Masayuki Tokuda

Regulation of Interleukin-6 Expression in Human Dental Pulp Cell Cultures Stimulated with Prevotella intermedia Lipopolysaccharide

Interleukin (IL)-6 expression in human dental pulp cell cultures after stimulation with Prevotella intermedia lipopolysaccharide (LPS) was investigated by Northern blot analysis, enzyme immunoassay, and bioassay. The IL-6 mRNA expression began to increase after 1 hr and continued after up to 8 hr of exposure on stimulation with 10 microg/ml of P. intermedia LPS. The bioactivity was dose-dependent on the concentration of P. intermedia LPS (0 to 100 microg/ml). The IL-6 mRNA expression was inhibited by actinomysin D and super-induced by cycloheximide. Anti-CD14 monoclonal antibody (MY4) inhibited the IL-6 mRNA expression when administered at a 0.5 microg/ml concentration before stimulation with P. intermedia LPS at 1 microg/ml. The immunoregulatory cytokines (interferon-gamma, IL-10, and IL-4) inhibited LPS-induced IL-6 production with a combined treatment. These results suggest the IL-6 expression by pulp cell cultures is CD14-dependent and regulated at the transcriptional level, and a combined treatment with immunoregulatory cytokines may be effective for control of pulpal inflammation due to P. intermedia LPS.

Interleukin-8 gene expression by human dental pulp fibroblast in cultures stimulated with Prevotella intermedia lipopolysaccharide.

Interleukin (IL)-8 mRNA expression was investigated in human dental pulp fibroblast cultures after stimulation with lipopolysaccharide (LPS) prepared from Prevotella intermedia and inflammatory cytokines. The expression of IL-8 mRNA and the release of IL-8 induced by P. intermedia LPS in pulpal fibroblast cultures were detected by Northern blot analysis and ELISA, respectively. The sufficient concentration of P. intermedia LPS on the IL-8 mRNA expression was 0.1 microgram/ml in pulpal fibroblast cultures. IL-8 mRNA levels began to increase after 2 h of exposure, reached a maximum at 4 to 8 h, and declined after 48 h, reaching the unstimulated level by 60 h. IL-8 production by the pulpal fibroblasts began to increase after 8 h of exposure upon stimulation with 10 microgram/ml of P. intermedia LPS. By contrast Salmonella LPS and synthetic lipid A did not increase IL-8 mRNA concentrations in pulpal fibroblast cultures. Recombinant human IL-1 alpha, beta, and tumor necrosis factor-alpha were capable of stimulating these cells to express IL-8 mRNA but natural human interferon-beta, gamma, and recombinant human IL-6 were incapable in our assay. These results suggest that pulpal fibroblasts are immunoresponsive cells and can elaborate IL-8 upon stimulation with P. intermedia LPS.

Dual Regulatory Effects of Interferon-α, -β, and -γ on Interleukin-8 Gene Expression by Human Gingival Fibroblasts in Culture upon Stimulation with Lipopolysaccharide from Prevotella intermedia, Interleukin-1α, or Tumor Necrosis Factor-α

In a previous study, we demonstrated that the amount of interleukin (IL)-8 mRNA expressed by human gingival fibroblasts stimulated with lipopolysaccharide (LPS) from Prevotella intermedia ATCC 25611 is increased by pre-treatment with beta or gamma interferon (IFN-β or --y). In the present study, we identified the regulatory effects of these IFNs on IL-8 mRNA expression and IL-8 production by human gingival fibroblasts. Priming with IFN-alpha (a), -β, or --y upregulated the IL-8 mRNA expression in response to P. intermedia LPS, whereas co-stimulation with these IFNs reduced the amount of mRNA expressed by the cells. The regulation of IL-8 mRNA expression induced by recombinant human tumor necrosis factor-a (rHuTNF-a) or rHuIL-1α was similar to that induced by LPS. The IL-8 mRNA expression in response to P. intermedia LPS was enhanced by IFN-γ independently of de novo protein synthesis, and was regulated, at least in part, at the transcriptional level. The IL-8 mRNA accumulation in response to P. intermedia LPS was inhibited by tosylphenyl-alanyl chloromethyl-ketone, an inhibitor of NF-KB activation, although the NF-KB activation itself was not altered by IFN-γ. These findings suggest that IFNs might be capable of both enhancing and inhibiting inflammatory responses in periodontal tissues through the dual regulation of IL-8 production by gingival fibroblasts in response to bacterial components and cytokines.

MK615 attenuates Porphyromonas gingivalis lipopolysaccharide-induced pro-inflammatory cytokine release via MAPK inactivation in murine macrophage-like RAW264.7 cells.

The Japanese apricot, known as Ume in Japanese, has been a traditional Japanese medicine for centuries, and is a familiar and commonly consumed food. The health benefits of Ume are now being widely recognized and have been strengthened by recent studies showing that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in the periodontal field remains unknown. Here, we found that MK615 significantly reduced the production of pro-inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) induced by Porphyromonas gingivalis lipopolysaccharide (LPS), a major etiological agent in localized chronic periodontitis, in murine macrophage-like RAW264.7 cells. MK615 markedly inhibited the phosphorylation of ERK1/2, p38MAPK, and JNK, which is associated with pro-inflammatory mediator release pathways. Moreover, MK615 completely blocked LPS-triggered NF-kappaB activation. The present results suggest that MK615 has potential as a therapeutic agent for treating inflammatory diseases such as periodontitis.

Ca2+ extrusion via Na+-Ca2+ exchangers in rat odontoblasts.

INTRODUCTION Intracellular Ca(2+) is essential to many signal transduction pathways, and its level is tightly regulated by the Ca(2+) extrusion system in the plasma membrane, which includes the Na(+)-Ca(2+) exchanger (NCX). Although expression of NCX1 isoforms has been demonstrated in odontoblasts, the detailed properties of NCX remain to be clarified. In this study, we investigated localization and ion-transporting/pharmacologic properties of NCX isoforms in rat odontoblasts. METHODS We characterized both the reverse and forward modes of NCX activity in odontoblasts in a dental pulp slice preparation. Ca(2+) influx by reverse NCX activity was measured by fura-2 fluorescence. Ca(2+) efflux by forward NCX activity elicited inward Na(+) current as measured by perforated-patch clamp recording. For immunohistochemical analysis, cryostat sections of incisors were incubated with antibodies against NCX. RESULTS Immunohistochemical observation revealed localization of NCX1 and NCX3 in the distal membrane of odontoblasts. Inward currents by forward NCX activity showed dependence on external Na(+). Fura-2 fluorescence measurement revealed that Ca(2+) influx by reverse NCX activity depended on extracellular Ca(2+) concentration, and that this influx was blocked by NCX inhibitor KB-R7943 in a concentration-dependent manner. However, Ca(2+) influx by NCX showed a slight sensitivity to SEA0400 (a potent NCX1 inhibitor), indicating that expression potencies in odontoblasts were NCX3 > NCX1. CONCLUSIONS These results suggest that odontoblasts express NCX1 and NCX3 at the distal membrane, and that these isoforms play an important role in the Ca(2+) extrusion system as well as in the directional Ca(2+) transport pathway from the circulation to the dentin-mineralizing front.

dpr and sod in Streptococcus mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H2O2.

ABSTRACT Large numbers of bacteria coexist in the oral cavity. Streptococcus sanguinis, one of the major bacteria in dental plaque, produces hydrogen peroxide (H2O2), which interferes with the growth of other bacteria. Streptococcus mutans, a cariogenic bacterium, can coexist with S. sanguinis in dental plaque, but to do so, it needs a means of detoxifying the H2O2 produced by S. sanguinis. In this study, we investigated the association of three oxidative stress factors, Dpr, superoxide dismutase (SOD), and AhpCF, with the resistance of S. sanguinis to H2O2. The knockout of dpr and sod significantly increased susceptibility to H2O2, while the knockout of ahpCF had no apparent effect on susceptibility. In particular, dpr inactivation resulted in hypersensitivity to H2O2. Next, we sought to identify the factor(s) involved in the regulation of these oxidative stress genes and found that PerR negatively regulated dpr expression. The knockout of perR caused increased dpr expression levels, resulting in low-level susceptibility to H2O2 compared with the wild type. Furthermore, we evaluated the roles of perR, dpr, and sod when S. mutans was cocultured with S. sanguinis. Culturing of the dpr or sod mutant with S. sanguinis showed a significant decrease in the S. mutans population ratio compared with the wild type, while the perR mutant increased the ratio. Our results suggest that dpr and sod in S. mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H2O2 in regulating the expression of Dpr.

Substance P activates p38 mitogen-activated protein kinase to promote IL-6 induction in human dental pulp fibroblasts.

Substance P (SP) induces the expression of proinflammatory cytokines, such as interleukin (IL)-6, which are implicated in pulp inflammation. To determine the signal pathway of SP-induced IL-6, we examined the activities of the mitogen-activated protein kinases (MAPKs) in human dental pulp cell (PF-10) cultures. SP induced the phosphorylation of p38 MAPK within 5 min; this activation persisted for up to 40 min and was independent of the activation of extracellular signal-related kinases (ERK-1 and ERK-2) that were induced after SP stimulation of PF-10 cells. As shown by electrophoretic mobility shift assay p38 MAPK was not involved in SP-induced activation of nuclear factor-kappa B (NF-κB). However, p38 MAPK mediated SP-induced IL-6 production, as shown by the use of specific inhibitors of this kinase. Our results suggest that the activation of p38 MAPK is important for NF-κB-independent regulator of neurogenic inflammation in dental pulp tissues.

Bending strength of zirconia/porcelain functionally graded materials prepared using spark plasma sintering

OBJECTIVES The purpose of this study was to fabricate functionally graded materials (FGMs) consisting of yttria-stabilised tetragonal zirconia polycrystal (Y-TZP) and porcelain using spark plasma sintering (SPS) and examine the influence of their microstructures and thermal stress on their bending strengths. METHODS Two types of four-layered Y-TZP/porcelain FGMs having a constant layer thickness and a varying layer thickness, Y-TZP/porcelain composite materials having a microstructure corresponding to each layer in FGMs and monolithic materials of Y-TZP and porcelain were fabricated by SPS. The Y-TZP/porcelain volume fraction of each layer in FGMs was varied over 100/0-70/30. Three-point bending test, X-ray diffraction, density measurement, microstructure observation, and thermal stress estimation were performed to characterise the materials. RESULTS The bending strength of the Y-TZP/porcelain composite materials decreased with the volume fraction of the porcelain. About FGMs, when the 100%Y-TZP layer was on the tensile stress side during the bending test, the bending strength was almost the same as that of the 100%Y-TZP monolithic material. On the other hand, when the 100%Y-TZP layer was on the compressive stress side, the bending strength of FGM having a constant layer thickness was almost the same as that of the 70%Y-TZP+30%porcelain composite material, while the bending strength of FGM with a varying layer thickness was significantly higher than that of the 70%Y-TZP+30%porcelain composite material. CLINICAL SIGNIFICANCE The FGMs prepared and analyzed in this research can potentially be used for crowns and bridges as well as for inlays and onlays. CONCLUSION The SPS method could effectively fabricate the Y-TZP/porcelain FGMs, and the bending strength results revealed that the graded structure was very efficient to raise the bending strength.

Temperature Triggered Shape Memory Effect of Transpolyisoprene-based Polymer

INTRODUCTION Pure gutta-percha (trans-1, 4-polyisoprene [TPI]) has been used extensively as a main component of gutta-percha for root canal filling. TPI has the interesting shape memory property by cross-linking, and this polymer was commercialized under the product name of SMP-2 (Kuraray Corp, Kashima, Japan). Therefore, the purpose of this study was to examine the thermal properties and the mechanism of the shape memory function of cross-linked SMP-2. METHODS The crystalline of the TPI was observed by x-ray diffraction. The effects of temperature on shape recovery, recovery stress, and relaxation modulus (Er[5]) were measured in cross-linked cylindrical specimens of SMP-2. Differential scanning calorimetry was used to monitor thermal events. RESULTS On heating, a pronounced increase in recovery stress, a marked decrease in Er(5), and endothermic DSC peaks were observed over the same temperature range (38°-51°C) with shape recovery. On the other hand, on cooling, a pronounced decrease in recovery stress, a marked increase in Er(5), and an exothermic DSC peak were observed over the same temperature range (27°-33°C). CONCLUSIONS The shape memory property of TPI is derived from its crystallinity and cross-linking ability. Fixing the deformed shape and shape recovery from the deformed shape to the original shape is relatively easy to achieve by changing the temperature of SMP-2. The shape memory function of the cross-linked SMP-2 was expected to be very useful as a root canal filling material by the modification of its some thermal properties.

Substance P Enhances Expression of Lipopolysaccharide-induced Inflammatory Factors in Dental Pulp Cells

To examine how substance P (SP) is related with dental pulp inflammation, we examined the effects of SP on expression of genes for inflammatory factors in human dental pulp cell cultures. Using reverse transcriptase-polymerase chain reaction, we found that Prevotella intermedia lipopolysaccharide (LPS) induced expression of SP and SP-receptor mRNAs, and that somatostatin inhibited the LPS-induced expression of SP mRNA. We also found that SP enhanced LPS-induced stimulation of NF-kappaB binding activity. In addition, SP induced expression of cyclooxygenase-2 and interleukin-10 receptor mRNAs. In contrast, SP inhibited expression of interferon-gamma receptor mRNA. These results suggest that SP may play a regulatory role in the immunological response of dental pulp tissue to pathogenic bacteria.