Shan-Ling Hung

The 9-kDa hydrophobic protein encoded at the 3' end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated.

Abstract The open reading frame potentially encoding a 78 amino acid, 9101 Da hydrophobic protein (HP) and, mapping at the 3′ end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome, was shown to be expressed during virus replication. The cloned HP gene was placed in a plasmid under control of the T7 RNA polymerase promoter and in vitro translation of transcripts generated in vitro yielded a 9.1-kDa protein that was immunoprecipitable with porcine hyperimmune anti-TGEV serum. Antiserum raised in rabbits against a 31 amino acid synthetic polypeptide that represented the central hydrophilic region of HP specifically immunoprecipitated HP from TGEV-infected cells. HP was further shown to become associated with microsomal membranes during synthesis in vitro and was found to be closely associated with the endoplasmic reticulum and cell surface membranes in infected cells. The intracellular location of HP suggests that it may play a role in the membrane association of replication complexes or in virion assembly.

Areca nut extracts increased expression of inflammatory cytokines, tumor necrosis factor-α, interleukin-1β, interleukin-6 and interleukin-8, in peripheral blood mononuclear cells

BACKGROUND AND OBJECTIVE Cytokines represent a central role in inflammatory tissue destruction and regulate the immune responses that may govern the progression of periodontal diseases. This study investigated the effects of areca nut extracts on the expression of inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8 in peripheral blood mononuclear cells. The role of oxidative stress of areca nut extracts was also examined using curcumin. MATERIAL AND METHODS The expression of cytokines in peripheral blood mononuclear cells treated with extracts of ripe areca nut or extracts of tender areca nut was analyzed using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. RESULTS Both extracts of ripe areca nut (< or = 40 microg/mL) and extracts of tender areca nut significantly enhanced the production of tumor necrosis factor-alpha and interleukin-1beta in peripheral blood mononuclear cells in a dose-dependent and time-dependent manner. The kinetics of mRNA expression of both cytokines was also enhanced by areca nut extracts. The stimulatory effects of areca nut extracts on the secretion of tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8 and on the mRNA expression of tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 at 4 h of incubation were reduced by curcumin (20-50 microm). However, the level of interleukin-8 transcripts was not affected by curcumin. Moreover, interleukin-1beta induction by extracts of tender areca nut, but not by extracts of ripe areca nut, was weakened by 10 microm curcumin. The inhibitory effects of curcumin may vary with different cytokines and with different areca nut extract treatments. CONCLUSION The complex cytokine profile induced by areca nut extracts-treated peripheral blood mononuclear cells implied the possibility of enhanced local inflammation and altered immune functions by the areca chewing habit. The inhibitory effects of curcumin on cytokine expression suggested that oxidative stress might be involved in areca nut extracts-associated immune alteration.

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.

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.

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.

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.

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.

Bacterial Penetration Through Antibiotic-Loaded Guided Tissue Regeneration Membranes

BACKGROUND This study compared bacterial penetration through guided tissue regeneration (GTR) membranes impregnated with antibiotics. METHODS Three barrier membranes, expanded polytetrafluoroethylene (ePTFE) membrane, collagen membrane, and glycolide fiber composite membrane, were loaded with amoxicillin or tetracycline. The penetration of Streptococcus mutans and Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) through the GTR membranes was achieved using a device consisting of an inner tube and an outer bottle filled with culture media. RESULTS The penetration of S. mutans or A. actinomycetemcomitans into the inner tubes significantly decreased with all of the antibiotic-loaded membranes compared to membranes without antibiotics. However, differences were found in the behavior of the three membranes. The antibiotic-loaded ePTFE membranes showed the best barrier effect. Moreover, the inhibitory effect of tetracycline on S. mutans was greater than that of amoxicillin for all GTR membranes. Furthermore, the inhibitory effect of tetracycline on A. actinomycetemcomitans was lower than that of amoxicillin with the glycolide fiber membrane. CONCLUSIONS The results showed that penetration of S. mutans and A. actinomycetemcomitans through amoxicillin- or tetracycline-loaded ePTFE membrane, glycolide fiber membrane, and collagen membrane was delayed and/or reduced. Thus, incorporation of an antibiotic into the membrane may be of value when controlling membrane-associated infection during GTR therapy.

Analysis of herpes simplex virus entering into cells of oral origin

The entry of herpes simplex virus (HSV) into an oral epithelial cell line, primary normal human oral keratinocytes (NHOK) and gingival fibroblasts (GF) was examined. Infection of these cells by HSV-1 and HSV-2 was blocked by heparin. Further examination indicated that heparin reduced viral attachment but not penetration. Moreover, neomycin inhibited HSV-1 infection more effectively than HSV-2 infection in GF, but not in NHOK. In conclusion, our results elucidated some aspects of the HSV entry process into oral cells and revealed some differences in HSV entering into NHOK and GF.