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CXCR4, a CXCL12 receptor, is expressed on epithelial cells, fibroblasts, and inflammatory cells. The CXCR4-CXCL12 interaction is related to the migration of neutrophils and monocytes/macrophages. Periodontitis, an inflammatory disease mainly caused by gram-negative bacteria, is characterized by infiltration of circulating inflammatory cells and alveolar bone (AB) loss. To investigate whether CXCR4 is involved in the distribution of neutrophils and monocytes/macrophages early after periodontitis induction, we examined the effects of AMD3100 (AMD), a CXCR4 antagonist, in ligature-induced periodontitis mice and LPS-injected air pouch mice. The periodontitis study was accomplished in control (C), periodontitis (P), and P + AMD groups. Periodontitis was induced by ligation of the mandibular first molar. AMD was intraperitoneally administered daily beginning the day before ligation until sacrifice on the third day after ligation. The air pouch study was accomplished in C, lipopolysaccharide (LPS), and LPS + AMD groups. Air pouches on mice backs were formed by subcutaneous injection of sterilized air. AMD was administered and then LPS was injected into the air pouch. For the detection of neutrophils and monocytes/macrophages in blood and air pouch exudates, flow cytometry was performed with anti-Ly6G/anti-CD11b antibodies (Abs) and anti-CD115 Ab, respectively. In periodontal tissue, Ly6G

Periodontitis is a bacteria-induced inflammatory disease associated with alveolar bone loss. Osteoclast is a macrophage-lineage cell that exhibits phagocytic activity; however, osteoclast phagocytic activity has not been demonstrated under pathological conditions. Diabetes is a pathological condition that exacerbates alveolar bone loss via periodontitis; therefore, we examined phagocytic osteoclasts in diabetic rats that had periodontitis. The rats were divided into the control (C), periodontitis (P), and diabetes with periodontitis (DP) groups. Diabetes and periodontitis were induced by streptozotocin injection and ligature of the mandibular first molars, respectively. On days 3 and 20 after the ligature, the rats were sacrificed, and osteoclasts containing inclusions were quantified by tartrate-resistant acid phosphatase staining. On day 3, there were more osteoclasts containing inclusions in the DP group than in the C group. Among inclusions, osteocyte-like cells and dense bodies were more frequently observed in the DP group than in the C group. Cytoplasm-like structures were elevated more in the DP group than in the C and P groups. However, no differences were observed on day 20. Interestingly, some osteoclasts were in contact with the osteocytes within the exposed lacunae and contained several inclusions within a large vacuole. Thus, the elevation of phagocytic osteoclasts in rats with diabetes and periodontitis provides insight into the role of osteoclast phagocytic activity under pathological conditions.

Backgrounds and objective Increased neutrophil infiltration and osteoclast formation are key characteristics of periodontitis. The effect of these neutrophils on osteoclast formation in periodontitis remains unclear. Therefore, we investigated the effects of neutrophils on osteoclast formation in a neutrophil-deficient mouse model of periodontitis. Methods Anti-Ly6G antibody (Ab) was used for neutrophil depletion in two mouse models: periodontitis and air pouch. In the periodontitis experiments, mice were divided into PBS-administered control (C), control Ab-administered periodontitis (P), and anti-Ly6G Ab-administered periodontitis (P + Ly6G) groups. Periodontitis was induced by ligature of mandibular first molars. In the air pouch experiments, mice were divided into PBS-administered (C), LPS and control Ab-administered (LPS), and LPS and anti-Ly6G Ab-administered (LPS + Ly6G) groups. Neutrophil migration into air pouches was induced by LPS injection. Flow cytometry was used to examine CD11b+ Ly6G+ neutrophils in the blood of periodontitis mice and CD11b+ Ly6G+ RANKL+ neutrophils in exudates of air pouch mice. In periodontal tissue, Ly6G+ neutrophil and RANKL+ cell numbers in periodontal ligament and alveolar bone areas were estimated using immunohistochemistry, osteoclast numbers were measured using TRAP assay, and alveolar bone loss was determined by H&E staining. Results In blood, CD11b+ Ly6G+ neutrophils were found in greater percentage in the P group than in the C group on days 3 and 7. However, the percentage of neutrophils was lower in the P + Ly6G group than in the C and P groups. In periodontal tissue, the numbers of Ly6G+ neutrophils and RANKL+ cells were lower in the P + Ly6G group than in the P group on day 3. Ly6G+ neutrophil numbers decreased more in the P + Ly6G group than in the P group on day 7, but RANKL+ cell numbers did not decrease in the P + Ly6G group. In exudates, the number of CD11b+ Ly6G+ RANKL+ neutrophils was greater in the LPS group than in the C and LPS + Ly6G groups. On days 3 and 7, the numbers of osteoclasts and alveolar bone loss were greater in periodontal tissue in the P and P + Ly6G groups than in the C group. Interestingly, there were fewer osteoclasts in the P + Ly6G group than in the P group on day 3. Conclusion Neutrophil deficiency caused a reduction in numbers of both RANKL+ cells and osteoclasts in periodontitis-induced tissues only on day 3. Furthermore, in the LPS-injected air pouch model, neutrophil deficiency reduced the influx of RANKL+ neutrophils. These findings suggest that the presence of neutrophils induces RANKL expression and could induce osteoclast formation in the early stages of periodontitis.

Objectives In dental clinics, dental hygienists are exposed to aerosolized pathologic bacteria, which can be transmitted to the oral cavity via lip cosmetics. Accordingly, such contamination poses a consistent health risk among staffs. Our study examined the bacterial contamination of lip cosmetics used by dental hygienists while in a clinic setting. Methods Sixteen dental hygienists were surveyed regarding their job assignments and habits associated with lip cosmetic. Subsequently, microorganisms were analyzed in collected samples of the hygienists' lip cosmetics using colony‐forming unit (CFU) assays, 16s‐rDNA polymerase chain reaction, and DNA sequencing. Results Notably, 81.3% of the submitted lip cosmetic samples were contaminated, with bacterial CFUs ranging from undetectable to innumerable. Many samples (43.8%) exceeded the microbial limits of cosmetic contamination. Of the lip cosmetic used for more than 6 months, 60% exceeded the microbial limit. When wearing a mask every time, only one of the six samples exceeded the microbial limit. More frequent dental mask changing was associated with a lower likelihood that the cosmetic sample would exceed the microbial limit. No samples from hygienists who changed their masks four times a day exceeded the microbial limit, compared to 33.3% from hygienists who only changed the mask when it became wet. Most isolated bacteria were gram‐positive, facultative anaerobic, asporogenic, and opportunistically pathogenic, and the most prevalent species were Staphylococcus aureus, Streptococcus salivarius, and Staphylococcus epidermidis. Conclusion Our findings indicate that dental staff, including dental hygienists, should exercise more careful workplace habits, particularly with regard to infection control and cosmetic use.

Increases of neutrophils and osteoclasts are pathological changes of periodontitis. RANKL is an osteoclast differentiation factor. The effect of periodontopathogen LPS on RANKL-expressing neutrophils has not been clarified yet. We evaluated numerical changes of RANKL-expressing neutrophils in air pouches of mice injected with LPSs of Fusobacterium nucleatum and Porphyromonas gingivalis. Mice with air pouches were assigned into saline (C)-, E. coli LPS- (Ec LPS)-, F. nucleatum LPS (Fn LPS)-, P. gingivalis LPS (Pg LPS)-, and Fn LPS and Pg LPS (Fn + Pg LPS)-injected groups. CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils in blood and air pouch exudates were determined by flow cytometry. In blood, compared to the C group, the Fn LPS group showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils whereas the Pg LPS group showed no significant differences. These increases in the Fn LPS group were not different to those in the Ec LPS group. In exudates, Fn LPS and Pg LPS groups showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils compared to the C group. Increased levels in the Fn LPS group were not different to those in the Ec LPS group, but Pg LPS group was lower than those in the Ec LPS group. In blood and exudates, the Fn + Pg LPS group showed no difference in levels of these neutrophils compared to the Ec LPS group. LPSs of F. nucleatum and P. gingivalis increased RANKL-expressing neutrophils although the degrees of increases were different. These suggest that periodontopathogen LPS can act as a stimulant to increase RANKL-expressing neutrophils.

To determine the effect of the tumor necrosis factor-α (TNF-α) in odontoclast formation, we administrated a TNF-α inhibitor in rats with diabetes rats with periodontitis. The rats included in the study were divided into three groups: control rats without diabetes or periodontitis (the C group), rats with periodontitis and diabetes (the PD group), and rats with periodontitis and diabetes treated by infliximab, the TNF inhibitor (the PD+infliximab group). The PD and PD+ infliximab groups received intravenous administrations of streptozotocin (STZ, 50 mg/kg) to induce diabetes. After 7 days of STZ injections, the mandibular first molars were ligatured to induce periodontitis. The PD+infliximab group was intrapenitoneally administrated by infliximab (5 mg/kg). On days 3 and 20 after the ligature administration, odontoclast formation along root surfaces was evaluated by tartrate resistant acid phosphatase (TRAP) staining and cathepsin K immunohistochemistry. On day 3, the number of TRAP- and cathepsin K-positive cells increased more so in the PD group than in the C group. The PD+infliximab group showed a lower number of positive cells than the PD group. There was no difference in all the groups on day 20. On day 3, the cathepsin-K positive multinucleated and mononucleated cells were higher in the PD group than in the C group. The number of cathepsin-K positive multinucleated cells was lower in the PD+infliximab group than in the PD group. The PD group showed more cathepsin K-positive cells in the furcation and distal surfaces than the c group. The Cathepsin K-positive cells of the PD+infliximab group were lower than that of the PD group in furcation. These results suggest that TNF-α stimulates odontoclast formation in diabetes with periodontitis.

The purpose of this study was to evaluate the effect of various polishing methods on surface roughness of zirconia-porcelain veneer and to correlate the findings with early bacterial adhesion. The study specimens were glazed (control group), glazed after fine polishing (glazed group) and polished with Exa Cerapol (Cerapol group) or with Shofu porcelain adjustment kit (Shofu group) (n =20). Surface roughness was then measured using profilometer and scanning electron microscope (SEM). After artificial saliva coating, the specimens were incubated in Streptococcus mitis suspension for 4 h at 37 °C. Adherent bacteria were quantified from SEM images. Streptococcal viability was assessed by LIVE/DEAD staining kit and fluorescent microscope. There were significant differences in surface roughness according to polishing method and surface material. Relatively smoother surfaces were found in zirconia surfaces and glazed porcelain surfaces. There were also significant differences in bacterial adhesion according to polishing method and surface material. Cerapol group showed minimal bacterial adhesion with more dead cells when compared to other groups. A positive correlation between surface roughness and bacterial adhesion was found in glazed porcelain surface and a negative correlation in zirconia surface of Cerapol group, both with no statistical significance. Within the limitations of in vitro study, surface roughness and bacteria adhesion were significantly influenced by polishing method and surface material. Also, there was a positive correlation and negative correlation between surface roughness and bacterial adhesion in glazed porcelain surface and in zirconia surface of Cerapol group, respectively.

The array of outer membrane proteins (OMPs) found in Helicobacter pylori provides a crucial component for persistent colonization within the gastric niche. Not only does H. pylori harbor a wide number of OMPs, but these OMPs often vary across strains; this likely contributes to immune evasion, adaptation during long term colonization, and potentially differential disease progression. Previous work from our group described OMP differences among the Bab family (babA, babB, and babC) and Hom family (homA and homB) from 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). In the current study, we expanded our investigation to include the less well characterized Hom family member, HomC.Overall, we identified and genotyped three homC variants: homC

Summary Background & aims Licochalcone (lico) F is a novel synthetic retrochalcone. In this study, we investigated the anti-inflammatory effects of lico F in vitro, and its effects on obesity-induced chronic inflammation, glucose intolerance, and fatty liver in vivo. Methods The inhibitory effects of lico F on TNFα-induced inflammation were investigated using NF-κB luciferase reporter assay and RT-PCR. Diet-induced obese mice were treated orally, once per day, with vehicle or lico F (10 mg/kg/day), for 3 weeks, and blood, liver, and adipose tissues were analyzed. Results Lico F inhibited TNFα-induced NF-κB activation and mRNA expression of TNFα, COX-2, IL-6, IL-1β, and NOS2. In obese mice, lico F administration significantly alleviated glucose tolerance without changes in body weight gain and food intake. Lico F reduced adipocyte size and macrophage infiltration into white adipose tissue and improved hepatic lesions, by decreasing fat droplets and glycogen deposition. The mRNA expression levels of TNFα, MCP-1, and CD68 in white adipose tissue also decreased markedly. Moreover, lico F enhanced Akt signaling, but reduced p38 MAPK signaling in white adipose tissue. Conclusions Lico F had anti-inflammatory effects and showed beneficial effects on glucose metabolism, which could be partially caused by activation of the Akt signal pathway and obesity-induced chronic inflammation, probably by downregulating p38 signal pathway. Moreover, lico F could be used as a potential novel therapeutic compound against type 2 diabetes and obesity-induced chronic inflammation without the deleterious effects of body weight gain and fatty liver.

Background and Objective Osteoclast precursors (OPs) re-migrate from the bone surface into blood vessels through sphingosine-1-phosphate receptor 1 (S1PR1) expression. T cells also express S1PR1, mediating their migration from the lymph nodes into blood vessels. OP and T-cell migration are one of the sequential steps related to osteoclast formation. To characterize the role of S1PR1 in osteoclast formation induced by periodontitis, we investigated the effect of S1PR1-binding molecule FTY720 (FTY) on the number of OPs and T cells in periodontal tissue and peripheral blood of rats with ligature-induced periodontitis. Material and Methods Rats were divided into four groups; control (Con), FTY, periodontitis (Peri), and periodontitis+FTY (Peri+FTY) groups. Ligatures were placed around the first molars in the left and right mandibles. The rats were intraperitoneally injected with vehicle or 3 mg/kg FTY daily until they were killed. The number of osteoclasts and cluster of differentiation (CD)11b, CD3 and receptor activator of NF-κB ligand (RANKL)-positive cells in first molar furcation were counted by tartrate-resistant acid phosphatase or immunohistochemistry staining. The number of CD11b- and CD3-positive cells in peripheral blood was estimated by flow cytometry. Results The number of osteoclasts in the Peri group was higher than Con, Peri+FTY and FTY groups (p < 0.05) and CD11b, CD3 and RANKL-positive cells were also higher in the Peri group than other groups in furcation (p < 0.05). While CD11b-positive cells in furcation of the Peri+FTY group were lower than the Peri group (p < 0.05), they were higher in peripheral blood (p < 0.05). Dissimilar to CD11b-positive cells, CD3-positive cells in the Peri+FTY group were lower in peripheral blood as well as furcation than the Peri group (p < 0.05). RANKL-positive cells in furcation of the Peri+FTY group were also lower than Peri group (p < 0.05). Conclusion These results indicate that FTY may facilitate re-migration of OPs from the alveolar bone surface into blood vessels, blocking T-cell migration from the lymph nodes into blood vessels and subsequently reducing osteoclast formation induced by periodontitis. This suggests that S1PR1-S1P binding may play a role in osteoclast formation of periodontitis by modulating OP and T-cell migration.

Background Periodontitis is an infectious disease that manifests as alveolar bone loss surrounding the roots of teeth. Diabetes aggravates periodontitis-induced alveolar bone loss via suppression of bone formation. Intermittent parathyroid hormone (PTH) administration displays an anabolic effect on bone. In this study, we investigated the effect of intermittent PTH administration on alveolar bone loss in type 1 diabetic rats with periodontitis. Methods Rats were divided into control (C), periodontitis (P), periodontitis treated with PTH (P + PTH), diabetes with periodontitis (DP), and diabetes with periodontitis treated with PTH (DP + PTH) groups. To induce type 1 diabetes, rats were injected with streptozotocin and periodontitis was induced bilaterally by applying ligatures to the mandibular first molars for 30 days. During the experimental period, the P + PTH and DP + PTH groups were subcutaneously injected with PTH (40 μg/kg) three times per week, whereas the C, P, and DP groups were injected with citrate buffer. To observe the mineralization of the alveolar bone, the DP and DP + PTH groups were injected with calcein on days 10 and 27, and with alizarin red on day 20. Thirty days after ligation, histological findings and fluorescence labeling were analyzed in the furcations of the mandibular first molars. Sclerostin-positive osteocytes were assessed by immunohistochemical analyses. Results The DP groups had smaller areas of alveolar bone than the other groups, and the DP + PTH group had a larger alveolar bone area than the DP group. The DP group had less osteoid formation than the C group, whereas the DP + PTH had greater osteoid formation than the DP group. Fluorescence labeling results revealed that the DP + PTH group had more mineral deposition on the alveolar bone than the DP group. The DP + PTH group exhibited lower percentage of sclerostin-positive osteocytes in alveolar bone than the DP group. Conclusions Intermittent PTH administration diminishes alveolar bone loss and sclerostin expression in osteocytes, but increases osteoid formation and mineralization, suggesting that intermittent PTH administration attenuates diabetes-aggravated alveolar bone loss by the induction of bone formation. PTH-induced bone formation may be related to the regulation of osteocytic sclerostin expression in type 1 diabetic rats with periodontitis.

Helicobacter pylori is associated with hypergastrinemia, which has been linked to the development of gastric diseases. Although the molecular mechanism is not fully understood, H. pylori is known to modulate the Erk pathway for induction of gastrin expression. Herein we found that an epidermal growth factor (EGF) receptor kinase inhibitor significantly blocked H. pylori-induced gastrin promoter activity, suggesting involvement of EGF receptor ligands. Indeed, H. pylori induced mRNA expression of EGF family members such as amphiregulin, EGF, heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α. Of these, specific siRNA targeting of HB-EGF significantly blocked H. pylori-induced gastrin expression. Moreover, H. pylori induced HB-EGF ectodomain shedding, which we found to be a critical process for H. pylori-induced gastrin expression. Thus, we demonstrate a novel role for human mature HB-EGF in stimulating gastrin promoter activity during H. pylori infection. Further investigation using specific siRNAs targeting each isoform of Raf, Mek, and Erk elucidated that the mechanism underlying H. pylori-induced gastrin expression can be delineated as the sequential activation of HB-EGF, the EGF receptor, C-Raf, Mek1, and the Erk2 molecules in the MAPK pathway. Surprisingly, whereas Erk2 acts as a potent activator of gastrin expression, siRNA knockdown of Erk1 induced gastrin promoter activity, suggesting that Erk1 typically acts as a repressor of gastrin expression. Elucidation of the mechanism of gastrin modulation by HB-EGF-mediated EGF receptor transactivation should facilitate the development of therapeutic strategies against H. pylori-related hypergastrinemia and consequently gastric disease development, including gastric cancers.

To determine the effect of diabetes on root resorption in periodontitis, we investigated odontoclast formation and root resorption in diabetic rats with periodontitis. Odontoclast formation was observed in three groups of F344 rats: Controls (C) were normal rats without diabetes or periodontitis; the periodontitis (P) group had mandibular first molars to be ligatured; the periodontitis with diabetes (PD) group was intravenously administered streptozotocin (50 mg/kg) to induce diabetes and had mandibular first molars to be ligatured. On days 3, 10, and 20 after ligature, tumor necrosis factor (TNF)-α and receptor activator of nuclear factor-κB ligand (RANKL) expression, odontoclast formation, and root resorption areas were evaluated by immunohistochemistry, tartrate-resistant acid phosphatase staining, and hematoxylin and eosin staining, respectively. The PD group showed frequent urination, weight loss, and hyperglycemia. Numbers of TNF-α- and RANKL-positive cells were higher in the P and PD groups than in the C group. It was more prevalent in PD group on day 3. Odontoclast formation was greater in the P and PD groups than in the C group on days 3 and 10, then decreased to same level as the C group by day 20. Root resorption in the PD and P groups showed increases on days 3 and 10, respectively, compared to the C group. These results suggest that diabetes may transiently increase root resorption on day 3 with high expression of TNF-α and RANKL after periodontitis induction. This study could aid the understanding of root resorption in diabetic patients with periodontitis.

Osteocytic sclerostin inhibits bone formation, and its expression is stimulated by tumor necrosis factor (TNF)-α. This study investigates sclerostin and TNF-α expression in rats with diabetes mellitus (DM) and periodontitis.Rats were divided into control (C), periodontitis (P), and DM + periodontitis (DP) groups. After induction of DM by streptozotocin, periodontitis was induced by ligature. At day 0 (control) and at days 3 and 20 after induction of periodontitis, alveolar bone, osteoclasts, osteoid area, and TNF-α and sclerostin expression were evaluated.The distance between the cemento-enamel junction and the alveolar bone crest of the DP group was longer than that of the P group at day 20 after induction of periodontitis, but the number of osteoclasts was not different. Osteoid area decreased in both the P and DP groups by day 3, but whereas sustained osteoid suppression was observed in the DP group at day 20, osteoid formation was increased in the P group. The number of sclerostin-positive osteocytes increased in both groups at day 3, but the increased number of sclerostin-positive osteocytes was maintained only in the DP group through day 20. The number of TNF-α-positive cells increased more in the DP group than in the P group.Enhanced alveolar bone loss, suppressed bone formation, and prevalent TNF-α expression were characteristic of the DP group compared with the P group. Suppressed bone formation in the DP group was observed simultaneously with increased sclerostin and TNF-α expression. These results suggest that upregulated osteocytic sclerostin expression in periodontitis accompanied by DM may play a role in suppressed bone formation.

Received: April 10, 2015, Accepted: May 6, 2015 ISSN 1598-4478 (Print) / ISSN 2233-7679 (Online) Correspondence to: Yun-Jung Yoo Department of Oral Biology, College of Dentistry, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea Tel: +82-2-2228-3060, Fax: +82-2-2227-7903, E-mail: yu618@yuhs.ac Correspondence to: Eun-Jung Bak Department of Oral Biology, College of Dentistry, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea Tel: +82-2-2228-3062, Fax: +82-2-2227-7903, E-mail: ejbak@yuhs.ac *These authors contributed equally to this work.

Infection with Helicobacter pylori is a major risk factor for development of gastric disease, including gastric cancer. Patients infected with H. pylori strains that express CagA are at even greater risk of gastric carcinoma. Given the importance of CagA, this report describes a new molecular mechanism by which the cagA copy number dynamically expands and contracts in H. pylori. Analysis of strain PMSS1 revealed a heterogeneous population in terms of numbers of cagA copies; strains carried from zero to four copies of cagA that were arranged as direct repeats within the chromosome. Each of the multiple copies of cagA was expressed and encoded functional CagA; strains with more cagA repeats exhibited higher levels of CagA expression and increased levels of delivery and phosphorylation of CagA within host cells. This concomitantly resulted in more virulent phenotypes as measured by cell elongation and interleukin-8 (IL-8) induction. Sequence analysis of the repeat region revealed three cagA homologous areas (CHAs) within the cagA repeats. Of these, CHA-ud flanked each of the cagA copies and is likely important for the dynamic variation of cagA copy numbers. Analysis of a large panel of clinical isolates showed that 7.5% of H. pylori strains isolated in the United States harbored multiple cagA repeats, while none of the tested Korean isolates carried more than one copy of cagA. Finally, H. pylori strains carrying multiple cagA copies were differentially associated with gastric disease. Thus, the dynamic expansion and contraction of cagA copy numbers may serve as a novel mechanism by which H. pylori modulates gastric disease development., IMPORTANCE Severity of H. pylori-associated disease is directly associated with carriage of the CagA toxin. Though the sequences of the CagA protein can differ across strains, previous analyses showed that virtually all H. pylori strains carry one or no copies of cagA. This study showed that H. pylori can carry multiple tandem copies of cagA that can change dynamically. Isolates harboring more cagA copies produced more CagA, thus enhancing toxicity to host cells. Analysis of 314 H. pylori clinical strains isolated from patients in South Korea and the United States showed that 7.5% of clinical strains in the United States carried multiple cagA copies whereas none of the South Korean strains did. This study demonstrated a novel molecular mechanism by which H. pylori dynamically modulates cagA copy number, which affects CagA expression and activity and may impact downstream development of gastric disease.

Type 1 diabetes with periodontitis shows elevated TNF-α expression. Tumor necrosis factor (TNF)-α stimulates the expression of receptor activator of nuclear factor-κB ligand (RANKL) and sclerostin. The objective of this study was to determine the effect of TNF-α expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis using infliximab (IFX), a TNF-α antagonist. Rats were divided into two timepoint groups: day 3 and day 20. Each timepoint group was then divided into four subgroups: 1) control (C, n = 6 for each time point); 2) periodontitis (P, n = 6 for each time point); 3) diabetes with periodontitis (DP, n = 8 for each time point); and 4) diabetes with periodontitis treated with IFX (DP+IFX, n = 8 for each time point). To induce type 1 diabetes, rats were injected with streptozotocin (50 mg/kg dissolved in 0.1 M citrate buffer). Periodontitis was then induced by ligature of the mandibular first molars at day 7 after STZ injection (day 0). IFX was administered once for the 3 day group (on day 0) and twice for the 20 day group (on days 7 and 14). The DP group showed greater alveolar bone loss than the P group on day 20 (P = 0.020). On day 3, higher osteoclast formation and RANKL-positive osteocytes in P group (P = 0.000 and P = 0.011, respectively) and DP group (P = 0.006 and P = 0.017, respectively) than those in C group were observed. However, there was no significant difference in osteoclast formation or RANKL-positive osteocytes between P and DP groups. The DP+IFX group exhibited lower alveolar bone loss (P = 0.041), osteoclast formation (P = 0.019), and RANKL-positive osteocytes (P = 0.009) than that of the DP group. On day 20, DP group showed a lower osteoid area (P = 0.001) and more sclerostin-positive osteocytes (P = 0.000) than P group. On days 3 and 20, the DP+IFX group showed more osteoid area (P = 0.048 and 0.040, respectively) but lower sclerostin-positive osteocytes (both P = 0.000) than DP group. Taken together, these results suggest that TNF-α antagonist can diminish osteocytic RANKL/sclerostin expression and osteoclast formation, eventually recovering osteoid formation. Therefore, TNF-α might mediate alveolar bone loss via inducing expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis.

Osteocytes are increasingly recognized as significant sources of osteoclast differentiation factor, receptor activator of nuclear factor-κB ligand (RANKL), and osteoblast differentiation inhibitory factor, sclerostin. In this study, RANKL and sclerostin expression of osteocytes is investigated in rats with ligature-induced periodontitis.Rats were divided into control and periodontitis groups, and periodontitis was induced by ligature on the mandibular first molars. At 1, 3, 10, and 20 days after ligature, histologic analyses of alveolar bone (AB) and osteoid areas in the molar furcation were performed. The numbers of osteoclasts and RANKL- and sclerostin-positive osteocytes were estimated by tartrate-resistant acid phosphatase staining and immunohistochemistry, respectively.The AB area gradually decreased at day 10 after ligature and increased at day 20. The number of osteoclasts markedly increased at day 3 and then decreased. Conversely, osteoid formation was suppressed up to day 3 and then showed a remarkable increase above control level at day 20. The number of RANKL-positive osteocytes increased at days 1 and 3 and then decreased. Sclerostin-positive osteocytes markedly increased at days 3 and 10 but decreased below control level at day 20.These results show that AB loss is accompanied by enhanced osteoclast formation and suppressed osteoid formation. Osteocytes express RANKL when osteoclast formation increases, and they express sclerostin when osteoid formation is suppressed. Conversely, osteocytic sclerostin expression decreases when osteoid formation increases. These findings suggest that osteocytes may be important in AB loss via RANKL and sclerostin expression in periodontitis.

Background and objectives Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts are formed in sequential steps: proliferation and differentiation of hematopoietic progenitors into quiescent osteoclast precursors (QOPs), followed by fusion of QOPs. In this study, we investigated whether enhancement of osteoclast formation by periodontitis is derived from the stimulation of proliferation of hematopoietic progenitors or the differentiation of QOPs into osteoclasts. Material and methods Ligatures were placed around the first molars in the left mandibles of Fischer 344 inbred rats. The rats received drinking water containing bromodeoxyuridine (BrdU) (which can be incorporated into dividing nuclei) after ligation during the experimental period. The number of inflammatory cells in the distal area was counted. Alveolar bone loss was histologically estimated by measuring the distance from the cementoenamel junction to the alveolar bone crest in the distal area and determining the percentage of periodontal ligament area in the furcation. The number of osteoclasts and percentage of BrdU+ nuclei in total osteoclasts nuclei were counted after TRAP and BrdU double labeling. Results The number of polymorphonuclear cells increased on day 1 and then rapidly decreased. The number of mononuclear cells increased in a time-dependent manner up to day 5 and remained the same until day 10. Alveolar bone loss of ligatured teeth increased in a time-dependent manner. The number of osteoclasts peaked on day 3 then gradually decreased. At peak, the percentage of BrdU+ nuclei in total osteoclasts nuclei in the distal and furcation areas were 7.9% and 4.1%, respectively. Conclusion These results indicate that most of the osteoclasts formed after periodontitis induction are derived from preformed QOPs, suggesting that enhancement of osteoclast formation by periodontitis might be mainly caused by stimulating the differentiation of QOPs into osteoclasts.

Gallic acid, a phenolic phytochemical, has been shown to exert a variety of effects, including anti-oxidative, anti- carcinogenic, anti-allergic, and anti-inflammatory effects. In this study, we attempted to determine whether gallic acid affects metabolic syndrome such as obesity and diabetes. Diet-induced obesity mice were treated intraperitoneally once per day with gallic acid (10 mg/kg/day). After 2 weeks of treatment, the mice were sacrificed to collect the blood for metabolic parameter assessments, and the adipose tissues and liver to weigh and analyze. The triglyceride concentrations were significantly improved in the gallic acid group relative to those measured in the control group. And most importantly, the blood glucose concentrations in the gallic acid group were significantly improved. In the epididymal white adipose tissue of the gallic acid group, adipocyte size was reduced, PPARγ expression was induced, and the Akt signaling pathway was activated. Our results demonstrate that gallic acid improves glucose tolerance and lipid metabolism in the obesity mice, thereby showing evidence of anti-hyperglycemic activity. The findings of an upregulation of PPARγ expression and Akt activation also contribute to our current understanding of the mechanisms underlying the effects of gallic acid on glucose metabolism.

This study examined the effect of the interaction between periodontitis and type 1 diabetes mellitus on alveolar bone, mandibular condyle and tibia in animal models.Rats were divided into normal, periodontitis, diabetic and diabetic with periodontitis groups. After injection of streptozotocin to induce diabetes, periodontitis was induced by ligation of both lower-side first molars for 30 days. Alveolar bone loss and trabecular bone volume fraction (BVF) of the mandibular condyle and tibia were estimated via hematoxylin and eosin staining and micro-computed tomography, respectively. Osteoclastogenesis of bone marrow cells isolated from tibia and femur was assayed using tartrate-resistant acid phosphatase staining.The cemento-enamel junction to the alveolar bone crest distance and ratio of periodontal ligament area in the diabetic with periodontitis group were significantly increased compared to those of the periodontitis group. Mandibular condyle BVF did not differ among groups. The BVF of tibia in the diabetic and diabetic with periodontitis groups was lower than that of the normal and periodontitis groups. Osteoclastogenesis of bone marrow cells in the diabetic groups was higher than that in the non-diabetic groups. However, the BVF of tibia and osteoclastogenesis in the diabetic with periodontitis group were not significantly different than those in the diabetic group.Type 1 diabetes mellitus aggravates alveolar bone loss induced by periodontitis, but periodontitis does not alter the mandibular condyle and tibia bone loss induced by diabetes. Alveolar bone, mandibular condyle and tibia may have different responses to bone loss stimuli in the diabetic environment.

Helicobacter pylori is an important factor of chronic gastritis, digestive ulcer, and stomach cancer. CagL, a virulence factor of H. pylori, is well-known as a pilus protein which acts as adhesion to host cell and a component of Type 4 secretion system. In this study, we evaluated the protective response of recombinant CagL protein (rCagL) using Mongolian gerbil animal model for H. pylori infection. The cagL gene was cloned from 26695 H. pylori followed by over-expression and purification of the protein in E. coli. Mongolian gerbils were immunized with rCagL protein mixed with aluminum adjuvant via intramuscular injections once a week during 4 weeks. At a week after the last immunization, the Mongolian gerbils were administrated with H. pylori 7.13 strain into the stomach and sacrificed to measure antibody titer on rCagL by ELISA and bacterial colonization in the stomach, and to examine the histopathological changes and cytokine expression at 6 week after challenge. Antibody titers on recombinant protein were significantly increased from a week after the first immunization. There was no significant change of the number of bacterial colony between control group and immunized group. The relative stomach weight was significantly decreased in immunized group, but the significant change of histopathological assessment was not observed in the stomach. Cytokine expression such as IL- and KC also was not significantly different between control and immunized groups. These results indicate that rCagL could effectively induce the formation of the specific IgG antibodies. However, bacterial colonization and histopathological lesions could not be inhibited by the immunization in the stomach, indicating not enough protection against H. pylori infection. We consider that along with CagL other adequate antigens could be needed stimulating immune response and inducing protective effects against gastric disease, and also a better adjuvant could be considered.

This study was conducted to develop a novel salad dressing composite recipe of natural seasoning containing the dried sea tangle(Laminaria japonica) that has a high preference. Sea tangle(Laminaria japonica) is included in the vitamins and minerals, magnesium, calcium, iodine, iron content, such as high, and contained in Sea tangle alginate is not a small conference known as dietary fiber. To manufacture salad dressing with sea tangle, dressing with 0%, 3%, 6%, 9%, and 12% added sea tangle were prepared and tested for quality. The pH tended to increase with the increased sea tangle in addition but in contrast, acidity showed. The ‘L’ color decreased with added sea tangle, whereas the ‘a’ and ‘b’ values increased. Brix measurements increase with added sea tangle. The strength texture results, 0% was the highest, lowest 9%. Bitterness and chewiness texture results, 9% was the highest, lowest 0%. According to the sensory test results, in the topic overall quality 3% was by 3.76 point the highest. But, during total nine clause, in clause six, by 6% was the highest.Key world: Laminaria japonica, salad dressing, vinegar, acidity, natural materials

Chana series are new chalcone derivatives. To evaluate the possibility of Chana series as therapeutic agents of type 2 diabetes, the inhibitory effects of Chana series on the activities of α-glucosidase and DPP-4 were investigated using in vitro enzyme assays, and their effects on adipocyte differentiation were investigated in C3H10T1/2 cells. Chana 1 and Chana 7 among the Chana series showed significant inhibition of α-glucosidase activity. In DPP-4 enzyme assay, Chana 1 exhibited the highest inhibitory activity while Chana 7 did not. In MTT assay, Chana 1 did not show significant cytotoxicity up to a concentration of 250 μM, whereas cytotoxicity was observed with Chana 7 at a concentration of 300 μM. In addition, Chana 1 induced adipocyte differentiation. Therefore, Chana 1 showed inhibitory effects on α-glucosidase and DPP-4 as well as a stimulatory effect on adipocyte differentiation, suggesting that Chana 1 may be a potential beneficial agent for the treatment of type 2 diabetes.

Gastritis, peptic ulcer disease, and gastric cancer are a few of the diverse disease manifestations that have been shown to be associated with infection by Helicobacter pylori . Why some individuals develop more severe forms of disease remains largely unknown. In this study, 225 South Korean strains were genotyped for vacA and then analyzed to determine if particular genotypes varied across disease state, sex, or cagA allele. Of these strains, 206 strains carried an s1/i1/m1 allele, 11 strains carried an s1/i1/m2 allele, and 8 strains carried an s1/i2/m2 allele. By using Fisher's exact test, a statistical association between variations in the cagA and vacA alleles was identified ( P = 0.0007), and by using log linear modeling, this variation was shown to affect the severity of disease outcome ( P = 0.027). Additionally, we present evidence that variation within the middle region of VacA contributes significantly to the distribution of vacA alleles across gender ( P = 0.008) as well as the association with disease outcome ( P = 0.011). In this South Korean population, the majority of H. pylori strains carry the vacA s1/i1/m1 allele and the CagA EPIYA-ABD allele. These facts may contribute to the high incidence of gastric maladies, including gastric cancer.

To evaluate the inhibitory activity of wogonin against lipopolysaccharide (LPS)-induced bone resorption, we investigated the effect of wogonin on osteoclastogenesis induced by LPS. Wogonin inhibited LPS-induced osteoclastogenesis in co-cultures of mouse calvaria-derived osteoblasts and bone marrow-derived pre-osteoclasts. Wogonin also suppressed osteoclastogenesis in LPS-injected mouse calvaria. In osteoblasts, the upregulation of receptor activator of nuclear factor-κB (RANKL) expression and the downregulation of osteoprotegerin (OPG) expression by LPS were inhibited by wogonin. Wogonin and NS-398, a COX-2 inhibitor, suppressed LPS-stimulated PGE2 production in osteoblasts. NS-398 inhibited the effect of LPS on RANKL and OPG expression in osteoblasts. These results suggest that wogonin acts as an inhibitor of LPS-induced osteoclastogenesis through downregulation of RANKL and upregulation of OPG expression via blockage of PGE2 production. Based on these results, wogonin has potential for use as a therapeutic agent in bacteria-induced bone resorption. Copyright © 2009 John Wiley & Sons, Ltd.

Adipocytokines, bioactive molecules secreted from adipose tissues, play important roles in physiology, development, and disease. Recently, heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified as an adipocytokine whose expression correlates with obesity. However, the biological role of fat-secreted HB-EGF is still unclear. In this study, we investigated the effects of HB-EGF on the adipocyte differentiation of C3H10T1/2 pluripotent mesenchymal cells. Upon adipogenic conversion of C3H10T1/2 cells, HB-EGF displayed dynamic changes in expression where an initial decrease was followed by increased levels of expression at later stages. HB-EGF treatment during adipogenic induction inhibited lipid accumulation and decreased the expression of adipocyte molecular markers (fatty acid-binding protein, peroxisome proliferator-activated receptor gamma, and CAAT enhancer-binding protein alpha) and lipogenic genes (glucose transporter, fatty acid synthetase, and lipoprotein lipase). Therefore, HB-EGF has an inhibitory effect on adipocyte differentiation. Administration of HB-EGF at various intervals during adipocyte differentiation revealed that HB-EGF acts during the early stages of adipocyte differentiation, but not at the later stages of differentiation. Furthermore, HB-EGF was able to block the commitment of pluripotent mesenchymal cells to the adipocyte lineage triggered by bone morphogenic protein 4 treatment. These data suggest that HB-EGF acts as a negative regulator of adipogenesis by inhibiting the commitment and early differentiation of the adipose lineage. The inhibitory role of HB-EGF on adipocyte differentiation of pluripotent mesenchymal cells sheds light on potential mechanisms that control adipose tissue homeostasis.

Periodontitis is an inflammatory disease that often leads to destruction of alveolar bone; a number of bacteria in subgingival plaque are associated with bone destruction in periodontitis. To understand the mechanism of how periodontopathogens induce osteoclastogenesis, we determined which mediators are involved in the osteoclastogenesis.We investigated effects of sonicates from three periodontopathic bacteria, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, on osteoclast formation in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. The osteoclast formation was determined by tartrate resistant acid phosphatase (TRAP) staining. The expression of the receptor activator of nuclear factor-kappa B ligand (RANKL), prostaglandin E(2) (PGE(2)) and osteoprotegerin (OPG) in mouse calvaria-derived osteoblasts was determined by immunoassay.Each bacterial sonicate induced the osteoclast formation in the co-culture system. These bacterial sonicates increased the expression of RANKL and PGE(2), and decreased the expression of OPG in osteoblasts. The addition of OPG, an inhibitor of RANKL, in the co-culture completely suppressed the osteoclastogenesis that was stimulated by each bacterial sonicate. Indomethacin, which is an inhibitor of PGE(2) synthesis, reduced more than 88% of the osteoclast formation induced by each bacterial sonicate. Indomethacin inhibited more than 80% of RANKL expression in osteoblasts induced by T. denticola and T. socranskii, and 59% by P. gingivalis. Indomethacin completely recovered the depression of OPG expression in osteoblasts by T. denticola and T. socranskii to the level of the untreated osteoblasts. Indomethacin recovered the reduction of OPG expression by P. gingivalis to 67%.These findings suggest that the osteoclastogenesis by P. gingivalis, T. denticola, and T. socranskii is mediated by a RANKL-dependent pathway and that PGE(2) is a main factor in the pathway by the enhancing of RANKL expression and the depression of osteoprotegerin, a RANKL inhibitor.

The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 micro M bumetanide reduced RANKL mRNA expression induced by 10 nM 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2-terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1alpha,25(OH)2D3-induced osteoclastogenesis partly via the phosphorylation of JNK.

Objectives: The purpose of this study was to evaluate a partially purified extract (elm extract) from the Ulmi cortex (Ulmi macrocarpa Hance) and its active ingredient, a mix of procyanidin oligomers (3 to 12 flavan-3-ol monomers, an average molecular weight of 1,518 with an average polymerization degree of 5.3) for a possible inhibitory effect against proteases. Background: Host-derived matrix metalloproteinases (MMPs) and bacterial proteases play important roles in the gingival tissue destruction that is a characteristic of periodontitis. The inhibitors of these proteases may be developed into therapeutic agents against periodontitis. Methods: The inhibitory effects were assessed by gelatin zymography. The MMPs tested were originated from the gingival crevicular fluid (GCF) of adult periodontitis patients and from the conditioned media of cultured periodontal ligament (PDL) cells, which provided the proMMP-2 and activated MMP-2 when treated with a periodontopathogen, Treponema lecithinolyticum. Bacterial enzymes tested were secreted forms from two major periodontopathogens, Porphyromonas gingivalis and Treponema denticola. In addition, the inhibitory effects on trypsin-like enzymes from these two periodontopathogens were assayed by the n-benzoyl-DL-arginine-naphthylamide (BANA) test. Results: The elm extract and the procyanidin oligomer (100-1,000 μg/ml) exhibited potent inhibitory effects on the MMPs in GCF (chiefly MMP-8 and MMP-9), the pro and active forms of MMP-2, and secreted and trypsin-like enzymes from T. denticola and P. gingivalis. Conclusions: These results suggest that elm cortex should be considered as a potential agent against periodontal diseases, due to its inhibitory action on MMPs and the proteases of periodontopathogens.

Periodontal tissue destruction is a characteristic of periodontitis. This can be caused by either bacterial enzymes or host cell-derived matrix metalloproteinases (MMPs). In order to elucidate the etiologic role of oral spirochetes, we investigated the effects of Treponema lecithinolyticum, a novel saccharolytic species, on MMP-2 activation.Gingival fibroblasts (GFs) and periodontal ligament (PDL) cells obtained from healthy human subjects were cultured to confluence in alpha-minimal essential medium (alpha-MEM) supplemented with 10% fetal bovine serum. After serum starvation for a day, the cultures were treated with whole cell sonicates, heat-denatured whole cell sonicates, outer membrane fraction (OMF) or formaldehyde-fixed cells of T. lecithinolyticum. Culture supernatants were collected after incubation for 24 to 48 hours and analyzed for MMP-2 activation by gelatin zymography. Collagenolytic activity was quantitatively measured using human [3H] type IV collagen as a substrate.Treatment of GFs and PDL cells with whole cell sonicates, formaldehyde-fixed whole cells, or the OMF of T. lecithinolyticum resulted in the production of MMP-2 partly in the fully active form with a molecular mass of 62 kDa, whereas non-treated control cultures and cultures treated with a heat-denatured fraction did not show the active form. Cultures exposed to T. lecithinolyticum had higher collagenolytic activity than non-treated cultures.Our results demonstrate that T. lecithinolyticum, possibly mediated by proteinaceous cell surface-associated components, may participate in extracellular matrix degradation by activation of MMP-2 during periodontal inflammation.

Objectives. Recent molecular approaches have revealed that fastidious organisms such as Bacteroides forsythus and oral treponemes were frequently found in root canals with apical periodontitis. The purpose of this study was to identify the isolates of oral spirochetes at the species level in endodontic infections and to determine their association with B forsythus and Porphyromonas gingivalis. Study design. Seventy-nine teeth with apical periodontitis were selected for this study. After sampling from the root canals aseptically, polymerase chain reaction amplification for the 16S rRNA gene was performed with eubacterial universal primers. Subsequently, dot-blot hybridization was performed with 8 species-specific oligonucleotide probes. The microbial associations were analyzed by using the odds ratio. Results. The most frequently found species was P gingivalis (27.4%), followed by Treponema maltophilum (26%), B forsythus (16.4%), and Treponema socranskii (2.7%). Other treponemes, including Treponema denticola, were not detected in our samples. Significant microbial associations were identified between T maltophilum, B forsythus, and P gingivalis by performing analysis with the odds ratio. Conclusion. Our results indicate that T maltophilum should be included in etiologic studies of endodontic diseases. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:329-34)

Alveolar bone destruction is a characteristic feature of periodontal diseases and multinucleated osteoclast cells derived from hemopoietic cells are responsible for bone resorption. Treponema lecithinolyticum is a novel oral spirochete isolated from the periodontal lesions.The effect of whole cell sonicates on the osteoclast differentiation was examined in a co-culture system of hemopoietic mouse bone marrow cells and calvaria derived-osteoblastic cells to clarify the role of T. lecithinolyticum in the alveolar bone destruction associated with periodontal diseases. The differentiated osteoclasts were confirmed by tartrate-resistant acid phosphatase (TRAP) staining.Sonicates of this bacterium stimulated the osteoclast formation in the co-culture system in a dose-dependent manner. The sonicates-induced osteoclast formation was partially inhibited by the heat treatment of sonicates. Indomethacin, which is a prostaglandin inhibitor, decreased the osteoclast formation induced by the bacterial sonicates.These findings suggest that T. lecithinolyticum induces osteoclast differentiation by a prostaglandin E2-dependent mechanism and that heat-labile components may be involved in this process.

Periodontitis is a very common oral inflammatory disease that results in the destruction of supporting connective and osseous tissues of the teeth. Although the exact etiology is still unclear, Gram-negative bacteria, especially Porphyromonas gingivalis in subgingival pockets are thought to be one of the major etiologic agents of periodontitis. Endothelin (ET) is a family of three 21-amino acid peptides, ET-1, -2, and -3, that activate G protein-coupled receptors, ETA and ETB. Endothelin is involved in the occurrence and progression of various inflammatory diseases. Previous reports have shown that ET-1 and its receptors, ETA and ETB are expressed in the periodontal tissues and, that ET-1 levels in gingival crevicular fluid are increased in periodontitis patients. Moreover, P. gingivalis infection has been shown to induce the production of ET-1 along with other inflammatory cytokines. Despite these studies, however, the functional significance of endothelin in periodontitis is still largely unknown. In this study, we explored the cellular and molecular mechanisms of ET-1 action in periodontitis using human gingival epithelial cells (HGECs). ET-1 and ETA, but not ETB, were abundantly expressed in HGECs. Stimulation of HGECs with P. gingivalis or P. gingivalis lipopolysaccharide increased the expression of ET-1 and ETA suggesting the activation of the endothelin signaling pathway. Production of inflammatory cytokines, IL-1β, TNFα, and IL-6, was significantly enhanced by exogenous ET-1 treatment, and this effect depended on the mitogen-activated protein kinases via intracellular Ca2+ increase, which resulted from the activation of the phospholipase C/inositol 1,4,5-trisphosphate pathway. The inhibition of the endothelin receptor-mediated signaling pathway with the dual receptor inhibitor, bosentan, partially ameliorated alveolar bone loss and immune cell infiltration. These results suggest that endothelin plays an important role in P. gingivalis-mediated periodontitis. Thus, endothelin antagonism may be a potential therapeutic approach for periodontitis treatment.

Although extensive microbial analyses have been performed from subgingival plaque samples of periodontitis patients, systematic analysis of subgingival microbiota has not been carried out in a Korean population so far. The purpose of this study was to describe the prevalence of major putative periodontopathogens in Korean patients by culture-independent methods.A total of 244 subgingival plaque samples (5 sites in each participant) were taken from 29 advanced adult periodontitis (AP) patients and 20 periodontally healthy subjects. AP samples were obtained from the 4 deepest periodontal pockets (or =6 mm probing depth [PD]) and 1 healthy site (or =3 mm PD) in each patient. Polymerase chain reaction (PCR) of 16S ribosomal DNA (rDNA) of subgingival plaque bacteria was performed with eubacterial primers. Aliquots of PCR products were then applied on nylon membranes and hybridized with specific oligonucleotide probes labeled with digoxigenin.All diseased sites harbored Fusobacterium sp., while Porphyromonas gingivalis, Treponema sp., and Bacteroides forsythus were detected in more than 96% of 116 diseased sites. Peptostreptococcus micros, Actinobacillus actinomycetemcomitans, and Prevotella intermedia were present in 82%, 74%, and 71% of diseased sites, respectively. In sites of periodontally healthy subjects, Fusobacterium sp. was present in the highest proportion (58%). Treponema sp., P. gingivalis, and B. forsythus were detected in 22%, 18%, and 18% of healthy sites, respectively. P. micros, P. intermedia, and A. actinomycetemcomitans were found in 8%, 2%, and 1% of healthy sites, respectively. The prevalence of the periodontopathogens, with the exceptions of Fusobacterium sp. and B. forsythus, was significantly higher in the healthy sites of periodontitis subjects than in the healthy sites of periodontally healthy subjects (P0.05).Using highly sensitive methods relying on 16S ribosomal RNA-based oligonucleotide probes, we confirmed the strong association of 7 putative periodontopathogens with AP patients in a Korean population. With the exceptions of Fusobacterium sp. and B. forsythus, all the periodontopathogens were significantly more associated with the healthy sites of periodontitis subjects than in the healthy sites of periodontally healthy subjects.

Background and Objective Epigallocatechin-3-gallate (EGCG) is known for its beneficial properties, including anti-inflammatory and anti-oxidative activities. Recently, reports have suggested that EGCG plays a pivotal role in regulating cytokine expression and osteoclastic activity. In the present study, we investigated whether orally administered EGCG has a therapeutic effect on ligature-induced periodontitis. Materials and Methods Forty-eight Sprague–Dawley rats were treated with EGCG or phosphate-buffered saline. Periodontitis was induced by tying a ligature for 7 d. After removing ligation, EGCG (200 mg/kg) or phosphate-buffered saline was administered via oral gavage on a daily basis. Rats were killed after 1, 2 and 4 wk of administration. Histologic and histomorphometric analyses, tartrate resistant acid phosphatase staining and immunohistochemistry were carried out. Results In the control group, bone loss did not recover even after the causative factor of periodontitis was eliminated. On the other hand, distance from cemento-enamel junction to alveolar bone crest, long junctional epithelium and collagen destruction were reduced in the EGCG group. Decreased interleukin (IL)-6 expression was shown from the early stage of EGCG administration, followed by reduced tumor necrosis factor (TNF) expression at week 4 EGCG group. The CT area showed a higher decrease of IL-6 expression between the control and EGCG group than alveolar bone area. Downregulation of TNF and IL-6 expression led to a decrease in osteoclast number and activity, which resulted in reduced bone loss. Conclusions Systemic administration of EGCG could have a therapeutic effect on damaged periodontal tissue. Inhibited cytokine expression, including TNF and IL-6 is responsible for the reduction in osteoclast formation, osteoclastic activity and collagen destruction.

The objective of this study was to investigate the ability of alendronate and taurine in inhibiting in vitro osteoclast differentiation induced by bacteria. Whole cell sonicates of Porphyromonas gingivalis were used as an osteoclast-stimulating factor in a mouse coculture system and differentiated osteoclasts were confirmed by tartrate-resistant acid phosphatase (TRAP) staining. Alendronate at the concentrations of 10(-7) M, 10(-6) M, and 10(-5) M and taurine at the concentrations of 4 mM, 8 mM, and 12 mM were used. The cytotoxic effects of alendronate and taurine were examined using methylthiazole-tetrazolium bromide (MTT) assay. The amounts of interleukin-6 (IL-6) in culture supernatants were also measured using ELISA. The sonicates of P. gingivalis at the concentration of 0.01-0.1 microg/ml significantly stimulated the formation of osteoclasts (p0.05). Alendronate (10(-5) M) and taurine (12 mM) significantly suppressed the sonicate-stimulated osteoclast formation. In MTT assay, no cytotoxic effects were evident in all concentrations of alendronate and taurine. Alendronate and taurine did not affect the amount of IL-6 induced by P. gingivalis sonicates. These data indicate that alendronate and taurine have inhibitory effects on bacteria-stimulated osteoclast formation in vitro and that this inhibitory mechanism is not related to the blocking of IL-6 production.