Enhancing production of herpes simplex virus type 1 in oral epithelial cells by co-infection with Aggregatibacter actinomycetemcomitans.

BACKGROUND/PURPOSE: The association between herpetic/bacterial co-infection and periodontal diseases has been reported. However, how interactions between herpesviruses and periodontal bacteria dampen periodontal inflammation is still unclear. This study determined effects of co-infection with oral bacteria, including Streptococcus sanguinis, Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans, in herpes simplex virus type 1 (HSV-1)-infected oral epithelial cells. METHODS: Cell viability was determined by detection the activity of mitochondrial dehydrogenase. Viral production was measured using the plaque assay. Levels of bacterial and viral DNA were determined by real-time polymerase chain reaction. Secretion of interleukin (IL)-6 and IL-8 was measured using the enzyme-linked immunosorbent assay. RESULTS: Viability was not further reduced by bacterial co-infection in HSV-1-infected cells. Co-infection with HSV-1 and S. sanguinis or F. nucleatum reduced the viral yield whereas co-infection with HSV-1 and A. actinomycetemcomitans significantly enhanced the viral yield in oral epithelial cells. The enhancing effect of A. actinomycetemcomitans was not affected by bacterial heat-inactivation. Co-infection with HSV-1/A. actinomycetemcomitans increased intracellular levels of both viral and bacterial DNA. Secretion of IL-6 and IL-8 stimulated by A. actinomycetemcomitans infection was partly reduced by co-infection with HSV-1 in oral epithelial cells. CONCLUSION: In contrast to S. sanguinis and F. nucleatum, A. actinomycetemcomitans enhanced the yield of HSV-1. Either HSV-1 or A. actinomycetemcomitans may be benefited from co-infection, in aspects of increases in production of viral and bacterial DNA as well as reductions in cytokine secretion. These findings echoed with previous clinical studies showing co-infection of HSV and A. actinomycetemcomitans in patients with aggressive periodontitis.

Modulation of proinflammatory mediators by viruses-bacteria synergism in human osteoblasts-an in vitro study.

BACKGROUND/PURPOSE: Viruses-bacteria synergistic interaction is associated with destructive periodontal diseases. However, the underlying mechanism for tissue destruction is not fully elucidated. In this study, lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS) and polyinosinic-polycytidylic acid (poly I:C) were used to simulate bacteria and viruses, respectively. The possible combined effects of both molecular patterns on secretion of interleukin (IL)-6 and prostaglandin E2 (PGE2) from osteoblasts were determined. The effects of povidone-iodine (PVP-I) on the secretion of IL-6 and PGE2 were also examined. METHODS: Viability of treated osteoblastic cells (MG63) was examined by detection the mitochondrial dehydrogenase activity. Secretion of IL-6 and PGE2 was detected using the enzyme-linked immunosorbent assay (ELISA). Mitogen-activated protein kinases (MAPKs) and cyclooxygenase-2 (COX-2) were determined using the Western blotting analysis. RESULTS: Pg-LPS or poly I:C significantly enhanced the production of IL-6 and PGE2 in MG63 cells. The additive/synergistic effects of Pg-LPS/poly I:C on production of IL-6 and PGE2 were evident. The levels of phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) and expression of COX-2 protein were enhanced by Pg-LPS and/or poly I:C. On the other hand, the level of phosphorylation of extracellular signal-regulated kinase (ERK) was reduced by Pg-LPS and/or poly I:C. The stimulatory secretion of PGE2 by poly I:C was significantly reduced by PVP-I. CONCLUSION: Concomitant infection of viruses and bacteria may be potentially harmful to the tooth supporting tissues by production of proinflammatory mediators. The results suggest the potential anti-inflammatory effect of PVP-I on bacterial or viral infection.

The role of fibroblasts in the modulation of dental pulp inflammation.

BACKGROUND/PURPOSE: Dental pulp fibroblasts can protect dental pulp from microbial invasion. However, little is known about the interaction between pulp fibroblasts and the immune cells. In this study, the production of proinflammatory cytokines related to inflammatory cell recruitment was evaluated in tumor necrosis factor (TNF)-α-stimulated human dental pulp fibroblasts (HDPFs). The role of TNF-α-stimulated HDPFs in the cell fusion under inflammatory process was determined with the cell co-culture with peripheral blood mononuclear cells (PBMCs). METHODS: HDPFs were stimulated with various concentrations of TNF-α, and the secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 was analyzed by the enzyme-linked immunosorbent assay. The mRNA expression levels of intercellular adhesion molecule-1 (ICAM-1), macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) were determined by real-time quantitative polymerase chain reaction. TNF-α-treated HDPFs were co-cultured with PBMCs for 21 days, and characteristics of cell differentiation were assessed. RESULTS: TNF-α induced IL-6, IL-8 and MCP-1 production in HDPFs. Moreover, mRNA expression levels of ICAM-1, M-CSF and OPG were significantly increased in TNF-α-treated HDPFs. Co-culture of TNF-α-treated HDPFs and PBMCs stimulated formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, and the F-actin rings were observed in these multinucleated cells. CONCLUSION: Our results indicate that under the stimulation of TNF-α, HDPFs may amplify inflammatory response by cytokines production, which in turn can modulate the differentiation of immune cells.

Effects of epigallocatechin-3-gallate and acyclovir on herpes simplex virus type 1 infection in oral epithelial cells.

BACKGROUND/PURPOSE: Herpes simplex virus type 1 (HSV-1) is the pathogenic agent of human diseases, including gingivostomatitis and herpes labialis. The anti-viral activities of the tea polyphenol, epigallocatechin-3-gallate (EGCG), have been demonstrated. This study examined the combined effects of EGCG and the antiviral drug, acyclovir (ACV), on infection of HSV-1 in oral epithelial cells. METHODS: Cell viability was examined using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide. Viral yields were determined using the plaque assay. Viral proteins were detected using Western blotting analysis or confocal laser scanning microscopy. Viral DNA was detected using the real-time polymerase chain reaction. RESULTS: Cytotoxic effects of HSV-1 on the viability of oral epithelial cells were evidently reduced in the presence of EGCG (25 µg/ml) or/and ACV (50 µg/ml). Viral yields were also significantly reduced by treatment of cells with EGCG or/and ACV. Expression of viral immediate early protein, infected cell protein 0 (ICP0), was greatly inhibited when cells were treated with EGCG. Combined effects of EGCG and ACV were more evident for the expression of viral thymidine kinase, ICP5 and glycoprotein D. EGCG, but not ACV, significantly reduced the levels of viral particles and viral DNA during viral entry phase. However, at 20 h post infection, the intracellular viral DNA was evidently reduced in HSV-1 infected cells treated with EGCG and ACV. Moreover, the stimulatory effects of HSV-1 on phosphorylation of c-Jun N-terminal kinase could be reduced by ACV. CONCLUSION: The results demonstrated the additive effects of EGCG and ACV on HSV-1 infection in oral epithelial cells.

Stimulatory effects of Porphyromonas gingivalis GroEL protein on interleukin-6 and interleukin-8 in human osteoblasts.

BACKGROUND/PURPOSE: Porphyromonas gingivalis is an oral pathogen associated with periodontal diseases. P. gingivalis GroEL protein is a stimulator of inflammatory cytokines in macrophages. This study inspected effects of P. gingivalis GroEL protein on production of interleukin (IL)-6 and IL-8 by human osteoblasts. METHODS: Viability of GroEL-treated osteoblasts was analyzed with 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide. Secretion of IL-6 and IL-8 was analyzed using the enzyme-linked immunosorbent assay. Levels of mRNA were analyzed using the reverse transcription and real-time polymerase chain reaction. The antioxidant (curcumin), the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) and the c-Jun N-terminal kinase (JNK) inhibitor (SP600125) were employed to elucidate possible signaling pathways involved. RESULTS: Treatment with GroEL did not affect morphology and viability of osteoblasts. GroEL significantly induced the secretion of IL-6 and IL-8 by osteoblasts in a concentration-dependent pattern. Moreover, the mRNA levels of IL-6 and IL-8 were stimulated by GroEL. The application of SP600125 (10 µM) significantly suppressed the induction of IL-6 and IL-8 by GroEL-treated cells. However, curcumin (20 µM) and SB203580 (20 µM) only down-regulated the stimulatory effects of GroEL on IL-6. CONCLUSION: GroEL protein stimulated the inflammatory reaction of osteoblasts, probably through the activation of p38 MAPK or JNK pathway. The findings suggest that P. gingivalis GroEL may influence the immune functions of osteoblasts and endanger the periodontal health.

Inhibition of Streptococcus mutans by a commercial yogurt drink.

BACKGROUND/PURPOSE: Studies have been focused on using probiotics to prevent caries. The lactobacillus probiotic bacteria in Yakult® (LcY) has been shown to inhibit the growth or biofilm formation of Streptococcus mutans. However, sucrose in Yakult® raised concerns. The purpose of this study was to determine effects of Yakult® on the growth and adhesion of S. mutans. MATERIALS AND METHODS: S. mutans was grown in serial diluted Yakult®, filtered Yakult® or 20% heated Yakult®. S. mutans was co-cultured with LcY in media with or without diluted filtered Yakult®, or in LcY grown in media with or without sugars. Colony forming units and pH values of bacterial cultures were determined. SYTO 9-stained adhered bacteria were observed. RESULTS: Yakult® inhibited the growth of S. mutans. Filtering or heating Yakult® reduced its inhibitory ability against S. mutans. The inhibitory effect of LcY against S. mutans was enhanced when cultured in the presence of 20% filtered Yakult®. LcY cultured in sucrose media for 24 h inhibited the growth of S. mutans, but this effect was less evident when LcY was grown for 48 h. LcY grown in glucose or lactose media similarly reduced S. mutans growth. Culturing S. mutans with LcY grown in sucrose or glucose media reduced bacterial adhesion. However, co-culturing S. mutans with LcY grown in the lactose media did not decrease bacterial adhesion. CONCLUSION: Yakult® and its probiotic content may inhibit S. mutans growth and the effect may be moderated by the type of sugar added for LcY cultivation.

Inhibitory effects of tea catechin epigallocatechin-3-gallate against biofilms formed from Streptococcus mutans and a probiotic lactobacillus strain.

OBJECTIVE: Effects of tea catechin epigallocatechin-3-gallate (EGCG) against biofilm formation by Streptococcus mutans and probiotic Lactobacillus casei in Yakult® (LcY) were examined. DESIGN: Biofilms were formed by S. mutans alone (Sm) and co-culture of S. mutans and LcY (Sm + LcY) in the absence or presence of EGCG. The biomass of biofilms, which were sonicated or not, was measured by the crystal violet assay. Biofilm morphology was observed by scanning electron microscopy. Bacterial viability and extracellular polysaccharides were determined by SYTO9/propidium iodide and dextran-conjugated fluorescein staining, respectively, and confocal microscopy. Gene expression of glucosyltransferase was determined by quantitative polymerase chain reaction. RESULTS: While 250 µg/ml EGCG significantly decreased the biomass and acid production of Sm biofilms, 500 µg/ml EGCG was required to inhibit Sm + LcY biofilm formation and acid production. EGCG decreased the amount of live bacteria present in both Sm and Sm + LcY biofilms. The level of dead bacteria in Sm + LcY biofilms was higher than in Sm biofilms when formed in the presence of 250 µg/ml EGCG. EGCG decreased levels of extracellular polysaccharides in Sm and Sm + LcY biofilms. The extent of biofilm removal by sonication was not different between Sm and Sm+LcY biofilms formed in the absence or presence of 62.5 or 125 µg/ml EGCG. The level of Sm gtfB and gtfD expression in Sm + LcY biofilms was higher than those in the Sm biofilms when formed in the presence of EGCG at 250 µg/ml. CONCLUSION: The results indicated that LcY might interfere the inhibitory effects of EGCG against biofilm formation by S. mutans.

Association between monocyte chemoattractant protein-1 -2518 A/G gene polymorphism and the outcome of the nonsurgical periodontal treatment.

BACKGROUND/PURPOSE: Elevated monocyte chemoattractant protein-1 (MCP-1) is related to severe periodontal destruction. Furthermore, MCP-1 -2518 A/G gene polymorphism affects MCP-1 after inflammatory stimuli. This study analyzed the association between MCP-1 -2518 gene polymorphism and the outcome of nonsurgical periodontal treatment. METHODS: Forty periodontal patients were recruited and MCP-1 -2518 A/G gene polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism assay. The clinical periodontal parameters, including probing depth (PD), clinical attachment level (CAL), gingival index (GI), bleeding index (BI) and plaque index (PI), were recorded before and six weeks after nonsurgical periodontal therapy. Patients were divided into chronic periodontitis (CP) or aggressive periodontitis (AP). Multiple linear regression analysis was performed to investigate certain predictors of the therapy outcome. RESULTS: The frequency of MCP-1 -2518 genotype-positive (carrying allele G) was 42.5%. Poor treatment outcome in PD, GI and BI improvement could be predicted with MCP-1 -2518 A/G genotype and aggressive periodontitis status as the predictor variables. In contrast, MCP-1 -2518 A/A genotype and aggressive periodontitis status could predict better treatment response in PD and BI improvement. However, MCP-1 -2518 genotype did not affect the treatment outcome in patients with chronic periodontitis. CONCLUSION: MCP-1 -2518 A/G genotype might be useful in predicting less favorable nonsurgical treatment outcome in patients with aggressive periodontitis. However, MCP-1 -2518 gene polymorphism may not play a role in patients with chronic periodontitis. This study suggests that MCP-1 -2518 genotype may influence the outcome of nonsurgical periodontal treatment in aggressive periodontitis patients.

Levels of high-mobility group box-1 in gingival crevicular fluid in nonsmokers and smokers with chronic periodontitis.

BACKGROUND/PURPOSE: High-mobility group box-1 (HMGB1), a proinflammatory cytokine, plays a role in inflammatory disorders. Smoking is a well-established risk factor for periodontal disease. The aim of this study was to compare the levels of HMGB1 in the gingival crevicular fluid from periodontally healthy nonsmokers, chronic periodontitis nonsmokers, and chronic periodontitis smokers. Furthermore, the relationship between levels of HMGB1 and periodontal parameters was examined. METHODS: Periodontal parameters of 17 nonsmokers with chronic periodontitis, nine smokers with chronic periodontitis, and nine periodontally healthy nonsmokers were examined. Gingival crevicular fluid samples were collected, and the levels of HMGB1 were analyzed using the enzyme-linked immunosorbent assay. RESULTS: The median level of HMGB1 was statistically significantly higher in chronic periodontitis nonsmokers (37.5 ng/mL) than in chronic periodontitis smokers (9.5 ng/mL) and periodontally healthy nonsmokers (3.7 ng/mL). There was no significant difference in the levels of HMGB1 between chronic periodontitis smokers and periodontally healthy nonsmokers. Levels of HMGB1 were positively correlated with plaque index, gingival index, probing depth, and clinical attachment level of nonsmokers. However, no significant correlations were found between levels of HMGB1 and all periodontal parameters examined in chronic periodontitis smokers. CONCLUSION: Chronic periodontitis nonsmokers had elevated levels of HMGB1 in gingival crevicular fluid. Moreover, the levels of HMGB1 were correlated with severity of periodontitis. Chronic periodontitis smokers exhibited lower levels of HMGB1 than chronic periodontitis nonsmokers. Further research is needed for understanding the role of HMGB1 in smoking and pathogenesis of periodontitis.

Bacterial adhesion to antibiotic-loaded guided tissue regeneration membranes - a scanning electron microscopy study.

BACKGROUND/PURPOSE: Bacterial contamination of sites undergoing guided tissue regeneration (GTR) therapy may reduce the efficiency of periodontal regeneration. This study compared bacterial adhesion onto various GTR membranes incorporated with antibiotics. METHODS: Three barrier membranes, including expanded polytetrafluoroethylene (ePTFE) membrane, collagen membrane, and glycolide fiber membrane, were loaded with tetracycline or amoxicillin. The adhesion of Streptococcus mutans and Aggregatibacter actinomycetemcomitans onto the GTR membranes with or without antibiotics was analyzed using the scanning electron microscopy (SEM) analysis. RESULTS: The SEM analysis showed no apparent alteration in the physical structure of the membranes loaded with antibiotics. Both S. mutans and A. actinomycetemcomitans attached best on the collagen membranes, followed by the ePTFE membranes, and then the glycolide fiber membranes without antibiotics. Moreover, higher numbers of bacteria were observed on the fibril areas than on the laminar areas of the ePTFE membranes. The amounts of attached bacteria on the GTR membranes increased after longer incubation. Incorporation of tetracycline or amoxicillin greatly reduced the adhesion of S. mutans and A. actinomycetemcomitans onto all of the GTR membranes examined. CONCLUSION: Incorporation of tetracycline or amoxicillin greatly reduced adhesion of S. mutans or A. actinomycetemcomitans on the ePTFE, glycolide fiber, or collagen membranes. This finding indicates that it is valuable and effective to use the antibiotic-loaded GTR membranes for periodontal regeneration therapy.

Porphyromonas gingivalis GroEL induces osteoclastogenesis of periodontal ligament cells and enhances alveolar bone resorption in rats.

Porphyromonas gingivalis is a major periodontal pathogen that contains a variety of virulence factors. The antibody titer to P. gingivalis GroEL, a homologue of HSP60, is significantly higher in periodontitis patients than in healthy control subjects, suggesting that P. gingivalis GroEL is a potential stimulator of periodontal disease. However, the specific role of GroEL in periodontal disease remains unclear. Here, we investigated the effect of P. gingivalis GroEL on human periodontal ligament (PDL) cells in vitro, as well as its effect on alveolar bone resorption in rats in vivo. First, we found that stimulation of PDL cells with recombinant GroEL increased the secretion of the bone resorption-associated cytokines interleukin (IL)-6 and IL-8, potentially via NF-κB activation. Furthermore, GroEL could effectively stimulate PDL cell migration, possibly through activation of integrin α1 and α2 mRNA expression as well as cytoskeletal reorganization. Additionally, GroEL may be involved in osteoclastogenesis via receptor activator of nuclear factor κ-B ligand (RANKL) activation and alkaline phosphatase (ALP) mRNA inhibition in PDL cells. Finally, we inoculated GroEL into rat gingiva, and the results of microcomputed tomography (micro-CT) and histomorphometric assays indicated that the administration of GroEL significantly increased inflammation and bone loss. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to osteoclastogenesis of PDL cells and resulting in periodontal disease with alveolar bone resorption.

Inhibitory effects of areca nut extract on expression of complement receptors and fc receptors in human neutrophils.

BACKGROUND: Chewing of areca quid increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the phagocytic activity of human neutrophils. This in vitro study investigates the effects of ANE on complement- and antibody-opsonized phagocytosis by neutrophils. Expression of complement receptors, Fc receptors, and F-actin in ANE-treated neutrophils is also analyzed. METHODS: The viability of ANE-treated neutrophils was determined using the propidium iodide staining method. The possible effects of ANE on the expression of complement receptors and Fc receptors were examined using an immunofluorescence staining method followed by flow cytometry and confocal laser scanning microscopy. The phagocytic activity of neutrophils against complement or immunoglobulin (Ig)G-opsonized fluorescent beads was analyzed using flow cytometry. Expression of F-actin was determined using confocal laser scanning microscopy. RESULTS: ANE significantly inhibited the production of complement receptors (CR1, CR3, and CR4) and Fc receptors (FcγRII and FcγRIII) in a concentration-dependent manner. Treatment of neutrophils with ANE significantly impaired their ability to phagocytose fluorescent beads. ANE also inhibited phagocytosis of fluorescent beads that were opsonized by complement or IgG. Moreover, expression of F-actin was inhibited after ANE treatment. CONCLUSIONS: ANE inhibits the complement- and IgG-mediated neutrophil phagocytosis that may result from reduction of the expression of complement receptors, Fc receptors, and F-actin formation after ANE treatment. The findings suggest that areca nut chewing may jeopardize the defensive functions of neutrophils and affect periodontal health.

Effects of areca nut extract on lipopolysaccharides-enhanced adhesion and migration of human mononuclear leukocytes.

BACKGROUND: Areca chewers have a higher prevalence of periodontitis than non-chewers. Cell adhesion and movement (migration) are important for leukocyte recruitment to inflammation sites. This study investigates the effects of areca nut extract (ANE) on the adhesion and migration abilities of the human immune cells, peripheral blood mononuclear cells (PBMCs). The combined effects of nicotine and lipopolysaccharides (LPS) were also analyzed. METHODS: Purified PBMCs obtained from healthy adults were treated with ANE, nicotine, and/or LPS. Cell adhesion ability was examined using fibronectin-coated microslides, Liu stain, and light microscopy. Cell migration ability was evaluated using the transwell system followed by staining and fluorescence microscopy. Statistical difference was analyzed using the Mann-Whitney U test. RESULTS: When compared with the media-treated control samples, PBMCs treated with ANE for 4 hours showed a significant reduction of the adherent cells on the microslides. Interestingly, LPS treatment increased cell adhesion, which could be reduced by simultaneous ANE plus nicotine treatment. The chemotactic migration of PBMCs was reduced by ANE treatment for 1, 4, or 24 hours in a dose-dependent manner. LPS treatment increased PBMC migration, which could be reduced by simultaneous treatment with ANE or with ANE plus nicotine. CONCLUSIONS: ANE reduced the adhesion and migration abilities of PBMC. ANEs, with or without nicotine, also attenuated the migration of LPS-stimulated PBMCs. The results implicated that the immune cell functions were impaired in areca chewers, which might increase the host susceptibility to oral and periodontal infection.

Stimulatory effects of glucose and Porphyromonas gingivalis lipopolysaccharide on the secretion of inflammatory mediators from human macrophages.

BACKGROUND: Hyperglycemia is widely considered to be the causal link between diabetes mellitus (DM) and diabetic complications. The purpose of this study is to determine the effects of high glucose in the presence of lipopolysaccharide (LPS) purified from the periodontal pathogen Porphyromonas gingivalis on human macrophages. METHODS: Macrophages (U937) were treated with various concentrations of P. gingivalis-LPS under normal (5.5 mM) or high (25 mM) glucose conditions. Mitochondrial dehydrogenase activity was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay. The levels of inflammatory mediators secreted were determined using the enzyme-linked immunosorbent assay and the competitive enzyme immunoassay. The intracellular calcium chelator was used to examine whether the intracellular calcium was involved. Statistical differences were assessed using a one-way analysis of variance and Tukey multiple-comparison intervals with α = 0.05. RESULTS: High glucose condition enhanced the mitochondrial dehydrogenase activity in macrophages. P. gingivalis-LPS induced the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and prostaglandin E(2) (PGE(2)) in a dose-dependent manner both in normal and high glucose conditions. The stimulatory effects by P. gingivalis-LPS were more evident when cells were cultured under high glucose conditions. Changes of intracellular calcium concentration were involved not only in high glucose-induced mitochondrial dehydrogenase activity but also in P. gingivalis-LPS-induced production of IL-6, TNF-α, or PGE(2), especially under the high glucose conditions. CONCLUSIONS: High glucose appeared to enhance the inflammatory response induced by the periodontal pathogen. The information generated may help to delineate the possible mechanisms by which hyperglycemia compromises the periodontal health of patients with DM.

Areca nut extracts increased the expression of cyclooxygenase-2, prostaglandin E2 and interleukin-1α in human immune cells via oxidative stress.

BACKGROUND AND OBJECTIVES: Areca nut has been identified as a carcinogen. Inflammation reveals a strong link with tumourigenesis. The aim of this study was to investigate the effects of areca nut on the expression of the key pro-inflammatory mediators involved in malignancy, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), interleukin (IL)-1α and nuclear factor-κB (NF-κB), by human immune cells. The role of oxidative stress was also examined. MATERIALS AND METHODS: Human peripheral blood mononuclear cells (PBMCs) were treated with extracts of ripe areca nut (rANE) or tender areca nut (tANE). Expression of pro-inflammatory mediators was assayed using Western blotting, reverse transcription-polymerase chain reaction, competitive enzyme immunoassay or enzyme-linked immunosorbent assay (ELISA). Activity of NF-κB was evaluated using an ELISA-based method. RESULTS: Both rANE and tANE enhanced the expression of COX-2, PGE2 and IL-1α by PBMCs. The secretion of PGE2 was induced by rANE (≤20-40µgml(-1)) and tANE (≤160µgml(-1)) significantly in a dose- and time-dependent manner. However, the above enhancing effects of ANEs could be attenuated by antioxidants. ANEs also increased the nuclear expression of the redox-sensitive factor NF-κB. CONCLUSIONS: The results demonstrate that ANEs induced the expression of pro-inflammatory mediators mainly through the induction of oxidative stress and implicate the possibility of using antioxidants for disease prevention.

Antiherpetic potential of 6-bromoindirubin-3'-acetoxime (BIO-acetoxime) in human oral epithelial cells.

Glycogen synthase kinase 3 (GSK-3) functions in the regulation of glycogen metabolism, in the cell cycle, and in immune responses and is targeted by some viruses to favor the viral life cycle. Inhibition of GSK-3 by 6-bromoindirubin-3'-acetoxime (BIO-acetoxime), a synthetic derivative of a compound from the Mediterranean mollusk Hexaplex trunculus, protects cells from varicella infection. In this study, we examined the effects of BIO-acetoxime against herpes simplex virus-1 (HSV-1) infection in human oral epithelial cells, which represent a natural target cell type. The results revealed that BIO-acetoxime relieves HSV-1-induced cytopathic effects and apoptosis. We also found that BIO-acetoxime reduced viral yields and the expression of different classes of viral proteins. Furthermore, addition of BIO-acetoxime before, simultaneously with or after HSV-1 infection significantly reduced viral yields. Collectively, BIO-acetoxime may suppress viral gene expression and protect oral epithelial cells from HSV-1 infection. These results suggest the possible involvement of GSK-3 in HSV-1 infection.

Up-regulation of nucleotide-binding oligomerization domain 1 in inflamed human dental pulp.

INTRODUCTION: The innate immune response is activated by recognition of microbial components through specific pattern recognition receptors including nucleotide-binding oligomerization domain (NOD)-like receptors. However, the regulation of NOD-1 in inflamed human dental pulp remains poorly understood. This study aimed to evaluate the expression of NOD-1 in healthy and inflamed human dental pulps. In addition, the secretion of chemokines induced by NOD-1 and the related signaling pathways were studied. METHODS: Samples of human dental pulp tissues were obtained from freshly extracted wisdom teeth. The protein localization of NOD-1 in the pulp tissues was detected by immunohistochemistry. In addition, human dental pulp fibroblasts were stimulated with NOD-1 agonist γ-D-glutamylmeso-diaminopimelic acid. Production of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) was determined by an enzyme-linked immunosorbent assay. The involvement of mitogen-activated protein kinase (MAPK) signaling pathways was examined by Western blot analysis, and the association of MAPK signaling with chemokine production was determined. RESULTS: The results demonstrated the expression of NOD-1 in normal dental pulp, and up-regulated NOD-1 expression was observed in inflamed dental pulp. On stimulation with NOD-1 agonist, production of IL-8 and MCP-1 was induced in a dose-dependent manner. Moreover, phosphorylation of p38 MAPK and Jun N-terminal kinase (JNK) was enhanced by stimulation of NOD-1. With the treatment of p38 MAPK and JNK inhibitors, the NOD-1-induced IL-8 production was suppressed. CONCLUSIONS: In response to microbial invasion, the expression of NOD-1 can be regulated in a ligand-inducible manner. NOD-1 might participate in pulp inflammation through chemokine production via MAPK signaling pathways.

PI3K/Akt signaling mediated apoptosis blockage and viral gene expression in oral epithelial cells during herpes simplex virus infection.

Phosphatidylinositol 3-kinases (PI3Ks) function in the anti-apoptotic pathway, and are commonly exploited by various viruses to accomplish the viral life cycle. This study examined the role of the PI3K pathway in human oral epithelial cells following herpes simplex virus type 1 (HSV-1) infection. The results showed that HSV-1 induced the phosphorylation of Akt and glycogen synthase kinase 3 (GSK-3). Phosphorylation of Akt, but not GSK-3, induced by HSV-1 was PI3K-dependent. The expression of HSV-1 immediate-early genes may be involved in the initial phosphorylation of Akt and GSK-3. Inhibition of HSV-1-induced PI3K activity increased DNA fragmentation and cleavage of poly ADP-ribose polymerase (PARP), caspase 3 and caspase 7 compared with infected alone. Inhibition of PI3K attenuated the expression of HSV-1-infected cell protein 0 (ICP0), but not thymidine kinase (TK) and viral replication. Collectively, these data suggested that, in oral epithelial cells, the HSV-1-induced PI3K/Akt activation was involved in the regulation of apoptosis blockage and viral gene expression.

Stimulatory effects of areca nut extracts on prostaglandin E2 production by human polymorphonuclear leukocytes.

BACKGROUND: Areca quid chewing increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the defensive functions of human polymorphonuclear leukocytes (PMNs). This in vitro study investigates the effects of ANE on the production of cyclooxygenase (COX)-2 and the inflammatory mediator prostaglandin E(2) (PGE(2)) by PMNs. METHODS: The possible effects of ANE on the production of COX-2 were examined using Western blotting analysis. The viability and production of PGE(2) of treated PMNs were determined using the propidium iodide staining method and the competition enzyme assay, respectively. The possible pathways involved were also examined using the COX-2 inhibitor (NS398), the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), and the extracellular signal-regulated protein kinase (ERK) inhibitor (U0126). The effects of ANE on the viability or PGE(2) production were statistically assessed using a one-way analysis of variance and Tukey multiple-comparison intervals with alpha = 0.05. RESULTS: ANE significantly induced the production of PGE(2) in a time- and concentration-dependent manner. This induction resulted from an increased expression of COX-2. Moreover, the application of BAPTA-AM, SB203580, and U0126 statistically significantly suppressed the induction of PGE(2). CONCLUSIONS: ANE induced the production of PGE(2). The activation of the intracellular calcium concentrations, p38 MAPK, and ERK may be involved in the inducing effects of ANE on PMNs. The findings suggest that areca nut chewing may induce an inflammatory response and affect the periodontal health of consumers.

Comparison of clinical outcomes following guided tissue regeneration treatment with a polylactic acid barrier or a collagen membrane.

This prospective, randomized, controlled clinical trial compared the clinical outcomes for a polylactic acid barrier versus a collagen membrane in conjunction with guided tissue regeneration (GTR). Thirty patients with chronic periodontitis and at least one intrabony defect of a minimum 4 mm were enrolled. Following full-mouth scaling, GTR using a collagen membrane or a polylactic acid barrier was performed at one site in each patient. At 1 week before and 6 months after surgery, probing pocket depth (PPD), clinical attachment level (CAL), marginal tissue recession, and bone fill were assessed. A significant reduction in PPD (2.50 +/- 1.35 mm for the polylactic acid barrier and 2.60 +/- 1.08 mm for the collagen membrane) was obtained, in addition to gains in CAL (2.40 +/- 1.17 mm for the polylactic acid barrier and 2.60 +/- 1.26 mm for the collagen membrane) and bone fill (0.33 +/- 1.89 mm for polylactic acid barrier and 2.57 +/- 1.64 mm for collagen membrane), for each group compared to baseline. Significantly, the results from 6 months after surgery showed that there was greater bone fill when the collagen membrane was used compared to the polylactic acid barrier.