Observation of self-polarization in BSA protected Au20 clusters.

Bovine serum albumin (BSA)-protected gold clusters (atomicity âˆ¼ 20), prepared using a wet chemical route, show strong dipolar radiative transition with a gap energy of 1.93 eV due to the high oscillator strength, as confirmed by the emission studies. Self-arrangement of the clusters with fixed atomicity yields a low dispersive dielectric and electric self-polarization nature. The electrical hysteresis loop measurements returned a remanent polarization of 0.05 µC cm-2, which can be correlated with the dipolar orientation (activation energy âˆ¼ 45.32 meV), originating from the structure-dependent deformation of the charge density.

A genome-wide association study of periodontitis in a Japanese population.

Periodontitis is a multifactorial disease in which bacterial, lifestyle, and genetic factors are involved. Although previous genetic association studies identified several susceptibility genes for periodontitis in European populations, there is little information for Asian populations. Here, we conducted a genome-wide association study and a replication study consisting of 2,760 Japanese periodontitis patients and 15,158 Japanese controls. Although single-nucleotide polymorphisms that surpassed a stringent genome-wide significance threshold (P < 5 × 10(-8)) were not identified, we found 2 suggestive loci for periodontitis: KCNQ5 on chromosome 6q13 (rs9446777, P = 4.83 × 10(-6), odds ratio = 0.82) and GPR141-NME8 at chromosome 7p14.1 (rs2392510, P = 4.17 × 10(-6), odds ratio = 0.87). A stratified analysis indicated that the GPR141-NME8 locus had a strong genetic effect on the susceptibility to generalized periodontitis in Japanese individuals with a history of smoking. In conclusion, this study identified 2 suggestive loci for periodontitis in a Japanese population. This study should contribute to a further understanding of genetic factors for enhanced susceptibility to periodontitis.

Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.

Assessment of the plasma/serum IgG test to screen for periodontitis.

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.

Inhibitory effect of 5,8,11-eicosatrienoic acid on angiogenesis.

INTRODUCTION: Cartilage contains high levels of n-9 eicosatrienoic acid (20:3n-9) but no blood vessels. 20:3n-9 might inhibit angiogenesis. MATERIALS AND METHODS: Angiogenesis was measured in human umbilical vein endothelial cells and diploid fibroblasts. Co-culture was performed with vascular endothelial growth factor-A (VEGF-A, 10 ng/mL) and fatty acids (0.1-10 µmol/L). After 10 days of incubation and immunostaining for endothelial cells, vessel areas were calculated with image analyser software. RESULTS: Addition of 20:3n-9 and n-3 eicosatrienoic acid (20:3n-3) dose dependently inhibited VEGF-A-stimulated angiogenesis (more than the positive control suramin). Arachidonic, eicosapentaenoic, dihomo-γ-linolenic (20:3n-6) and oleic acids did not affect VEGF-A-stimulated angiogenesis even at 10 µmol/L. Arachidonic and dihomo-γ-linolenic acids enhanced angiogenesis without VEGF-A. DISCUSSION AND CONCLUSIONS: We suggest that the presence of 20:3n-9 in cartilage may be related to its vessel-free status and that 20:3n-9 may be useful for the treatment of disorders with excessive vasculature. ACKNOWLEDGEMENTS: This work was partly supported by Polyene Project, Inc.

Evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis.

OBJECTIVE: A new loop-mediated isothermal amplification (LAMP) test kit, including a simple DNA extraction device for the detection of Mycobacterium tuberculosis complex, was developed for commercial use and evaluated for its usefulness in diagnosing tuberculosis (TB). DESIGN: The LAMP test was performed using untreated and N-acetyl-L-cysteine (NALC) NaOH-treated sputum specimen. The efficiency of the kit was compared with other conventional laboratory examinations, including other nucleic acid amplification (NAA) tests. RESULTS: The sensitivity of LAMP using raw sputum (direct LAMP) in smear- and culture-positive specimens was 98.2% (95%CI 94.9-99.4), while the sensitivity in smear-negative, culture-positive specimens was 55.6% (95%CI 43.4-68.0). The diagnostic sensitivity of direct LAMP for the diagnosis of individuals with TB was 88.2% (95%CI 81.4-92.7). The sensitivity values of direct LAMP were slightly, but not statistically significantly lower than those of Cobas Amplicor MTB and TRC Rapid MTB, while the sensitivity of the LAMP test using NALC-NaOH treated sputum was significantly lower than other NAA tests (P < 0.05) for smear-negative, culture-positive specimens. The new commercial version of the LAMP kit was easy to handle and yielded results within 1 h of receiving sputum specimens. CONCLUSIONS: This test is considered a promising diagnostic tool for TB, even for peripheral laboratories with limited equipment, such as those in developing countries.

Fifty-two week chronic toxicity of enzymatically decomposed rutin in Wistar rats.

The chronic toxicity of enzymatically decomposed rutin, which consists mainly of isoquercitrin, was evaluated in male and female Wistar rats with dietary administration at concentrations of 0%, 0.04%, 0.2%, 1% and 5% for 52 weeks. No toxicological findings were found in the mortality, body weights, food consumption, hematology, clinical biochemistry or organ weights in either sex. Obvious clinical signs were chromaturia that could be attributed to the color of test substance in the 5% groups of both sexes. Coloration of the urine collected over 24h in the 1% and 5% groups of both sexes was noted. Increased daily urinary calcium excretion was observed in the 5% groups of both sexes and an increase in urinary calcium concentration was observed in the male 5% group. On histopathological examination, incidences of mineralization, inflammatory cell debris, inflammatory cell infiltration and/or transitional cell hyperplasia in the renal pelvis were increased in the 5% male group, whereas treated females showed no apparent difference in these incidences. Based on the above findings, the no observed adverse effect level (NOAEL) was estimated to be 1% in both sexes (542.4 mg/kg body weight/day for males and 674.0mg/kg body--weight/day for females).

A Novel Therapeutic and Prophylactic Vaccine (HVJ-Envelope / Hsp65 DNA + IL-12 DNA) against Tuberculosis Using the Cynomolgus Monkey Model.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65 + IL-12/HVJ). An IL-12 expression vector (IL-12DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine on the basis of C.F.U of number of TB, survival, an induction of the CD8 positive CTL activity and improvement of the histopathological tuberculosis lesions. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) (prolongation of survival time and the decrease in the number of TB in the lung) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. All monkeys in the control group (saline) died within 8 months, while 50% of monkeys in the HSP65+hIL-12/HVJ group survived more than 14 months post-infection (the termination period of the experiment). Furthermore, the BCG priming and HSP65 + IL-12/HVJ vaccine (booster) by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% survival). Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials.

Characteristics and function of Alcaligenes sp. NBRC 14130 esterase catalysing the stereo-selective hydrolysis of ethyl chrysanthemate.

AIMS: Alcaligenes sp. NBRC 14130 was found as a strain hydrolysing a mixture of (+/-)-trans- and (+/-)-cis ethyl chrysanthemates to (1R,3R)-(+)-trans-chrysanthemic acid. The Alcaligenes cells also have hydrolytic activity for 6-aminohexanoate-cyclic dimer (6-AHCD, 1,8-diazacyclotetradecane-2,9-dione). The correlation of function on the enzyme from the Alcaligenes strain with hydrolysis activities for both ethyl chrysanthemate and 6-AHCD was demonstrated. METHODS AND RESULTS: The esterase was purified to homogeneity. The purified esterase hydrolysed 20 mmol l(-1) ester including the four stereoisomers to the corresponding (+)-trans acid with a 37% molar conversion of ethyl (+)-trans chrysanthemate. The esterase showed high hydrolytic activity for various short-chain fatty acid esters, n-hexane amide and 6-AHCD. The amino acid sequence of the Alcaligenes esterase was identical to that of Arthrobacter 6-AHCD hydrolase (EC 3.5.2.12) and similar to that of fatty acid amide hydrolase (EC 3.5.1.4) from Rattus norvegicus, having both serine and lysine residues of the catalytic site and the consensus motif Gly-X-Ser-X-Gly. CONCLUSION: The stereo-selective hydrolytic activity was found in Alcaligenes sp. NBRC14130 by screening of ethyl chrysanthemate-hydrolysing activity in micro-organisms, and the purified esterase also acted on fatty acid esters and amides. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated that there are great differences in the enzymatic properties, amino acid sequence and catalytic motif of esterases in both Alcaligenes and Arthrobacter globiformis with excellent stereo-selectivity for (+)-trans-ethyl chrysanthemate, but the amino acid sequence of Alcaligenes esterase is identical to that of Arthrobacter 6-AHCD hydrolase.

Regulatory roles of beta-catenin and AP-1 on osteoprotegerin production in interleukin-1alpha-stimulated periodontal ligament cells.

BACKGROUND: Periodontitis is a chronic inflammatory disease characterized by the enhanced expression of inflammatory mediators leading to alveolar bone resorption. Osteoprotegerin (OPG) plays a suppressive role in cytokine-induced osteoclastogenesis. In osteoblasts, OPG expression is upregulated by beta-catenin but downregulated by the transcription factor activator protein-1 (AP-1; c-fos/c-jun). The purpose of this study was to examine the roles of beta-catenin and AP-1 in interleukin-1alpha (IL-1alpha) -induced OPG production in human gingival fibroblasts (hGFs) and periodontal ligament (PDL) cells. METHODS: Expression of c-fos and c-jun messenger RNA was measured by reverse transcription-polymerase chain reaction and OPG production was analysed by enzyme-linked immunosorbent assay. The nuclear AP-1 activity was quantified using an AP-1 microplate assay. The effect of the Wnt canonical pathway on OPG production was evaluated using small interfering (si) RNA for beta-catenin and the effect of AP-1 on OPG production was evaluated using the AP-1 inhibitor curcumin. RESULTS: Levels of c-fos messenger RNA and nuclear AP-1 activity were higher in PDL cells than in hGFs. When stimulated with IL-1alpha, PDL cells had significantly higher c-fos expression and lower OPG production compared with hGFs. The siRNA for beta-catenin suppressed the IL-1alpha-induced OPG production in both PDL cells and hGFs, whereas the AP-1 inhibitor curcumin augmented the IL-1alpha-induced OPG production in PDL cells, but not in hGFs. CONCLUSION: The present study suggests that beta-catenin enhances IL-1alpha-induced OPG production in both PDL cells and hGFs, whereas AP-1 suppresses IL-1alpha-induced OPG production in PDL cells. Higher expression of c-fos in PDL cells than in hGFs may implicate a role of PDL cells in alveolar bone resorption in periodontitis.

Extracorporeal membrane oxygenation in a patient with propionic acidaemia: a therapeutic option for cardiac failure.

We report a Japanese girl affected with a neonatal-onset form of propionic acidaemia (PA). She developed severe metabolic crisis after dehydration at 2 years of age. Bradycardia with complete atrioventricular block responded to haemodiafiltration, but severe cardiac failure was refractory to inotropic treatment. She was diagnosed with acute cardiac dysfunction caused by PA-induced metabolic crisis. Extracorporeal membrane oxygenation (ECMO), a technique for providing mechanical circulatory support, was required. This is the first case report of a PA patient who recovered from a life-threatening metabolic crisis with cardiac failure by ECMO. Cardiac failure may be a cause of death, but it is occasionally an under-recognized complication. Mitochondrial dysfunction in the myocardium due to propionyl-CoA could contribute to the pathomechanism of cardiac complications of PA. We believe that ECMO should be attempted in PA patients with cardiac failure, in addition to haemodiafiltration and other therapeutic measures, because doing so may lead to the recovery of cardiac dysfunction, as was evident in our patient. In conclusion, prompt investigations and management of cardiac complications should be performed immediately during PA-induced metabolic crises.

Caspase activation is involved in early megakaryocyte differentiation but not in platelet production from megakaryocytes.

To elucidate whether caspase activation is involved in megakaryopoiesis, we characterized megakaryocytes (MKs) in vav-bcl-2 transgenic (Tg) mice, in which Bcl-2 is overexpressed in hematopoietic cells. To exclude the effect of splenomegaly in Tg mice on megakaryopoiesis, splenectomy was performed. After splenectomy, basal platelet counts in peripheral blood were not significantly different between Tg and wild-type (WT) mice. However, when experimental thrombocytopenia was induced by injecting 5-fluorouracil into splenectomized mice, overshoot of platelet counts during the recovery phase was hardly observed in Tg mice. Analyses of MK ploidy during the recovery phase showed that MKs less than 16 N ploidy were significantly decreased in Tg mice, suggesting that MK supply from progenitors is impaired. Supporting this, differentiation of CD34-/c-kit+/Sca-1+/Lineage- stem cells into MKs was significantly hampered in Tg mice, whereas megakaryocyte-erythroid progenitors (MEPs) normally differentiated into MKs. It suggests that differentiation into MKs is impaired in Tg mice before the stage of MEP. Furthermore, MK colony formation in WT cells was dose-dependently inhibited in the presence of a caspase inhibitor. Contrary, Bcl-2-overexpressing MKs showed normal ability for in vitro platelet production. We thus believe that caspase activation is involved in the differentiation of progenitors into megakaryocytic lineage but not in platelet production.

Multi-center intervention study on glycohemoglobin (HbA1c) and serum, high-sensitivity CRP (hs-CRP) after local anti-infectious periodontal treatment in type 2 diabetic patients with periodontal disease.

The purpose of this study was to examine whether periodontal treatment incorporating topical antibiotic therapy affects on levels of glycohemoglobin (HbA1c) and serum high-sensitivity C-reactive protein (hs-CRP) in type 2 diabetic patients with periodontal disease, and to explore the relationship between CRP and glycemic control. The whole intervention group (n=32), which underwent anti-infectious periodontal treatment, showed only transient reduction in HbA1c levels without any change in hs-CRP, while the control group (n=17) did not show any changes in HbA1c or hs-CRP. Multiple regression analysis of all subjects revealed that BMI and change in hs-CRP correlated significantly with the reduction of HbA1c at 6 months after the periodontal treatment. Based on the results of multiple regression analysis, the intervention group was subdivided into two groups: those in which hs-CRP levels decreased (CRP-D group), and those in which hs-CRP levels unchanged or increased (CRP-N group) (n=16, respectively), and re-analysis was conducted based upon these subgroups. In the CRP-D subgroup, HbA1c was significantly reduced at the end of the study, but it did not decrease in the CRP-N subgroup. The decrease of HbA1c in the CRP-D subgroup following periodontal treatment was significantly greater than that in the CRP-N subgroup. BMI of each group remained unchanged in this study at the end of the study. Thus, the results suggested that periodontal treatment with topical antibiotics improves HbA1c through reduction of CRP, which may relate to amelioration of insulin resistance, in type 2 diabetic patients with periodontal disease.

Latitudinal variation in egg size and number in anadromous masu salmon Oncorhynchus masou.

Latitudinal variation in egg size and number in anadromous masu salmon Oncorhynchus masou was examined. Relatively greater variation in egg size occurred among rivers than among females within rivers or within females. Egg size was generally greater and egg number generally lower at more northerly latitudes.

Molecular mimicry of Aggregatibacter actinomycetemcomitans with beta2 glycoprotein I.

INTRODUCTION: beta2-Glycoprotein I (beta 2GPI) is important in the suppression of coagulation, and antibodies against TLRVYK peptides on the beta 2GPI molecule are related to thrombosis. According to the Swiss-Prot database, Aggregatibacter actinomycetemcomitans leukotoxin c has sequences (SIRVYK) that are homologous to the TLRVYK peptides. The aim of this study was to investigate the effects of A. actinomycetemcomitans infection on the antibody response against SIRVYK peptides in patients with periodontitis. METHODS: Serum immunoglobulin G (IgG) antibody and IgG subclass antibody titers against SIRVYK or TLRVYK peptides were measured by enzyme-linked immunosorbent assay in 46 patients with aggressive periodontitis (eight with localized disease, 38 with generalized disease), 28 patients with chronic periodontitis, and 20 periodontally healthy subjects. The presence of A. actinomycetemcomitans in plaque and saliva samples was determined using polymerase chain reaction. RESULTS: The level of anti-SIRVYK antibodies was significantly higher in patients who were A. actinomycetemcomitans-positive than in A. actinomycetemcomitans-negative patients (P < 0.05) in the chronic periodontitis group. A similar trend was found in the antibody response to TLRVYK peptide; however, no statistically significant difference was seen between A. actinomycetemcomitans-positive and -negative patients. The A. actinomycetemcomitans-positive patients displayed significantly higher levels of anti-SIRVYK IgG2 and IgG3 antibodies than A. actinomycetemcomitans-negative patients (P < 0.05 and P < 0.05, respectively). The level of IgG2 was highest among the four IgG subclasses and it predominantly increased in patients who were A. actinomycetemcomitans-positive. Anti-TLRVYK antibody levels were significantly correlated with anti-SIRVYK IgG antibody levels. CONCLUSION: The results suggest that A. actinomycetemcomitans infection may elicit anti-SIRVYK IgG antibodies and modify the anti-TLRVYK antibody response in patients with periodontitis by molecular mimicry with beta2GPI.

Shortened lifespan of paroxysmal nocturnal haemoglobinuria-affected RBC estimated from differences in ratios of CD59-negative populations between reticulocytes and whole RBC.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haemolytic disease characterized by complement-sensitive red blood cells (RBC). PNH-affected RBC (PNH-RBC) should have a shortened mean lifespan (MLS); however, direct measurement is difficult. We have recently developed a sensitive flow cytometric assay to analyse PNH-affected reticulocytes that may closely correspond to the PNH clone-derived erythropoiesis. Naturally, the CD59-negative populations in reticulocytes were larger than those in whole RBC in PNH. We estimated the MLS of PNH-RBC in six PNH patients from the differences in the ratios of CD59-negative populations between reticulocytes and whole RBC. The MLS of PNH-RBC was calculated using the following formula: W/100 = R x M/[(100 - R) x 120 + R x M], where W, percentage CD59-negative whole RBC; R, percentage CD59-negative reticulocytes; M, MLS (days) of CD59-negative RBC. The MLS of PNH-RBC, estimated as 16-45 days in the PNH patients, showed a weak positive and a weak negative relation with RBCs and percentage reticulocytes, respectively, among the patients. The MLS, in individual patients, altered irrespective of RBC and percentage reticulocytes. The MLS calculated from our methods may be a parameter that evaluates the haemolytic conditions in PNH.

Oxygen consumption in nonexercising muscle after exercise.

Little is known about the effect of exercise intensity on post-exercise oxygen consumption in nonexercising muscle. This study examined the effect of exercise intensity on muscle oxygen consumption (VO2mus) in nonexercising forearm flexor muscles (nonexVO2mus) after cycling exercise. Eight healthy male subjects performed 20 min of cycling exercise at 30%, 50%, and 70% of maximal oxygen consumption (%VO2max) on separate days. The nonexVO2mus values at rest, at the end of exercise, and during recovery after exercise were measured by near-infrared spectroscopy. VO2mus was determined using the rate of decrease in oxygenated hemoglobin during arterial occlusion. The nonexVO2mus at the end of exercise significantly increased by 1.3 +/- 0.1, 2.0 +/- 0.3, and 2.2 +/- 0.3-fold over resting values at 30%, 50%, and 70% VO2max, respectively. NonexVO2mus returned to the resting value after 3 - 5 min of recovery and then showed no significant change for 120 min after exercise at all exercise intensities. NonexVO2mus at the end of exercise at 70% VO2max was significantly higher than that after exercise at 30% VO2max. These results show that 20 min of cycling exercise induced an increase in nonexVO2mus and that higher intensity exercise produces a larger increase in nonexVO2mus after exercise.

Periodontitis may increase the risk of peripheral arterial disease.

OBJECTIVES: The aim of this case control study was to evaluate whether periodontitis was associated with peripheral arterial disease (PAD). SUBJECTS AND METHODS: Twenty-five patients diagnosed with aorto-iliac and/or femoro-popliteal occlusive disease and thirty-two generally healthy control subjects were enrolled in this study. Polymerase chain reaction (PCR) was used to identify Porphyromonas gingivalis, Treponema denticola, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Cytomegalovirus (CMV), Chlamydia pneumoniae, and Helicobacter pylori in tissue specimens taken from the anastomotic site of distal bypasses. Periodontal status was evaluated; serum IgG titres against the four listed bacteria were measured. RESULTS: Periodontopathic bacteria were detected in 13/25 (52%) atherosclerotic specimens. CMV or C. pneumoniae was detected in 1/25 (4%) specimens; H. pylori was not detected from any of these specimens. Fontaine grade III or IV patients showed higher detection frequency of P. gingivalis than Fontaine grade II patients (57.1% vs 22.2%, P=0.09). After adjusting for age, gender, diabetes and smoking, periodontitis increased 5-fold the risk of having PAD (OR 5.45). There were preliminary indications that periodontitis was associated with increased serum IL-6 and TNF-alpha concentrations. CONCLUSIONS: This study suggests that periodontitis may be associated with an increased risk of PAD. This association could result from the increased concentration of serum inflammatory cytokines in those with periodontitis.

Internal prostaglandin synthesis augments osteoprotegerin production in human gingival fibroblasts stimulated by lipopolysaccharide.

Periodontitis is an inflammatory bone disease caused by Gram-negative anaerobic bacteria. Osteoclast differentiation is regulated by the balance between receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG). The purpose of this study was to examine the mechanism of OPG production in human gingival fibroblasts (HGF) stimulated by lipopolysaccharide (LPS) from periodontopathic bacteria. The expressions of Toll-like receptor 2 (TLR-2) and TLR-4 in HGF were examined using flow-cytometry. HGF were stimulated with whole cell extracts or LPS from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis with or without polymyxin B, a LPS inhibitor. In addition, HGF were stimulated with LPS, prostaglandin E(2) (PGE(2)), various agonists of PGE receptors (EP1, EP2, EP3 and EP4 agonists) with or without indomethacin (IND), a prostaglandin synthesis inhibitor. OPG and PGE(2) production was measured using an enzyme-linked immunosorbent assay (ELISA). HGF expressed both TLR-2 and TLR-4. Both A. actinomycetemcomitans and P. gingivalis LPS augmented OPG expression in HGF. Whole cell extracts from A. actinomycetemcomitans and P. gingivalis augmented OPG production by HGF; the augmentation was suppressed by polymyxin B. IND suppressed OPG production in LPS-stimulated HGF. PGE(2) stimulated HGF to produce OPG. EP1 and EP2 agonists, but not EP3 and EP4 agonists, increased OPG production by HGF. These results suggest that LPS-induced OPG production by HGF is regulated via EP1 and/or EP2 receptors by endogenously generated PGE(2).