Elevated Foxo3a and Fas-ligand expression in human periapical granulomas as a potential treatment target.

OBJECTIVE: Periapical granuloma is a common periodontitis type involving chronic inflammation; however, the efficacy of current therapies is limited. Its molecular pathogenesis also remains obscure. Forkhead box transcription factor class o3a (Foxo3a) and Fas-ligand (FasL) are associated with chronic inflammation. Therefore, in this study, we aimed to clarify the roles of Foxo3a and FasL in periapical granuloma pathophysiology. SUBJECTS AND METHODS: Periapical lesions were obtained from patients during endodontic surgery and tooth extraction; those diagnosed with periapical granulomas using haematoxylin and eosin staining were further analysed. Immunohistochemical analysis was performed for Foxo3a and FasL, and real-time polymerase chain reaction was performed for FOXO3A, FASL and interleukin (IL)-1ß. Healthy gingival tissues were also examined as controls. RESULTS: Neutrophils, lymphocytes and plasma cells in the periapical granulomas, but not healthy tissues, expressed Foxo3a. Dual-colour immunofluorescence imaging revealed Foxo3a and FasL co-expression in leukocytes. FOXO3A, FASL and IL-1ß mRNA levels in healthy gingival tissues were significantly lower than those in the periapical granulomas. Additionally, FOXO3A and IL-1ß expressions were negatively correlated. CONCLUSIONS: Phosphorylated Foxo3a may reduce IL-1ß release by inhibiting apoptosis through FasL in periapical periodontitis and prevent exacerbation. Thus, Foxo3a is a potential therapeutic agent for periapical periodontitis.

Epstein-Barr Virus Promotes the Production of Inflammatory Cytokines in Gingival Fibroblasts and RANKL-Induced Osteoclast Differentiation in RAW264.7 Cells.

Periodontitis is an inflammatory condition that causes the destruction of the supporting tissues of teeth and is a major public health problem affecting more than half of the adult population worldwide. Recently, members of the herpes virus family, such as the Epstein-Barr virus (EBV), have been suggested to be involved in the etiology of periodontitis because bacterial activity alone does not adequately explain the clinical characteristics of periodontitis. However, the role of EBV in the etiology of periodontitis is unknown. This study aimed to examine the effect of inactivated EBV on the expression of inflammatory cytokines in human gingival fibroblasts (HGFs) and the induction of osteoclast differentiation. We found that extremely high levels of interleukin (IL)-6 and IL-8 were induced by inactivated EBV in a copy-dependent manner in HGFs. The levels of IL-6 and IL-8 in HGFs were higher when the cells were treated with EBV than when treated with lipopolysaccharide and lipoteichoic acid. EBV induced IκBα degradation, NF-κB transcription, and RAW264.7 cell differentiation into osteoclast-like cells. These findings suggest that even without infecting the cells, EBV contributes to inflammatory cytokine production and osteoclast differentiation by contact with oral cells or macrophage lineage, resulting in periodontitis onset and progression.

Role of macrophage-mediated Toll-like receptor 4-interleukin-1R signaling in ectopic tongue pain associated with tooth pulp inflammation.

BACKGROUND: The existence of referred pain and ectopic paresthesia caused by tooth pulp inflammation may make definitive diagnosis difficult and cause misdiagnosis or mistreatment; thus, elucidation of that molecular mechanism is urgent. In the present study, we investigated the mechanisms underlying ectopic pain, especially tongue hyperalgesia, after tooth pulp inflammation. METHODS: A rat model with mandibular first molar tooth pulp exposure was employed. Tooth pulp exposure-induced heat and mechanical-evoked tongue hypersensitivity was measured, and immunohistochemical staining for Iba1, a marker of active macrophages, IL-1ß, IL-1 type I receptor (IL-1RΙ), and toll-like receptor 4 in the trigeminal ganglion was performed. In addition, we investigated the effects of injections of liposomal clodronate Clophosome-A (LCCA), a selective macrophage depletion agent, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS, a toll-like receptor 4 antagonist), IL-1ß, or heat shock protein 70 (Hsp70, a selective agonist of toll-like receptor 4), to examine changes in tongue hypersensitivity and in the regulation of IL-1RΙ, toll-like receptor 4, and transient receptor potential vanilloid 1 (TRPV1) biosynthesis. RESULTS: At day 1 after tooth pulp exposure, obvious tooth pulp inflammation was observed. Tooth pulp exposure-induced heat and mechanical tongue hypersensitivity was observed from days 1 to 3 after tooth pulp exposure. The production of IL-1ß in activated macrophages and toll-like receptor 4 and IL-1RΙ expression were significantly increased in trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of LCCA significantly suppressed tongue hypersensitivity; however, toll-like receptor 4 and IL-1RΙ expression in trigeminal ganglion neurons innervating the tongue was not significantly altered. Intra-trigeminal ganglion injection of LPS-RS significantly suppressed tongue hypersensitivity and reduced IL-1RΙ expression in the trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of recombinant Hsp70 significantly promoted tongue hypersensitivity and increased IL-1RI expression in trigeminal ganglion neurons innervating the tongue in naive rats. Furthermore, intra-trigeminal ganglion injection of recombinant IL-1ß led to tongue hypersensitivity and enhanced TRPV1 expression in trigeminal ganglion neurons innervating the tongue in naive rats. CONCLUSIONS: The present findings suggest that the neuron-macrophage interaction mediated by toll-like receptor 4 and IL-1RI activation in trigeminal ganglion neurons affects the pathogenesis of abnormal tongue pain following tooth pulp inflammation via IL-1RI and TRPV1 signaling in the trigeminal ganglion. Further research may contribute to the establishment of new therapeutic and diagnostic methods.

Mirogabalin as a Therapeutic Option for Neuropathic Pain Emerging Post-endodontic Treatment: A Two-Case Report.

INTRODUCTION: Occlusal and percussion pain may manifest occasionally following endodontic treatment, influencing retreatment decisions. Two cases of periapical neuropathic pain, classified as post-traumatic trigeminal neuropathic pain according to the International Classification of Orofacial Pain, are presented. Although mirogabalin is effective in managing neuropathic pain, there is a lack of clinical reports on its use for occasional post-traumatic trigeminal neuropathic pain after endodontic treatment. These cases highlight clinical symptoms and successful treatment with mirogabalin for post-traumatic trigeminal neuropathic pain after endodontic treatment, providing clinicians a "take-away" lesson for improving patient condition. METHODS: The patients, referred by their primary dentist due to postendodontic abnormal pain, found no relief with antibiotics or nonsteroidal anti-inflammatory drugs. Although no findings including swelling or periapical radiolucency were observed around the tooth, they experienced occlusal and percussion pain. Local anesthetic testing showed that the pain originated from the peripheral area around the tooth rather than from central sensitization. Dental radiography and cone-beam computed tomography revealed no abnormal findings. Root canal retreatment was performed by a specialist in endodontic treatment. Although endodontic retreatment drastically decreased visual analog scale pain score, pain persisted. Based on the International Classification of Orofacial Pain criteria, diseases other than post-traumatic trigeminal neuropathic pain were excluded. Mirogabalin (10 mg/d) was prescribed once daily before bedtime. RESULTS: Visual analog scale scores gradually and drastically decreased 2 weeks after mirogabalin therapy. Several months later, no recurrence of postendodontic pain was observed after tapering off and discontinuing mirogabalin. CONCLUSIONS: These findings suggest the possibility of a new treatment method for post-traumatic trigeminal neuropathic pain after endodontic treatment.

Utility of biphasic calcium phosphate cement as a seal for root-end filling.

The recently developed biphasic calcium phosphate cement (BCPC) consists of α-tricalcium phosphate-tetracalcium phosphate as the solid phase and calcium phosphate solution as the liquid phase. BCPC powder is composed of a single solid solution with a monomodal size distribution. Here, we used a bacterial leakage model to examine the utility of BCPC as a seal for root-end filling. We prepared large (median particle size=9.96 µm; BCPC-L) and small (median particle size=4.84 µm; BCPC-S) BCPC powders. In total, 45 single-rooted teeth were instrumented, resected at the root-end, and retrofilled with experimental materials. Mineral trioxide aggregate (MTA) was used as the control. After visual confirmation of BCPC powder size and retrofilling quality by microscopy, bacterial leakage tests were conducted using Enterococcus faecalis. The bacterial leakage tests did not reveal any significant differences between BCPC-S and MTA. Our findings suggest that BCPC-S is useful for root-end filling.

IL-6 and soluble IL-6 receptor stimulate the production of MMPs and their inhibitors via JAK-STAT and ERK-MAPK signalling in human chondrocytes.

Elevated concentrations of IL-6 (interleukin-6) and sIL-6r (soluble IL-6 receptor) in the synovial fluid and serum of patients with arthritis have been implicated in joint cartilage destruction. This study examined the effects of IL-6 and sIL-6r on the expression of MMPs (matrix metalloproteinases), TIMPs (tissue inhibitor of metalloproteinases), the plasminogen activation system including tPA (tissue-type PA), uPA (urokinase-type PA) and PAI-1 (PA inhibitor type 1) using chondrocytes derived from normal human femur cartilage. The cells were cultured with or without 50 ng/ml IL-6 and/or 30 ng/ml sIL-6r in the presence or absence of the JAK3 (Janus kinase 3) inhibitor WHI-P131 or the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular signal protein kinase) kinase] inhibitor PD98059 for up to 28 days. The expression of MMPs, TIMPs, uPA, tPA and PAI-1 was investigated at the mRNA and protein levels. MMP protein expression and pSTAT3 (phosphorylation of signal transducer and activator of transcription 3) and pERK (phosphorylation of ERK) were also measured. Treatment with both IL-6 and sIL-6r markedly increased the expression of MMP-1, MMP-13, TIMP-1 and PAI-1, while significantly decreasing the expression of tPA and uPA and stimulating pSTAT3 and pERK. Adding WHI-P131 or PD98059 decreased IL-6 and sIL-6r enhancement of MMP-1, -3 and -13. The results suggest that IL-6 and sIL-6r stimulate the production of MMPs and their inhibitor via JAK-STAT and ERK-MAPK signalling in chondrocytes.

Role of S100A4 in the Pathogenesis of Human Periapical Granulomas.

BACKGROUND/AIM: S100A4 expression is associated with the pathology of chronic inflammatory diseases. In this study, we investigated the role of S100A4 and four inflammatory mediators (IL-1ß, IκB, IL-10, and TNF-α) in human periapical granulomas (PGs). MATERIALS AND METHODS: S100A4 expression in PGs obtained by apicoectomy was examined by immunohistochemistry. Further, the expression of S100A4 and four inflammatory mediators was compared between PGs and healthy gingival tissues (HGTs) using real-time PCR. RESULTS: In the PGs, S100A4 was found to be expressed in endothelial cells and fibroblasts. Furthermore, real-time PCR revealed that the expression of S100A4 and IL-1ß in PGs was significantly higher than that in HGTs. Although a correlation between the expression of S100A4 and IκB or IL-10 was not detected, a positive correlation between the expression of S100A4 and IL-1ß or TNF-α was observed. CONCLUSION: The expression of S100A4 correlates with the pathogenesis of PGs.

The use of micro-computed tomography to determine the accuracy of electronic working length with two apex locators.

PURPOSE: This study evaluated the precision of electronic working length by microcomputed tomography using two electronic apex locators (EALs). METHODS: Twenty single-rooted permanent teeth without caries or restorations were selected as the subject teeth. The positions of the minor apical constriction (AC) and major apical foramen (AF) were measured by electronic root canal length, and microcomputed tomography was performed with the file inserted and fixed in the root canal. All teeth were measured individually and independently by two operators. The Mann-Whitney U-test was used to statistically test the AC and AF values using two EALs; P < 0.05 was defined as statistically significant. RESULTS: This was 65.0% within 1.5 mm in the case of two EALs on AC. This was more than 90.0% within 1.0 mm in the case of two EALs on AF. Comparison of the differences between the respective AC and AF of the measurements obtained using the two EALs revealed no significant difference. CONCLUSION: The two EALs are devices that can greatly improve the accuracy of WL control.

Efficacy of prototype endodontic obturators for novel root canal obturation techniques using a resin-based sealer in various powder-liquid ratios.

PURPOSE: This study aimed to examine novel techniques using prototype endodontic obturators to obturate a resin-based sealer. METHODS: Powder-liquid ratios of MetaSEAL Soft were changed to obtain suitable root canal sealing, and the physical properties for various powder-liquid ratios were analyzed according to ISO-6876. Tensile bond strength was also examined. Prototype endodontic obturators with a combination of thread numbers and pitch angles were analyzed for sealing ability after MetaSEAL Soft was obturated in simulated root canals. RESULTS: Powder-liquid ratios of 1.0:1, 1.1:1, 1.2:1, and 1.3:1 showed suitable physical properties; however, flow for 1.4:1 was below a standard value. Tensile bond strength increased gradually when the powder-liquid ratio changed from 1.0:1 to 1.3:1, and 1.3:1 and 1.4:1 showed the highest and lowest bond strengths, respectively. Sealing ability increased when pitch angles of the obturators were 5°, 8°, and 11°; 11° showed the best results. Similarly, sealing ability increased when the thread number was 12, 17, and 22 pitches; 22 showed the best results. CONCLUSION: These findings suggest that the prototype endodontic obturator can be useful for obturating MetaSEAL Soft, and a powder-liquid ratio of 1.3:1 MetaSEAL Soft may be the most suitable for achieving excellent sealing.

Butyric Acid in Saliva of Chronic Periodontitis Patients Induces Transcription of the EBV Lytic Switch Activator BZLF1: A Pilot Study.

BACKGROUND/AIM: Epstein-Barr virus (EBV) associates with human chronic periodontitis (CP) progression. We previously demonstrated that butyric acid (BA), produced by periodontopathic bacteria, induced EBV lytic switch activator BZLF1 expression. We investigated whether short chain fatty acids (SCFAs) in CP patients' saliva enabled EBV reactivation. MATERIALS AND METHODS: Saliva was collected from seven CP patients and five periodontally healthy individuals. SCFAs were quantified using HPLC. BZLF1 mRNA and its pertinent protein ZEBRA were determined with Real-time PCR and western blotting. Histone H3 acetylation (AcH3) was further examined. RESULTS: BZLF1 mRNA expression and transcriptional activity in EBV-infected Daudi cells were induced only when treated with the CP saliva. Among SCFAs, BA alone correlated significantly with the BZLF1 transcription (r=0.88; p<0.02). As expected, CP patients' saliva induced AcH3. CONCLUSION: BA in saliva may play a role in EBV reactivation and hence contribute to EBV-related disease progression in CP patients.

EBV LMP1 in Gingival Epithelium Potentially Contributes to Human Chronic Periodontitis via Inducible IL8 Production.

BACKGROUND/AIM: Human chronic periodontitis is a major health problem. Although some oral bacteria have been reported to be putative pathogens, Epstein-Barr virus (EBV) is reported to be associated with the progression of periodontitis. However, the role of EBV in the aetiology of periodontitis is unknown. Therefore, we investigated periodontal pathogenesis of EBV to confirm whether EBV-encoded latent membrane protein 1 (LMP1) induces Interleukin-8 (IL8) production in human gingival cells. MATERIALS AND METHODS: Real-time polymerase chain reaction, luciferase assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were performed for determining IL8 mRNA expression, nuclear factor kappa B (NF-ĸB) transcription, IL8 production, and the phosphorylation of NF-ĸB p65 and Inhibitor of kappa B alpha (IĸBα), respectively, in Ca9-22 human gingival epithelial cells. Two LMP1 mutants lacking C-terminal activating region (CATR) domains responsible for activating NF-ĸB were used. RESULTS: Extremely high IL8 production was induced by LMP1 in time- and dose-dependent manner, where simultaneous phosphorylation of NF-κB p65 and IĸBα and transcription of NF-ĸB were observed. On the contrary, IL8 production and NF-ĸB transcription were drastically inhibited by dominant negative mutant of IĸBα. Moreover, the LMP1 mutants failed to induce IL8 production. CONCLUSION: Our findings suggest that due to CATR domains, LMP1 contributes to the progression of periodontitis via IL8 production attributable to NF-ĸB activation.

Profiling of radicular cyst and odontogenic keratocyst cytokine production suggests common growth mechanisms.

The aim of this study was to compare the cytokine expression profiles of cyst fluids (CFs) and tissue culture supernatants (SUPs) from 7 radicular cysts (RCs) and 7 odontogenic keratocysts (OKCs) by using Human Cytokine Antibody Array to identify the specific cytokines involved in formation and expansion of RCs and OKCs, respectively. There were significant differences in relative expression levels of IL-1 beta, MCP1, MIP1 beta, FGF-9, GDNF, HGF, IGFBP-3, Ang, IP-10, MIF, OPG, and TGF-beta2 between RC-CF and OKC-CF (P < .05). On the other hand, the cytokine expression patterns of RC-SUP (HGF, IL-8, NAP-2, IL-6, TIMP-1 and 2, GRO, IP-10, and Ang) were similar to those of OKC-SUP. Only the relative expression level of GRO differed between RC-SUP and OKC-SUP (P < .05). The similarities of cytokine production by tissue cultures derived from RC and OKC indicate that the expansion mechanisms of RC and OKC might involve similar biologic mechanisms other than infection.

Expression of silent information regulator 2 homolog 1 (SIRT1) in periapical granulomas.

Silent information regulator 2 homolog 1 (SIRT1) inhibits oxidative injury and has anti-inflammatory effects. SIRT1 may be involved in healing of periapical periodontitis; however, SIRT1 expression in periapical periodontitis lesions has not been investigated. This study evaluated SIRT1 expression and a marker of oxidative stress-8-hydroxy-2'-deoxyguanosine (8-OHdG)-in periapical granulomas. First, we used real-time polymerase chain reaction to determine whether U-937 monocytes express SIRT1. U-937 cells treated with the SIRT1 activator resveratrol exhibited the highest SIRT1 mRNA level after 6-h incubation. By contrast, treating cells with the SIRT1 inhibitor sirtinol returned SIRT1 expression level to that of the control. In addition, immunocytochemical analysis using cytospin specimens showed that U-937 cells co-expressed SIRT1 and Ki-67. Dual-color immunofluorescence imaging showed that round cells in periapical granulomas co-expressed SIRT1 and 8-OHdG; however, neither was expressed in healthy gingival tissues. The number of 8-OHdG-expressing cells was significantly greater than the number of SIRT1-expressing cells. Our findings suggest that macrophages express SIRT1 and that wound healing in periapical granulomas is enhanced by a SIRT1-mediated reduction in the level of oxidative stress.

Expression of the Forkhead box transcription factor Foxo3a in human periapical granulomas.

It has been reported that Forkhead box transcription factor class O3a (Foxo3a) is expressed in rheumatoid arthritis, a chronic inflammatory condition accompanied by bone resorption, and plays a role in its pathology. However, it has remained unclear whether Foxo3a is involved in the pathogenesis of periapical granulomas. The present study was performed to compare the expression of Foxo3a in periapical granulomas and healthy gingival tissues. Samples were obtained surgically from patients, and subjected to hematoxylin-eosin staining for histopathologic diagnosis. Two-color immunofluorescence staining was also performed using antibodies against Foxo3a and markers for three types of inflammatory cells: neutrophils, T lymphocytes, and B lymphocytes. This revealed that Foxo3a was expressed in all three cell types in periapical granulomas but not in healthy gingival tissues. Foxo3a was expressed in 82.1%, 78.3%, and 77.5% of neutrophils, T lymphocytes, and B lymphocytes, respectively, and statistical analysis using the Kruskal-Wallis test followed by the Steel-Dwass test showed no significant difference of Foxo3a expression among the three cell types. Our results suggest that Foxo3a transcription factors may be involved in the pathogenesis of periapical granulomas.

Microcomputed tomographic evaluation of techniques for warm gutta-percha obturation.

Transparent epoxy resin root canal models were used to evaluate vertical condensation techniques for obturating lateral canals. The root canal model was configured with a straight main root canal and four right-angled lateral canals at 1.0 and 3.0 mm from the apex. Root canal obturation was performed with Thermafil, Obtura II, or NT condenser. Obturation volume in lateral canals was measured by three-dimensional microcomputed tomography, and one-way analysis of variance was used to analyze differences between groups. Lateral canals at 1.0 and 3.0 mm were uniformly filled by all obturation methods. Among the three obturation methods, Thermafil resulted in the highest obturation volumes for all lateral canals.

Receptor activator of NF-kappaB ligand induces the expression of carbonic anhydrase II, cathepsin K, and matrix metalloproteinase-9 in osteoclast precursor RAW264.7 cells.

Interleukin-1 (IL-1) is a proinflammatory cytokine that is a potent stimulator of bone resorption and an inhibitor of bone formation, whereas macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB (RANK) ligand (RANKL) are essential and sufficient for osteoclast differentiation. Recently, we showed that IL-1alpha affects mineralized nodule formation in vitro and halts bone matrix turnover. We also showed that IL-1alpha stimulates osteoclast formation via the interaction of RANKL with RANK by increasing M-CSF and prostaglandin E(2) and decreasing osteoprotegerin. Here, we examined the effects of IL-1alpha or RANKL and/or M-CSF in the presence of IL-1alpha on the expression of carbonic anhydrase II (CAII), cathepsin K, matrix metalloproteinase-9 (MMP-9), RANK, M-CSF receptor (c-fms), and c-fos transcription factor using RAW264.7 cells as osteoclast precursors. Cells were cultured for up to 14 days in 0 or 100 U/ml IL-1alpha and either 50 ng/ml RANKL, 10 ng/ml M-CSF, or 50 ng/ml RANKL+10 ng/ml M-CSF in the presence of 100 U/ml IL-1alpha. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase staining. Expression of the genes coding for the six proteins of interest was determined using real-time PCR, and the expression of the three enzymes was examined using Western blotting or ELISA. In the presence of IL-1alpha, expression of CAII, cathepsin K, and MMP-9 was markedly increased in cells cultured with RANKL or M-CSF+RANKL, whereas expression was difficult to detect in cells cultured with IL-1alpha alone and M-CSF. RANK and c-fos expression was also increased in cells cultured with RANKL or M-CSF+RANKL in the presence of IL-1alpha, whereas c-fms expression did not change. These results indicate that the expression of CAII, cathepsin K, and MMP-9 in RAW264.7 cells is not induced by M-CSF, but by RANKL in the presence of IL-1alpha.

Involvement of inducible nitric oxide synthase and receptor for advanced glycation end products in periapical granulomas.

We examined the presence of the receptor for advanced glycation end products (RAGE) in periapical granulomas and analyzed the interaction between RAGE and inducible nitric oxide synthase (iNOS) to elucidate inflammatory reaction mechanisms. Periapical lesions were surgically removed from 37 patients with chronic periapical periodontitis and halved. Paraffin sections were prepared from half of each lesion and stained with hematoxylin and eosin, whereas cryostat sections were prepared from the other half. Based on the histological evaluation, 33 of the lesions were diagnosed as periapical granulomas. These were examined by immunohistochemistry using serial cryostat sections probed with anti-human iNOS or RAGE antibodies. Macrophages, lymphocytes, and endothelial cells expressed RAGE and these cell types, in addition to plasma cells, exhibited anti-iNOS immunoreactivity. Serial cryostat sections demonstrated the infiltration of RAGE-expressing cells around iNOS-producing cells, suggesting that these molecules may be important in the tissue injury associated with periapical periodontitis.

Lipopolysaccharide stimulates the production of prostaglandin E2 and the receptor Ep4 in osteoblasts.

Previous studies have indicated that one of the causes of alveolar bone destruction with periodontitis is lipopolysaccharide (LPS) from the cell wall of gram-negative bacteria in plaque, and that prostaglandin E(2) (PGE(2)) is one of the bone resorption factors that stimulate osteoclast formation through an intercellular interaction between osteoblasts and osteoclast precursors. The present study was undertaken to determine the effect of LPS on cell growth, alkaline phosphatase (ALPase) activity, the production of PGE(2), and the expression of receptors by PGE(2), cyclooxygenase (COX)-1, and COX-2, using human osteosarcoma cell line Saos-2 as osteoblasts. The cells were cultured with 0, 1, or 10 microg mL(-1) of LPS for up to 14 days. The production of PGE(2) and the gene expression of COX-1, COX-2, and PGE(2) receptors, including Ep1, Ep2, Ep3, and Ep4, were determined using enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), respectively. With the addition of LPS, cell growth and ALPase activity decreased by day 5 of the culture, while PGE(2) production increased in a dose-dependent manner throughout the entire 14-day culture period. LPS-reduced ALP activity and LPS-induced PGE(2) production returned to the control level by the addition simultaneously with indomethacin. The expression of COX-1, Ep1, Ep2, and Ep3 receptors decreased on day 14 of the culture, whereas the expression of COX-2 and Ep4 receptors increased significantly with the addition of LPS. These results suggest that LPS promotes PGE(2) production by increasing the expression of COX-2, and that LPS promotes the production of Ep4 receptors in osteoblasts. These results also indicate that LPS-induced PGE(2) may combine with osteoblast Ep4 receptors in autocrine or paracrine modes, and may promote the formation of osteoclasts.

Epstein-Barr virus infection in chronically inflamed periapical granulomas.

Periapical granulomas are lesions around the apex of a tooth caused by a polymicrobial infection. Treatment with antibacterial agents is normally performed to eliminate bacteria from root canals; however, loss of the supporting alveolar bone is typically observed, and tooth extraction is often selected if root canal treatment does not work well. Therefore, bacteria and other microorganisms could be involved in this disease. To understand the pathogenesis of periapical granulomas more precisely, we focused on the association with Epstein-Barr virus (EBV) using surgically removed periapical granulomas (n = 32). EBV DNA was detected in 25 of 32 periapical granulomas (78.1%) by real-time PCR, and the median number of EBV DNA copies was approximately 8,688.01/µg total DNA. In contrast, EBV DNA was not detected in healthy gingival tissues (n = 10); the difference was statistically significant according to the Mann-Whitney U test (p = 0.0001). Paraffin sections were also analyzed by in situ hybridization to detect EBV-encoded small RNA (EBER)-expressing cells. EBER was detected in the cytoplasm and nuclei of B cells and plasma cells in six of nine periapical granulomas, but not in healthy gingival tissues. In addition, immunohistochemical analysis for latent membrane protein 1 (LMP-1) of EBV using serial tissue sections showed that LMP-1-expressing cells were localized to the same areas as EBER-expressing cells. These data suggest that B cells and plasma cells in inflamed granulomas are a major source of EBV infection, and that EBV could play a pivotal role in controlling immune cell responses in periapical granulomas.

The periodontal pathogen Porphyromonas gingivalis induces the Epstein-Barr virus lytic switch transactivator ZEBRA by histone modification.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that usually results in latent infection of B cells. The EBV BZLF1 gene product ZEBRA is a master regulator of the transition from latency to the lytic replication cycle. In the latent state, hypoacetylation of histone proteins in the BZLF1 promoter by histone deacetylases (HDACs) is primarily involved in maintaining EBV latency. Although the mechanism that regulates the switch between latency and lytic replication has been a central research focus in EBV infection, the causal link between HDAC inhibition and the disruption of viral latency is not well understood. Periodontal disease is a complex chronic inflammatory disease caused by subgingival infection with oral anaerobic bacteria, typically Porphyromonas gingivalis. Periodontal disease occurs worldwide and is among the most prevalent microbial diseases in humans. In this study, we examined the biological effect of P. gingivalis infection on EBV reactivation and found that P. gingivalis induced expression of ZEBRA. This activity was associated with supernatant from bacterial culture, but not with other bacterial components such as lipopolysaccharide or fimbriae. We demonstrated that culture supernatant from P. gingivalis, which contained high concentrations of butyric acid, inhibited HDACs, thus increasing histone acetylation and the transcriptional activity of the BZLF1 gene. Chromatin immunoprecipitation assays revealed that HDACs were present in the BZLF1 promoter during latent state and that they were dissociated from the promoter concomitantly with the association of acetylated histone H3, upon stimulation by culture supernatant from P. gingivalis. Thus, P. gingivalis induced EBV reactivation via chromatin modification, and butyric acid-a bacterial metabolite-was responsible for this effect. These findings suggest that periodontal disease is a risk factor for EBV reactivation in infected individuals and might therefore contribute to progression of EBV-related diseases.