems concerning the oral health of Lao children. The study subjects were 59 school children who lived in Pakkading District. Dental caries, gingivitis malocclusions, temporomandibular joint (TMJ) disorders, dental plaque, and calculus were examined. We observed an average of 1.6 decayed, missing, and filled teeth (DMFT) and 4.1 decayed and filled deciduous teeth (dft) per child. 25.4% had gingivitis scores from 16 to 20 on the papillary, marginal, and attached (PMA) index; 29.6% had one or more occlusal abnormality; and 0% had signs of TMJ disorders. 93.5% of the children had at least one buccal or lingual tooth surface with plaque covering more than two thirds of the surface; 32.6% had dental calculus. Oral health promotion programs for children should prioritise prevention and treatment of caries. It is likely that the high rate of gingivitis in Lao children is due mainly to unsuccessful plaque control in daily life. In addition to descriptive epidemiological studies of dental diseases in other areas, the influence of sociological and behavioural factors on oral health should be analyzed epidemiologically to promote child health.

Nicotine and lipopolysaccharide stimulate the formation of osteoclast-like cells by increasing macrophage colony-stimulating factor and prostaglandin E2 production by osteoblasts.

Several studies have indicated that one of the causes of alveolar bone destruction with periodontitis is lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria in plaque and that tobacco smoking may be an important risk factor for the development and severity of periodontitis. The present study was undertaken to determine the effect of nicotine and LPS on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E2 (PGE2) in osteoblasts, and the indirect effect of nicotine and LPS on the formation of osteoclast-like cells. Saos-2 cells were cultured with 10(-3) M nicotine, or 1 or 10 microg/ml LPS and 10(-3) M nicotine, for up to 14 days. The gene and protein expression of M-CSF and OPG were determined using real-time PCR and ELISA, respectively. PGE2 expression was determined using ELISA. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from nicotine and LPS-treated Saos-2 cells and the soluble receptor activator of NF-kappaB ligand (RANKL). M-CSF and PGE2 expression increased markedly in cells cultured with nicotine and LPS compared with those cultured with nicotine alone. OPG expression increased in the initial stages of culture with nicotine and LPS but decreased in the later stages of culture. The conditioned medium containing M-CSF and PGE2 produced by nicotine and LPS-treated Saos-2 cells with soluble RANKL increased the TRAP staining of osteoclast precursors compared with that produced by nicotine treatment alone. These results suggest that nicotine and LPS stimulate the formation of osteoclast-like cells via an increase in M-CSF and PGE2 production and that the stimulation is greater than with nicotine treatment alone.

The effect of IL-1beta on the expression of inflammatory cytokines and their receptors in human chondrocytes.

Cytokines released at sites of inflammation and infection can alter the normal processes of cartilage turnover, resulting in pathologic destruction or formation. Interleukin (IL)-1beta plays a central role in the pathophysiology of cartilage damage and degradation in arthritis. In the present study, we examined the effect of IL-1beta on the expression of IL-1beta, IL-6, IL-8, IL-11, tumor necrosis factor-alpha (TNF-alpha), and their receptors in human chondrocytes. The cells were cultured either with or without 100 U/ml of IL-1beta for up to 28 days. The level of expression of the cytokines and their receptors was estimated by determining mRNA levels using real-time PCR or by determining protein levels using ELISA. The expression of IL-1beta, IL-8, and TNF-alpha markedly increased in the presence of IL-1beta after day 14 of culture. The expression of IL-6 and IL-11 increased greatly in the presence of IL-1beta on day 1 and after day 14 of culture. The expression of IL-1beta, IL-8, IL-11, and TNF-alpha receptors significantly decreased in the presence of IL-1beta after day 14 of culture, whereas the expression of IL-6 receptor significantly increased. The expression of these cytokines, except for IL-6, decreased with the addition of human IL-1 receptor antagonist. These results suggest that IL-1beta promotes the resolution system of cartilage matrix turnover through an increase in inflammatory cytokine production by chondrocytes and that it also may promote the autocrine action of IL-6 through an increase in IL-6 receptor expression in the cells.

Employing dmft score as a risk predictor for caries development in the permanent teeth in Japanese primary school girls.

The aim of this study was to examine the significance of evaluating dmft for predicting the incidence of permanent teeth caries in Japanese girls. The subjects were two birth cohorts in a Primary girls' school: cohort I composed of 45 girls born in 1981 and 1982 and cohort II composed of 53 girls born in 1989 and 1990. In both cohorts, there was a significant correlation between the dmft score in the first grade and the DMFT score in the sixth grade. The validity of employing dmft score was examined by receiver operating characteristic (ROC) curve and risk ratios. The shape of the curve and areas under the curve were similar in the two cohorts. In cohort II, sensitivity and specificity for the optimal cut-off level (dmft >or= 5) were 0.519 and 0.923, respectively. The positive and negative predictive values for that level were 0.875 and 0.649, respectively. In cohort I, sensitivity and specificity were 0.741 and 0.722 for that level and positive and negative predictive values were 0.800 and 0.650, respectively. In both cohorts, the risk ratio for permanent teeth caries was significant for many cut-off levels of dmft. The results indicated that the dmft score is a useful predictor of the permanent teeth caries in Japanese primary school girls.

The effect of IL-1beta on the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human chondrocytes.

Interleukin-1 (IL-1) plays key roles in altering cartilage matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). In the present study, we examined the effect of IL-1beta on cell proliferation, alkaline phosphatase (ALPase) activity, and the expression of MMPs, and TIMPs in chondrocytes derived from normal human femoral cartilage. The cells were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum and 0, 1, 10, or 100 U/ml of IL-1beta for up to 28 days. The level of expression of MMPs and TIMPs was estimated by determining mRNA levels using real-time PCR and by determining protein levels using an enzyme-linked immunosorbent assay. Cell proliferation decreased in the presence of IL-1beta after day 21 of culture. ALPase activity decreased significantly in the presence of IL-1beta after day 10 of culture. The expression of MMP-1, -2, and -3 increased markedly in the presence of IL-1beta after day 21 of culture. MMP-13 expression increased markedly in the presence of IL-1beta on day 1 of culture, but decreased markedly after day 7. The expression of TIMP-1 increased significantly after day 14 of culture. The expression of TIMP-2 decreased significantly on day 1, but increased significantly from day 3 to day 14 of culture. These results suggest that IL-1beta may stimulate cartilage matrix turnover by increasing mainly MMP-13 production by the cells.

Nicotine affects mineralized nodule formation by the human osteosarcoma cell line Saos-2.

Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10(-4) M, and 10(-3) M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.

Nicotine affects bone resorption and suppresses the expression of cathepsin K, MMP-9 and vacuolar-type H(+)-ATPase d2 and actin organization in osteoclasts.

Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10(-5), 10(-4), or 10(-3) M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1-5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization.

Interleukin-17A induces cathepsin K and MMP-9 expression in osteoclasts via celecoxib-blocked prostaglandin E2 in osteoblasts.

Interleukin-17 (IL-17) is a cytokine secreted primarily by T(H)-17 cells that can stimulate the development of osteoclasts (osteoclastogenesis) in the presence of osteoblasts. IL-17, through osteoblasts, has indirect effects on the expression of bone resorption-related enzymes in osteoclasts, which have not been well clarified. Here, using MC3T3-E1 cells and RAW264.7 cells as osteoblasts and osteoclast precursors, we aimed to clarify these effects of IL-17A. MC3T3-E1 cells were cultured in the presence or absence of IL-17A for 72 h and the conditioned media collected (in the presence of soluble receptor activator of NF-кB ligand) and used to culture RAW264.7 cells. To assess osteoclast differentiation, adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). Our analyses demonstrated that the number of TRAP-positive multinucleated cells increases after 3 days of culture in conditioned medium from IL-17A-treated cells compared to untreated controls. In addition, we observed that the levels of cathepsin K and MMP-9 increase in the conditioned medium from IL-17A-treated cells, whereas CA II expression levels remain unaffected. PGE(2) production from MC3T3-E1 cells increased in the presence of IL-17A. Celecoxib, a specific inhibitor of cyclooxygenase-2 (COX-2), blocked both the IL-17A-stimulated increase in TRAP-positive multinucleated cells and the expression of cathepsin K and MMP-9. Furthermore, when MC3T3-E1 cells were transformed with small interfering RNA to silence COX-2 expression before IL-17A treatment, the resulting conditioned medium was less effective at inducing cathepsin K and MMP-9 expression in RAW264.7 cells. These results suggest that IL-17A induces the differentiation and function of osteoclasts via celecoxib-blocked prostaglandin, mainly PGE(2), in osteoblasts.

A cohort study on the association between periodontal disease and the development of metabolic syndrome.

BACKGROUND: An association between periodontal disease and metabolic syndrome based on cross-sectional and case-control studies was recently reported, but their causal relationship has not been fully clarified. The objective of this cohort study is to investigate the association between periodontal disease and changes in metabolic-syndrome components to accumulate evidence of the causal relationship between the two conditions. METHODS: The study subjects consisted of 1,023 adult employees (727 males and 296 females; mean age: 37.3 years) who underwent medical and dental checkups between 2002 and 2006 and in whom all metabolic-syndrome components were within the standard values in 2002. The association between the presence of periodontal pockets and the positive conversion of metabolic-syndrome components was investigated using multiple logistic-regression analysis, odds ratios (ORs), and 95% confidence intervals (CIs). RESULTS: The presence of periodontal pockets was associated with a positive conversion of one or more metabolic components during the 4-year observation period (OR: 1.6; 95% CI: 1.1 to 2.2). The ORs for a positive conversion of one component and two or more components were 1.4 (95% CI: 1.0 to 2.1) and 2.2 (95% CI: 1.1 to 4.1), respectively, and the difference was significant for two or more positive components. Of the metabolic-syndrome components, positive conversions of blood pressure and the blood-lipid index were significantly associated with the presence of periodontal pockets. CONCLUSION: The presence of periodontal pockets was associated with a positive conversion of metabolic-syndrome components, suggesting that preventing periodontal disease may prevent metabolic syndrome.

Effects of nicotine and lipopolysaccharide on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in human osteoblasts.

OBJECTIVE: Lipopolysaccharide (LPS) from periodontopathic bacteria can initiate alveolar bone loss through the induction of host-derived cytokines. Smoking increases the risk and severity of periodontitis. We examined the effects of nicotine and LPS on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors, including tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1), in osteoblasts. METHODS: The cells were cultured with or without 10(-4) M nicotine and 100 ng/ml LPS for 12 days or with 100 microg/ml polymyxin B, 10(-4) M D-tubocurarine, 10 micromol/ml NS398, or 10(-6) M celecoxib in the presence of either nicotine or LPS for 12 days. The gene and protein expression levels for MMPs, PAs, TIMPs, and PAI-1 were examined using real-time PCR and ELISAs, respectively. PGE(2) production was determined using an ELISA. RESULTS: The addition of nicotine and/or LPS to the culture medium increased the expression of MMP-1, -2, and -3 and tissue-type PA (tPA); decreased the expression of TIMP-1, -3, and -4; and did not affect expression of TIMP-2 or PAI-1. In the presence of d-tubocurarine or polymyxin B, neither nicotine nor LPS stimulated the expression of MMP-1. In the presence of NS398 or celecoxib, the stimulatory effects of nicotine and LPS on MMP-1 expression were unchanged, but they were unable to stimulate PGE(2) production. CONCLUSION: These results suggest that nicotine and LPS stimulate the resorption process that occurs during turnover of osteoid by increasing the production of MMPs and tPA and by decreasing the production of TIMPs. Furthermore, they suggest that the stimulatory effect of nicotine and LPS on PGE(2) production is independent of their stimulatory effect on MMP-1 expression.

Effects of IL-6 and soluble IL-6 receptor on the expression of cartilage matrix proteins in human chondrocytes.

Elevated concentrations of interleukin (IL)-6 and soluble IL-6 receptor (sIL-6Ralpha) in synovial fluid have been implicated in joint cartilage destruction. We examined the effect of IL-6 and sIL-6Ralpha on cell growth, alkaline phosphatase (ALPase) activity, and the expression of Sox-9, type II collagen, aggrecan core, link protein, BMP-7, and BMP receptors in human chondrocytes. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Ralpha, whereas ALPase activity decreased markedly. The expression of Sox-9 and aggrecan core did not change in the presence or absence of IL-6 and sIL-6Ralpha, whereas the expression of type II collagen, link protein, BMP-7, and BMP receptors increased in the presence of both IL-6 and sIL-6Ralpha. These results suggest that IL-6 and sIL-6Ralpha suppress the differentiation of chondrocytes and induce the repair of arthrodial cartilage through an increase in the expression of cartilage matrix proteins, BMP-7, and BMP receptors in the cells.

Nicotine treatment induces expression of matrix metalloproteinases in human osteoblastic Saos-2 cells.

Tobacco smoking is an important risk factor for the development of severe periodontitis. Recently, we showed that nicotine affected mineralized nodule formation, and that nicotine and lipopolysaccharide stimulated the formation of osteoclast-like cells by increasing production of macrophage colony-stimulating factor (M-CSF) and prostaglandin E2 (PGE2) by human osteoblastic Saos-2 cells. In the present study, we examined the effects of nicotine on the expression of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), the plasminogen activation system including the component of tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PA inhibitor type 1 (PAI-1), alpha7 nicotine receptor, and c-fos. We also examined the effect of the nicotine antagonist D-tubocurarine on nicotine-induced expression of MMP-1. Gene expression was examined using real-time polymerase chain reaction (PCR) to estimate mRNA levels. In addition, expression of the MMP, TIMP, uPA, tPA, and PAI-1 proteins was determined by Western blotting analysis. Nicotine treatment caused expression of MMP-1, 2, 3, and 13, but not MMP-14, to increase significantly after 5 or 10 d of culture; MMP-14 expression did not change through day 14. Enhancement of MMP-1 expression by nicotine treatment was eliminated by simultaneous treatment with D-tubocurarine. In the presence of nicotine, expression of uPA, PAI-1, or TIMP-1, 2, 3, or 4 did not change over 14 d of culture, whereas expression of tPA increased significantly by day 7. Nicotine also increased expression of the alpha7 nicotine receptor and c-fos genes. These results suggest that nicotine stimulates bone matrix turnover by increasing production of tPA and MMP-1, 2, 3, and 13, thereby tipping the balance between bone matrix formation and resorption toward the latter process.