Ayako Kato

Higher Prevalence of Epstein–Barr Virus DNA in Deeper Periodontal Pockets of Chronic Periodontitis in Japanese Patients

Periodontitis, a complex chronic inflammatory disease caused by subgingival infection, is among the most prevalent microbial diseases in humans. Although traditional microbiological research on periodontitis has focused on putative bacteria such as Porphyromonas gingivalis, the herpes virus is proposed to be involved in the pathogenesis of periodontitis because bacterial etiology alone does not adequately explain various clinical aspects. In this study, we established for the first time, more Epstein–Barr virus (EBV) DNA is found deeper in periodontal pockets of chronic periodontitis in Japanese patients. Subgingival samples were collected from 85 patients with chronic periodontitis having two periodontal sites with probing depths (PD) of ≤3 mm (shallow) or ≥5 mm (deep) and were subjected to a nested polymerase chain reaction. EBV DNA was more frequently detected in patients with deeper PD sites (66%) than in those with shallow PD sites (48%) or healthy controls (45%). Coexistence of EBV DNA and P. gingivalis was significantly higher in patients with deeper PD sites (40%) than in those with shallow PD sites (14%) or healthy controls (13%). Although no difference in clinical index for periodontitis, the odds ratio of EBV DNA in patients with deeper PD sites was 2.36, which was 2.07-fold higher than that in those with shallow PD sites. Interestingly, the odds of acquiring chronic periodontitis (PD ≥5 mm) were higher in the presence of both EBV DNA and P. gingivalis compared with either EBV DNA or P. gingivalis only. In addition, we also observed that EBV-encoded small RNA (EBER) in positive cells of human gingival tissues. These results would suggest that EBV DNA may serve as a pathogenic factor leading to chronic periodontitis among Japanese patients.

Tumor necrosis factor-α regulates human follicular dendritic cell-secreted protein gene transcription in gingival epithelial cells

Follicular dendritic cell‐secreted protein (FDC‐SP) is a secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium. To elucidate the transcriptional regulation of the human FDC‐SP gene by tumor necrosis factor‐α (TNF‐α), we conducted real‐time PCR, Western blotting, transient transfection analyses with chimeric constructs of the FDC‐SP gene promoter linked to a luciferase reporter gene, gel mobility shift and chromatin immunoprecipitation assays using Ca9‐22 gingival epithelial cells. TNF‐α (10 ng/ml) induced FDC‐SP mRNA and protein levels at 3 hr and reached maximum at 12 hr. In transient transfection assays, TNF‐α (12 hr) increased the LUC activities of constructs between −116FDCSP and −948FDCSP including the human FDC‐SP gene promoter. Transcriptional stimulations by TNF‐α were partially inhibited in the −345FDCSP constructs that included 3‐bp mutations in the YY1, GATA, CCAAT enhancer‐binding protein 2 (C/EBP2) and C/EBP3. Transcriptional activities induced by TNF‐α were inhibited by tyrosine kinase, MEK1/2 and phosphoinositide 3‐kinase inhibitors. The results of ChIP assays showed that YY1, GATA and C/EBPβ transcription factors interacted with the YY1, GATA, C/EBP2 and C/EBP3 elements that were increased by TNF‐α. These studies show that TNF‐α stimulates human FDC‐SP gene transcription by targeting YY1, GATA, C/EBP2 and C/EBP3 in the FDC‐SP gene promoter.

Prevalence of Epstein-Barr virus DNA and Porphyromonas gingivalis in Japanese peri-implantitis patients

BackgroundPeri-implantitis (PI) is an inflammatory reaction associated with functional deterioration of supporting bones around the dental implant. Recent studies suggested Epstein–Barr virus (EBV) is involved in the pathogenesis of periodontitis. We investigated the association between EBV and Porphyromonas gingivalis in Japanese PI patients.MethodsFifteen periodontally healthy individuals, 15 healthy implant patients and 15 PI patients were recruited. Forty five subgingival plaque samples were collected from the deepest probing pocket depth (PPD) site from each patient. Real-time PCR was used to detect EBV DNA and P. gingivalis.ResultsEBV and P. gingivalis were detected in 7 and 3 PPD sites of the healthy controls, in 9 and 4 PPD sites of the healthy implants, and in 13 and 14 PPD sites of the PI patients. P. gingivalis and coexistence of EBV and P. gingivalis were detected significantly higher in the PI patients than healthy controls and healthy implant patients. EBV was detected significantly higher in the PI patients than healthy controls.ConclusionsHigher levels of EBV and P. gingivalis were detected in PPD sites of PI patients. These results suggest that coexistence of EBV and P. gingivalis may serve pathogenic factors cause for PI in Japanese dental patients.

IL-1β and TNF-α regulate mouse amelotin gene transcription in gingival epithelial cells

Amelotin (AMTN) is an enamel protein expressed in maturation-stage ameloblasts and junctional epithelium. To clarify the transcriptional regulation of the AMTN gene by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with luciferase constructs including various lengths of the mouse AMTN gene promoter, and gel shift and chromatin immunoprecipitation assays using mouse gingival epithelial GE1 cells. The levels of AMTN mRNA and protein in GE1 cells were increased after 6 h of stimulation with IL-1β (1 ng/mL) and TNF-α (10 ng/mL). IL-1β and TNF-α induced luciferase activities of the constructs between -116AMTN and -705AMTN including the mouse AMTN gene promoter. Transcriptional activation by IL-1β and TNF-α was partially inhibited in -460AMTN including 3-bp mutations in the CCAAT-enhancer-binding protein 1 (C/EBP1), C/EBP2 and Yin Yang 1 (YY1) elements. Transcriptional activities induced by IL-1β and TNF-α were inhibited by tyrosine kinase, MEK1/2 and PI3-kinase inhibitors. Results of ChIP assays showed that IL-1β and TNF-α increased C/EBPβ and YY1 binding to the C/EBP1, C/EBP2 and YY1 elements. These results demonstrate that IL-1β and TNF-α increase AMTN gene transcription via the C/EBP1, C/EBP2 and YY1 elements in the mouse AMTN gene promoter.