Epstein-Barr virus reactivation by persistent apical periodontal pathogens.

AIM: To assess whether Epstein-Barr virus (EBV) reactivation is triggered by persistent apical periodontitis-related microbes using in vitro and ex vivo methodologies. METHODOLOGY: Surgically removed human periapical granulomas (n = 50) and healthy gingival tissues (n = 10) were analysed to determine the presence of EBV and seven persistent apical periodontitis-related microbes. In addition, real-time polymerase chain reaction was used to detect the mRNA expression of BZLF-1, an immediate-early gene of EBV. Expression of latent membrane protein (LMP)-1 and ZEBRA, an early lytic protein of EBV encoded by BZLF-1, was also examined using triple-colour immunofluorescence staining. n-Butyric acid produced by the microbes was quantified, and luciferase assays were performed in association with bacterial lysates. In addition, Daudi cells were cultured with bacterial lysates, and the expression levels of BZLF-1 mRNA and ZEBRA protein were determined. RESULTS: EBV DNA and BZLF-1 mRNA were detected in 47 out of 50 periapical granulomas, but not in healthy gingival tissues. The EBV DNA copy number and the number of Fusobacterium nucleatum were significantly positively correlated with BZLF-1 expression in periapical granulomas. The number of Prevotella intermedia was slightly correlated with BZLF-1 expression; however, the other microbes were not. CD79a-positive B cells in periapical granulomas, but not those in healthy gingival tissues, expressed both LMP-1 and ZEBRA. n-Butyric acid production was the highest in F. nucleatum and the lowest in P. intermedia. Enterococcus faecalis, Candida albicans and the other tested microbes did not produce n-butyric acid. An F. nucleatum lysate exhibited significantly increased BZLF-1-luciferase activity in the same manner of commercial butyric acid, whereas P. intermedia did not. F. nucleatum also induced the expression of BZLF-1 mRNA and ZEBRA protein by Daudi cells, indicating that EBV reactivation was induced. CONCLUSION: Among the persistent apical periodontitis-related bacteria that were tested, F. nucleatum most strongly reactivated latent EBV, whereas E. faecalis and C. albicans as well as the other microbes did not.

Porphyromonas endodontalis reactivates latent Epstein-Barr virus.

AIM: To determine whether Porphyromonas endodontalis can reactivate latent Epstein-Barr virus (EBV). METHODOLOGY: The concentrations of short-chain fatty acids (SCFAs) in P. endodontalis culture supernatants were determined using high-performance liquid chromatography. A promoter region of BamHI fragment Z leftward open reading frame 1 (BZLF-1), which is a transcription factor that controls the EBV lytic cycle, was cloned into luciferase expression vectors. Then, the luciferase assay was performed using P. endodontalis culture supernatants. Histone acetylation using Daudi cells treated with P. endodontalis culture supernatants was examined using Western blotting. BZLF-1 mRNA and BamHI fragment Z EB replication activator (ZEBRA) protein were also detected quantitatively using real-time polymerase chain reaction (PCR) and Western blotting. Surgically removed periapical granulomas were examined to detect P. endodontalis, EBV DNA, and BZLF-1 mRNA expression using quantitative real-time PCR. Statistical analysis using Steel tests was performed. RESULTS: The concentrations of n-butyric acid in P. endodontalis culture supernatants were significantly higher than those of other SCFAs (P = 0.0173). Using B-95-8-221 Luc cells treated with P. endodontalis culture supernatants, the luciferase assay demonstrated that P. endodontalis induced BZLF-1 expression. Hyperacetylation of histones was also observed with the culture supernatants. BZLF-1 mRNA and ZEBRA protein were expressed by Daudi cells in a dose-dependent manner after the treatment with P. endodontalis culture supernatants. P. endodontalis and BZLF-1 in periapical granulomas were also detected. The expression levels of BZLF-1 mRNA were similar to the numbers of P. endodontalis cells in each specimen. CONCLUSIONS: n-butyric acid produced by P. endodontalis reactivated latent EBV.

A potential role for the silent information regulator 2 homologue 1 (SIRT1) in periapical periodontitis.

AIM: To investigate the role played by silent information regulator 2 homologue 1 (SIRT1) during angiogenesis of periapical periodontitis. METHODOLOGY: Periapical granulomas were subjected to dual-colour immunofluorescence imaging and real-time polymerase chain reactions assaying the expression levels of SIRT1, vascular endothelial growth factor (VEGF) and VE-cadherin. The association between Ki-67 and SIRT1 expression was also examined. Human umbilical vein endothelial cells (HUVECs) were treated with a combination of lipopolysaccharide and resveratrol (a SIRT1 activator) or sirtinol (a SIRT1 inhibitor); and the levels of mRNAs encoding SIRT1, VEGF and VE-cadherin were determined. HUVEC tube formation was assayed in the presence of resveratrol or sirtinol. The Mann-Whitney U-test or the Tukey-Kramer test was used for statistical analysis. RESULTS: Ki-67-expressing cells, including endothelial cells, lay adjacent to SIRT1-expressing cells in periapical granulomas. In addition, SIRT1-expressing cells were detected adjacent to VEGF-expressing cells and VEGF- or VE-cadherin-expressing endothelial cells. SIRT1, VEGF and VE-cadherin mRNA expression levels in periapical granulomas were significantly higher (P = 0.0054, 0.0090 and 0.0090, respectively) than those in healthy gingival tissues. HUVECs treated with resveratrol exhibited significantly higher expression of mRNAs encoding SIRT1, VEGF and VE-cadherin (P = 0.0019, 0.00005 and 0.0045, respectively) compared with controls, but sirtinol inhibited such expression. Resveratrol caused HUVECs to form tube-like structures, whilst sirtinol inhibited this process. CONCLUSIONS: These findings suggest that SIRT1 may stimulate angiogenesis in periapical granulomas by triggering the proliferation of endothelial cells and inducing VEGF and VE-cadherin expression.

Characterization of human dental pulp-derived cell lines.

AIM: To establish and characterize different types of fibroblastic cell lines derived from dental pulp tissue. METHODOLOGY: Human dental pulp tissue-derived cells were transfected with SV40 large T antigen by Lipofectamine transfection method. Geneticin (G418)-resistant cells were selected and different cell lines were established by a limiting dilution method. To characterize the lineages of cells, each clone was immunofluorescently stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. Total RNA was extracted from each clone and subjected to a differential display experiment. RESULTS: By transfecting SV40 large T antigen, nine different cell clones were obtained. All these cell clones were positively stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. With differential display experiment, eight different genes, the expression levels of these genes were varied amongst each cell clone, were detected. After sequencing and database search, one gene was revealed to be identical to T-cell marker, Thy-1. Thy-1 expression in dental pulp tissue was confirmed by immunohistochemical staining. CONCLUSION: Fibroblastic cell lines derived from human dental pulp tissue possessed different gene expression profiles suggesting the existence of subpopulations.

Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation of cultured human dental pulp cells by low-power gallium-aluminium-arsenic laser irradiation.

AIM: To examine whether low-power laser irradiation (LPLI) promotes cellular proliferation of human dental pulp-derived fibroblast-like cells (dental pulp cells). METHODOLOGY: Dental pulp cells were obtained by primary culture of human dental pulp tissues from extracted third molar teeth. The phosphorylation of the mitogen-activated protein kinase (MAPK) family after LPLI of these cells was investigated by Western blotting. By using a specific MAPK/ERK kinase (MEK) inhibitor (PD098059), the specific effect of LPLI on the MAPK pathway was also investigated by Western blotting as described above. The incorporation of [3H]thymidine into the cells after LPLI was determined, and statistical analysis was performed by Wilcoxon signed-ranks test. RESULTS: Extracellular signal-regulated protein kinase (ERK) 1/2 was phosphorylated between 5 and 30 min after LPLI. Moreover, PD098059 inhibited LPLI-mediated ERK1/2 activation. LPLI did not affect p38 MAPK or c-Jun N-terminal kinase (JNK) phosphorylation. But LPLI did not stimulate [3H]thymidine incorporation into these cells. CONCLUSIONS: These results indicated that LPLI activated MAPK/ERK, a signal for proliferation, differentiation and survival, but did not activate the stress signals p38 MAPK and JNK in human dental pulp cells.

Behaviour of bone marrow osteoblast-like cells on mineral trioxide aggregate: morphology and expression of type I collagen and bone-related protein mRNAs.

AIM: To investigate the in vitro behaviour of rat bone marrow cells (RBM) on mineral trioxide aggregate (MTA) (ProRoot, MTA Root Canal Repair Material; Dentsply Tulsa, Tulsa, OK, USA) compared with intermediate restorative materials (IRM) (Dentsply Caulk, Milford, DE, USA). METHODOLOGY: RBM were obtained from rat femur and were primary cultured and then subcultured. Cells were then seeded on three dishes of each material, and cultured for 3 days, after which they were evaluated morphologically using scanning (SEM) and transmission (TEM) electron microscopy. Furthermore, the calcium released from hydrated material, the cell proliferation ratio and alkaline phosphatase (ALP) activity were analysed, and the expression of type I collagen and bone-related protein mRNAs were evaluated. The data were averaged and analysed via one-way analysis of variance (anova) and were then compared by the Scheffe's test. RESULTS: SEM showed that RBM attached to MTA and had a flattened appearance without nuclear protrusions and microspikes. TEM showed that the cells attached in the same manner as the control group, but gaps larger than 2 microm were frequently seen. The calcium released from hydrated MTA was about 130 ppm after 3 days of immersion in saline. The ALP activity was similar to the control group. Cell proliferation and expression of type I collagen mRNA was significantly lower, while the expression of osteopontin mRNA was significantly higher than the control group at the third day of culture. In IRM groups, a few rounded cells were observed on the material but no living cells were seen. CONCLUSIONS: MTA is a material of low toxicity which does not inhibit cell growth, but does suppress the differentiation of osteoblast-like cells.

Inducible nitric oxide synthase activity by interferon-gamma-producing cells in human radicular cysts.

AIM: In this study, the interaction of interferon-gamma-(IFN-gamma) and inducible nitric oxide synthase (iNOS)-producing cells in human radicular cysts were investigated. METHODOLOGY: Inflamed periapical tissues were obtained from patients at the time of endodontic surgical treatments and were cut into two pieces. After fixing with acetone or 4% paraformaldehyde in phosphate-buffered saline, 5-m-thick paraffin and cryostat sections were prepared. The paraffin sections of the inflamed tissues were evaluated histologically with haematoxylineosin stains. The specimens diagnosed as radicular cysts were then examined by immunostaining. Immunohistochemistry for iNOS and fluoresence microscopy for IFN-gamma using the cryostat sections were performed with a mixture of affinity purified human iNOS antiserum and human IFN-gamma monoclonal antibodies. RESULTS: The results revealed that iNOS-gamma producing cells localized adjacent to IFN-gamma-producing cells. In addition, some of iNOS-producing cells exhibited immunoreactive IFN-gamma. On the other hand, epithelial cells showed significant levels of iNOS production, but not IFN-gamma. CONCLUSIONS: The data would suggest the possibility that iNOS production could be precisely controlled by autocrine or paracrine effects of IFN-gamma producing cells in radicular cysts and might play a pivotal role in periapical lesions. These findings are consistent with a hypothesis suggesting that NO inhibitors could be used through the root canals as a pharmacological treatment for periapical lesions.

Fibroblasts inhibit mineralised bone nodule formation by rat bone marrow stromal cells in vitro.

The ability of rat skin fibroblasts (RSF) and human periodontal ligament fibroblasts (HPL) to inhibit the formation of mineralised bone nodules in rat bone marrow stromal cell (BMSC) cultures was studied. Co-culture of HPL or RSF with BMSC resulted in a large reduction of bone nodule formation when compared with controls. Conditioned medium from HPL or RSF cultures inhibited bone nodule formation in a dose-dependent manner. HPL-conditioned medium depressed cell proliferation and alkaline phosphatase expression in BMSC cultures. These effects were not due to increased cytotoxicity or nutrient depletion. Inhibitory activity was recovered in a fraction of less than 1 kD following ultrafiltration and was insensitive to freeze-thawing. The inhibitory activity was blocked when HPL cultures were grown in the presence of 10(-5) M indomethacin. Dose-dependent inhibiton of bone nodule formation was also observed in cultures incubated with prostaglandins E2 (at 10(-6) M) or F2 alpha (at 10(-7) M). The results indicate that fibroblasts may inhibit osteoblast differentiation and function in part by release of soluble factors including prostaglandins.