Yen-Ting Chen

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.

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.

Direct Binding of Herpes Simplex Virus Type 1 Virions to Complement C3

Glycoprotein C (gC) of type 1 herpes simplex virus (HSV-1) binds the human complement C3 as purified proteins, or when expressed on the surface of infected cells. However, it is not clear whether the purified HSV virion binds directly to C3. In this study, direct binding of purified virions, HSV-1(KOS) or HSV-1(hrR3), to C3-coated plate was demonstrated by an enzyme-linked immunosorbent assay (ELISA). Captured virions on C3-coated plates were still infectious as determined by adding Vero cells to allow for infection to occur. The binding of virions to C3 was abolished if C3 was heat-inactivated, confirming a requirement for complement. In addition, the interaction was inhibited by preincubation of purified virions with heparin. In conclusion, a direct interaction of C3 with the HSV-1 virions was demonstrated.

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.

Acrolein induces ribotoxic stress in human cancer cells regardless of p53 status

Acrolein (Acr) cytotoxicity contributes to chemotherapeutic activity of cyclophosphamide via metabolism of the anticancer drug. Our previous studies have shown that Acr causes ribosomal DNA (rDNA) damages, thus shuts down ribosomal RNA (rRNA) synthesis and leads to ribosomal stress in human cancer cells. Ribosome senses stress in 28S rRNA and induces subsequent activation of mitogen-activated protein kinase (MAPK) pathway which triggers ribotoxic stress response (RSR). Here, we report that cells harboring p53 or not responds differently to Acr-induced RSR. Our results show that Acr induced rRNA cleavage via the activated caspases in cancer cells with wild type p53, but not in cells with deficient p53. Furthermore, MAPK pathways were activated by Acr in cancer cells regardless of p53 status. Acr induced apoptosis in cells with wild type p53, while it induced G2/M cell cycle arrest in cancer cells with deficient p53. In conclusion, the presence of functional p53 plays a significant role in the mechanisms of Acr-induced rRNA cleavage and cell fates. Our results enhance our understanding of the molecular mechanisms of Acr-mediated antitumor activity which helps develop better therapeutic strategies for killing cancer cells with different p53 status.