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Primary immune responses are initiated by dendritic cells (DC) that inform naive T helper cells about invading pathogens. DC undergo sequential events leading to irreversible maturation upon bacterial stimulation. To investigate the responses of DC during periodontal infection, we studied the effects of LPS from Porphyromonas gingivalis on DC. DC generated from human peripheral monocytes by culture with IL-4 and GM-CSF were incubated with P. gingivalis LPS (Pg LPS) or Escherichia coli LPS (Ec LPS). Flow cytometry and real-time quantitative RT-PCR analysis revealed that Pg LPS, but not Ec LPS, preferentially up-regulated CD14 and CD16 expression at protein and mRNA levels. Furthermore, Pg LPS preferentially induced the secretion of soluble CD14. CD1a, HLA-DR and CD54 were highly expressed on DC stimulated with both kinds of LPS; however, CD40, CD80, CD83 and CD86 expression on Pg LPS-stimulated DC was lower than on Ec LPS-stimulated DC. With regard to IL-6, IL-8, IL-10, IL-12 and RANTES production from DC and allogeneic T cell proliferation, Pg LPS was a weaker stimulator than Ec LPS. These results suggested that Pg LPS triggers maturation of DC with unique characteristics, which exhibited weak immunostimulatory activity and may contribute to induction of chronic inflammation at the site of periodontal infection.

BACKGROUND AND OBJECTIVES CD73/5'-nucleotidase (5'-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5'-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. METHODS AND RESULTS We demonstrated that CD73/5'-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5'-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5'-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5'-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5'-AMP but not adenosine. CONCLUSIONS These findings suggested that CD73/5'-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5'-AMP to adenosine.

Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells.

CD40 is a crucial element in the process of fibroblast activation. We demonstrated that treatment of human gingival fibroblast (HGF) with human leukocyte elastase (HLE), a neutrophil serine protease, down-regulated the expression of CD40 and binding to the CD40 ligand (CD40L) using flow cytometry. The other neutrophil serine proteases, cathepsin G and proteinase 3, exhibited markedly less activity for CD40 reduction. The CD40 reduction by HLE was also observed in skin and lung fibroblasts, but not in monocytes, macrophages, and dendritic cells. The reduction resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD40 on fixed HGF and also on cell lysates and membranes. HLE treatment of HGF decreases interleukin (IL)-8 and macrophage chemoattractant protein-1 production by HGF when stimulated by CD40L, but not by IL-1α, suggesting that HLE inhibited a CD40-dependent cell activation. These results suggest that HLE possesses an anti-inflammatory effect for the HGF-mediated inflammatory process.

The cytokine-inducing activities of fungal polysaccharides were examined in human monocytes in culture, with special reference to CD14 and Toll-like receptors (TLRs). Tumor necrosis factor alpha (TNF-alpha) production by monocytes was markedly induced in a dose-dependent manner upon stimulation with cell walls from Candida albicans and mannan from Saccharomyces cerevisiae and C. albicans, although relatively high concentrations (10 to 100 microg/ml) of stimulants were required for activation as compared with the reference lipopolysaccharide (LPS) (1 to 10 ng/ml). The yeast form C. albicans and its mannan and cell wall fractions exhibited higher TNF-alpha production than respective preparations from the hyphal form. Only slight TNF-alpha production was induced by the S. cerevisiae glucan. The TNF-alpha production triggered by reference LPS and purified fungal mannans required the presence of LPS-binding protein (LBP), and these responses were inhibited by anti-CD14 and anti-TLR4 antibodies, but not by anti-TLR2 antibody. In contrast to the activity of LPS, the activity of purified S. cerevisiae mannan was not inhibited by polymyxin B. These findings suggested that the mannan-LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.

Arginine-specific cysteine proteinases (gingipains-R) from periodontopathicPorphyromonas gingivaliscleaved CD14, a bacterial pattern recognition receptor, on human gingival fibroblasts (HGF). Consequently, gingipains-R reduced lipopolysaccharide-induced interleukin-8 production by HGF, indicating that gingipains-R inhibited CD14-dependent HGF activation and are involved in immune evasion by the bacterium in periodontal tissues.

Dental calculus is calcified plaque composed primarily of calcium phosphate mineral salts, and there is a clear association between the presence of calculus and the initiation/progression of periodontitis. However, it is still inconclusive whether dental calculus can be a direct causative factor. The authors examined the effect of nano/microsized calcium phosphate particles, which may be generated in the process of early precipitation and/or dissolution of calcium phosphate mineral, on the expression of interleukin (IL)-8 in human gingival epithelial cells.Primary human gingival epithelial cells and/or a human gingival carcinoma cell line (Ca9-22) were stimulated with calcium phosphate particles. Gene and protein levels were assessed by real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay, respectively. The activity of nuclear factor (NF)-κB signaling was measured by an immunofluorescence assay to evaluate NF-κB p65 nuclear translocation.The results show that nano/microsized particles stimulate IL-8 expression in human gingival epithelial cells at gene and protein levels. The activity to induce IL-8 expression depends on the particle size: particles with a diameter of 200 nm are more effective than those of 40-nm and 5-μm diameters. Calcium phosphate particles (diameter 200 nm) stimulated NF-κB activity. Pretreatment with BMS-345541, an NF-κB signaling inhibitor, inhibited the particle-mediated IL-8 gene induction, suggesting a requirement for the NF-κB signaling pathway.These findings suggest that calcium phosphate particles, which may be related to calculus development, may act as a direct causative factor in the pathogenesis of gingival epithelium.

Activated polymorphonuclear leukocytes (PMNs) release various types of proteases and express them on the cell surface. The proteases play important roles in PMN-mediated events. In the present study, flow cytometric analysis revealed that CD14 expression on human gingival fibroblasts (HGF) was markedly reduced by PMA-activated PMNs in a coculture system. We found that this reduction was caused by both secreted and cell surface proteases produced by activated PMNs. A protease responsible for the reduction was found to be human leukocyte elastase (HLE) secreted from the activated PMNs by use of various protease inhibitors, although HLE was only partially involved in CD14 reduction caused by cell-bound molecule(s) on fixed PMNs. Analysis with purified HLE revealed a time- and dose-dependent reduction of CD14 on HGF, and complete reduction was observed by 20 μg/ml HLE treatment for 30–60 min, but the other molecules such as CD26, CD59, CD157, and MHC class I on HGF were only slightly reduced. This reduction of CD14 resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD14 on fixed HGF and also on purified cell membranes. As a result of CD14 proteolysis, IL-8 production by HGF was suppressed when triggered by 10 ng/ml LPS, but not by IL-1α, indicating that HLE inhibited a CD14-dependent cell activation. These findings suggested that activated PMNs have a potential negative feedback mechanism for HGF function at the inflammatory site, particularly in periodontal tissues.

A protein mono-ADP-ribosyltransferase (ADPRT), anchored in the cell membrane as a glycosylphosphatidylinositol (GPI)-anchored cell-surface enzyme, was recently described on murine cytotoxic T cells (CTL). Expression of this enzyme was shown to exert regulatory functions on CTL proliferation and cytotoxic activity, presumably by modulating activity of the protein tyrosine kinase p56(lck), which is associated with the CTL co-receptor CD8. Here we report on the molecular cloning and expression of this important regulatory enzyme. The ADPRT coding sequence was derived by making use of ADPRT sequence homologies from different vertebrate species. A cDNA fragment of the enzyme coding sequence was generated by reverse transcription polymerase chain reaction (RT-PCR) from murine T-cell lymphoma SL12, which expresses the cell-surface ADPRT. The cDNA fragment was found to share extensive homology with the corresponding sequences of human and rabbit muscle ADPRT. In Northern blot hybridization, this cDNA fragment generates a strong hybridization signal with RNA from murine heart and skeletal muscle. Weak signals are seen with SL12, thymus, and spleen. Therefore, a murine skeletal muscle cDNA library was used to identify and obtain the coding sequence of the ADPRT gene. It is shown that the nucleic acid open reading frame sequence of the murine skeletal muscle gene shares 80.3% and 76.3% homology with the sequences of the human and rabbit muscle genes, respectively. Semiquantitative RT-PCR with intron-spanning primers shows that the ADPRT mRNA is present in lymphoid organs, cytotoxic T cells, and T-cell lines. Transfection of the ADPRT coding sequence into EL4 cells results in expression of the enzyme as a functional GPI-anchored cell-surface protein, able to ADP-ribosylate the arginine analog agmatine as well as cell-surface molecules.

In this paper, a new analytical method for unsteady-state problems of heat conduction in anisotropic materials is presented. Under the assumption that the form of the heat conduction equation in anisotropic materials is invariant under a congruence matrix transformation, the equation of heat conduction can be transformed to an equation of the same form as that for the isotropic case. For this matrix transformation, the temperature gradient and heat flux must satisfy the continuity coditions at a boundary between two different media. This method is applicable to the treatment of thermal resistance of composite slabs, the determination of the Green function for the three-dimensional equation of heat conduction in an anisotropic conductor, and the analysis of temperature distribution in the composite regions of infinite anisotropic heat-conducting solids.

We reported previously that cementoblasts are provided with sensing mechanisms for extracellular Ca2+ and that elevated extracellular Ca2+ increases fibroblast growth factor-2 (FGF-2) gene and protein expression levels via a cyclic AMP/protein kinase A (PKA) dependent pathway. In the present study, we found that stimulation of murine cementoblasts with 10 mM CaCl2 induced cyclooxygenase-2 (COX-2) gene expression and prostaglandin E2 (PGE2) biosynthesis. NS-398, a COX-2 inhibitor, significantly reduced CaCl2-induced increase in Fgf-2 gene expression, indicating that PGE2 synthesized by COX-2 may be involved in FGF-2 induction. The inhibitory effect of NS-398 was restored completely by the addition of PGE2 receptor 4 (E-prostanoid receptor 4, called EP4) agonist, but not agonists for EP1, EP2, and EP3. Furthermore, EP4 antagonist significantly reduced CaCl2-induced Fgf-2 induction, suggesting that it is mediated by EP4 activation. However, stimulation with EP4 agonist alone in the absence of CaCl2 had no effect on the Fgf-2 induction, indicating that EP4 signaling alone is not sufficient. CaCl2 also upregulated gene expression levels of Ep4 and Cox-2, as well as Fgf-2 and induction of these genes was abolished by pretreatment with BMS-345541, a nuclear factor-κB (NF-κB) inhibitor, indicating that NF-κB signaling triggered by CaCl2 is indispensable for FGF-2 induction. Furthermore, CaCl2-induced Fgf-2 induction was synergistically enhanced by the addition of EP4 agonist. This indicates that the signaling triggered via CaCl2 and its combination with EP4 agonist may be useful as a novel strategy for periodontal regeneration.

We propose a new method of measuring the principal thermal conductivity of a two-dimensional anisotropic heat conductor using a steady heat source and sink. The theory of this new thermal conductivity measurement is based on congruent matrix transformation from nonuniform space of an anisotropic heat conductor to the uniform space of an isotropic one. When we introduce the hypothesis of symmetry in the heat conduction law, it leads to an equivalent relation of the thermal conductivity tensor between the anisotropic tensor Λμ and the uniform space tensor Λν. This new measuring method can provide the two principal thermal conductivities λ1, 2 and the principal rotation angle φ from the temperature detecting axis. We have measured the temperature dependence of two principal thermal conductivities λ1, 2 and the principal rotation angle φ of Y1Ba2Cu3O7-y. Anomalies of the two principal thermal conductivities and the principal rotation angle are clearly observed at the superconductive transition temperature Tc.

The cytokine interleukin-6 (IL-6) is released from working skeletal muscles and reportedly plays key roles in their glucose homeostasis. However, it is unclear whether IL-6 plays such roles in the masseter muscle (MM), which is important in normal and pathological chewing behaviors, such as bruxism and/or prolonged clenching. When restrained (R+) in a narrow cylinder blocked at the front end with a thin plastic strip, a mouse gnaws away (G+) the strip to escape. The absolute weight of plastic gnawed away serves as an index of MM activity. Using this model, we examined the roles of IL-6 in MM with the following results. R+G+ increased the expression levels of IL-6 and glucose transporter 4 (Glut4) mRNAs in MM and the serum level of IL-6 protein. IL-6-deficient mice exhibited about 60% less gnawing activity than wild-type mice at 3-4 h after the start of R+G+, slower recovery of glycogen levels (indicating poorer glucose supply) in MM after R+G+, and no significant change in Glut4 mRNA in MM upon R+G+. During an R+G+ test conducted after "training" (repeated R+G+ sessions), wild-type mice exhibited greater gnawing activity than untrained controls, but no increase in IL-6 mRNA in MM. IL-6 mRNA increased in MM when hard food was eaten by mice raised on soft food for 3 weeks from weaning, but not in those raised on (accustomed to) hard food. Thus, IL-6 may maintain glucose homeostasis in MM in support of unusually strenuous activity, but not of accustomed activity levels.

Extracellular phosphate (Pi) has been shown to act as a signaling molecule capable of altering gene expression and cellular function during osteoblastic differentiation. In this study, we investigated whether Pi regulates expression of bone morphogenetic protein (BMP)-2, a crucial regulator of osteogenic differentiation, and explored the signaling pathway involved using human dental pulp cells and human periodontal ligament cells. We found that Pi increased BMP-2 gene expression. Experiments using signaling inhibitors revealed that the increase in BMP-2 expression was dependent on the cAMP/protein kinase A signaling pathway. We also showed that Pi activates the ERK1/2, and treatment with PD98059, an ERK1/2 inhibitor, suppressed Pi-mediated BMP-2 increase, indicating that activation of the ERK1/2 pathway is required for this Pi-mediated response. These findings demonstrate a novel ability of Pi to stimulate BMP-2 via cAMP/protein kinase A and ERK1/2 pathways.

A resonance method was previously proposed to measure the frequency change of piezoelectric quartz crystals [S. Kurosawa et al., Anal. Chim. Acta, 274 (1993) 209]. The ordinary method which incorporates the crystal into an oscillator circuit is here referred to as the oscillation method. Viscosity changes during the depolymerization of actomyosin can be detected by a piezoelectric quartz crystal because its frequency change is sensitive to the viscosity in a solution. This change is not detectable by the oscillation method (using TTL circuit), but may be detected by the resonance method using a ‘spectrum analyzer’, which shortens the analysis time, compared with the technique described earlier.

Frequency changes of a piezoelectric quartz crystal were observed with the resonance method (S. Kurosawa et al., Anal. Chim. Acta, 274 (1993) 209–217), when actomyosin from fresh carp was depolymerized by addition of ATP (adenosine 5′-triphosphate). On the other hand, no frequency changes were observed when long-stored actomyosin was used, suggesting that the present system may be applicable for a meat-freshness sensor. We failed to observe the frequency changes with the ordinary method (oscillation method) which incorporates the crystal into an oscillator circuit.

The paper deals with the characteristics of boiling heat transfer phenomena on the metal surfaces where gravitational acceleration between 0g and 1g acts. To conduct the experiment in the field where the gravitational acceleration between 1g and 0g acted accurately, we produced the Atwood machine that was able to obtain the fixed gravitational acceleration field known by physics well. The metallic materials used by the experiment were brass, stainless steel, aluminum, copper and these materials were processed to 10mm in the diameter, and we put these samples in liquid nitrogen and experimented on the boiling phenomenon. As a result, it has been understood that there is the feature shown next in boiling heat transfer phenomena on the metal surface in gravitational acceleration field between 0g and 1g. (1) When brass, copper, stainless steel, and aluminum of the sample were put in the liquid nitrogen, the temperature differentiation coefficient on the sample surface showed the tendency to decrease in proportion to gravitational acceleration changed from 1g into 0g. (2) In boiling heat flux curve in these metals (brass, stainless steel, aluminum and copper), it was clarified for gravitational acceleration 1g to indicate maximum heat flux value qmax .Copyright © 2011 by ASME

The resonant frequency of a piezoelectric crystal was determined by transmission measurements. The simple principle of this resonance method is that a transmission is measured at varying frequencies with a two-port transmission line system containing parallel resistance. Previous workers formulated the resonant frequency of a quartz crystal and the equation derived indicated that the frequency is dependent on (ρη) 1 2 and F 3 2 b, where ρ, η and Fb are the density and viscosity of the solution and the fundamental frequency of the quartz crystal, respectively. The resonance method was applied to solutions of varying (ρη) 1 2 , and it is concluded that the equation holds for a wide range of different solutions. The resonance method is advantageous in solutions over the widely used conventional oscillation method because the range of (ρη) 1 2 for possible measurements is much wider.

When a piezoelectric quartz crystal is dipped into a solution, viscosity ambient to the crystal greatly affects resonant frequency. Resonant frequency was determined by a spectrum analyzer for various solutions whose viscosity was altered by the addition of sucrose. A proportional relationship between change in frequency at series resonant point ΔFs and √ρη up to 25.45 [g1/2·cm-3/2·cp1/2] was demonstrated. Beyond this value, some deviation from linearity was observed. ρ is the density of a solution and η, viscosity. In the present method in contrast to the previous resonance method [Kurosawa et al., Anal. Chim. Acta, 274, (1993)] the linear relation of ΔFs with √ρη holds over a wider range, thus reducing the time required for analysis and assuring greates accuracy of results.

Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. Exposure to Wnt3a inhibited the expression of the osteocalcin (OCN) gene. This effect was accompanied by decreased gene expression of Runx2. Pretreatment with Dickkopf-1 attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of Runx2. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.

We investigated the mechanism and impact of various inflammatory stimuli on dendritic cell (DC)-gingival fibroblast (GF) adhesion. Human immature (im) DCs were generated from monocytes by culturing with interleukin (IL)-4 and granulocyte macrophage-colony stimulating factor (GM-CSF). GFs were outgrown from the human gingival specimen. DCs were co-cultured with GFs with/without pretreatment with various stimuli. Adhered cells were measured by fluorometer. Expression of adhesion molecules was analyzed by flow cytometry. Pretreatment of GFs with tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and Escherichia coli (Ec) lipopolysaccharides (LPS) significantly increased the adhesion to imDCs and enhanced intercellular adhesion molecule (ICAM)-1 expression. A signifi-cantly increased DC-GF adhesion was also observed when imDCs were pretreated with Ec LPS, Porphyromonas gingivalis (Pg) fimbriae, and peptideglycan but not with Pg LPS. Expression of LFA-1 and Mac-1 on DCs was not altered by the pretreatment with these stimuli. However, LFA-1 and Mac-1 blockade of imDC signifi cantly reduced the adhesion to TNF-α-stimulated GFs. These results showed that inflammatory stimuli increased the imDC-GF adhesion via lymphocyte function- associated antigen (LFA)-1/Mac-1-ICAM-1 ligation. Adhesion of DC to GFs may be important not only for the localization of DCs in the inflammatory periodontal lesion, but also for the modulation of immune responses.

Although cementoblasts express Toll-like receptors (TLR)-2 and -4, little is known regarding the possible participation of cementoblasts in the inflammatory response. We investigated the effects of Porphyromonas gingivalis lipopolysaccharide (LPS), tetra- and penta-acylated lipid A species (designated PgLPS1435/1449 and PgLPS1690, respectively), on gene expression of osteoclastogenesis-associated molecules in murine cementoblasts. Real-time quantitative RT-PCR analysis revealed that receptor activator of NF-κB ligand (RANKL), interleukin-6, Regulated on activation, normal T-cell expressed, and secreted (RANTES), macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 were rapidly and dramatically induced upon stimulation with PgLPS1690, but only slightly induced with PgLPS1435/1449. Osteoprotegerin, which was expressed constitutively, was not altered significantly. ELISA demonstrated synthesis of corresponding proteins. PgLPS1690 significantly induced transcripts for NF-κB, and this activation was inhibited by pre-treatment with anti-TLR-2 but not with TLR-4 antibodies. These results suggest that cementoblasts participate in the recruitment of osteoclastic precursor cells by up-regulation of chemokines/cytokines.

BACKGROUND AND OBJECTIVES Retinoic acid (RA) exerts a wide variety of effects on development, cellular differentiation and homeostasis in various tissues. However, little is known about the effects of RA on the differentiation of periodontal ligament cells. In this study, we investigated whether RA can affect the dexamethasone-induced differentiation of periodontal ligament cells. METHODS AND RESULTS Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate for mineralized nodule formation, as characterized by von Kossa staining. Continuous treatment with all-trans-RA inhibited the mineralization in a dose-dependent manner, with complete inhibition over 1 microm RA. Other RA analogs, 9-cis-RA and 13-cis-RA, were also effective. Furthermore, addition of RA for just the first 4 days completely inhibited the mineralization; however, as RA was added at later stages of culture, the inhibitory effect was diminished, suggesting that RA had a phase-dependent inhibition of mineralization. RA receptor (RAR)-alpha agonist (AM-580), but not retinoid X receptor agonist (methoprene acid), inhibited the mineralization, and reverse transcription-polymerase chain reaction analysis revealed that RAR-alpha was expressed on the cells, suggesting that RAR-alpha was involved in the inhibitory mechanism. This inhibition was accompanied by inhibition of alkaline phosphatase activity; however, neither expression of platelet-derived growth factor (PDGF) receptor-alpha, PDGF receptor-beta, or epidermal growth factor (EGF) receptor, nor phosphorylation of extracellular signal-regulated kinases triggered by PDGF-ascorbic acid or PDGF-BB was changed, as assessed by flow cytometry or western blot analyses. CONCLUSIONS These findings suggest that RA is a potential negative regulator for differentiation of human periodontal ligament cells.

Cysteine proteinases (gingipains) from Porphyromonas gingivalis are considered key virulence factors of severe periodontitis and host immune evasion. Since expression of intercellular adhesion molecule-1 (ICAM-1) on gingival epithelium is indispensable in polymorphonuclear leukocyte (PMN) migration at the site of periodontitis, we examined the effects of gingipains on the expression of ICAM-1 on human oral epithelial cell lines (KB and HSC-2) by flow cytometry and Western blotting. We found that three purified forms of gingipains efficiently reduced ICAM-1 expression on the cells in a time- and dose-dependent manner. Gingipains reduced the expression on fixed cells and degraded the ICAM-1 in the cell membranes, indicating that the reduction resulted from direct proteolysis. They then disturbed the ICAM-1-dependent adhesion of PMNs to the cells. These results indicate that gingipains cleave ICAM-1 on oral epithelial cells, consequently disrupting PMN-oral epithelial cell interaction, and are involved in immune evasion by the bacterium in periodontal tissues.

Cysteine proteinases (gingipains) elaborated from Porphyromonas gingivalis exhibit enzymatic activities against a broad range of host proteins and are considered key virulence factors in the onset and development of adult periodontitis and host defense evasion. In this study, we examined the ability of arginine-specific gingipains (high molecular mass Arg-specific gingipain (HRGP) and Arg-specific gingipain 2) and lysine-specific gingipain (KGP) to cleave monocyte CD14, the main receptor for bacterial cell surface components such as LPS. Binding of anti-CD14 mAb MY4 to human monocytes was almost completely abolished by 0.3 μM HRGP and KGP treatments for 15 min, and 1 μM RGP2 for 30 min. In contrast, the expressions of Toll-like receptor 4, and CD18, CD54, CD59, and HLA-A, -B, -C on monocytes were slightly increased and decreased, respectively, by 0.3 μM HRGP and KGP. This down-regulation resulted from direct proteolysis, because 1) gingipains eliminated MY4 binding even to fixed monocytes, and 2) CD14 fragments were detected in the extracellular medium by immunoblot analysis. Human rCD14 was degraded by all three gingipains, which confirmed that CD14 was a substrate for gingipains. TNF-α production by monocytes after HRGP and KGP treatments was decreased at 1 ng/ml, but not at 20 μg/ml LPS, indicating that gingipains inhibited a CD14-dependent cell activation. These results suggest that gingipains preferentially cleave monocyte CD14, resulting in attenuation of the cellular recognition of bacteria, and as a consequence sustain chronic inflammation.

Protein mono-(ADP-ribosyl)transferases (ADPRTs) catalyze transfer of the ADP-ribose moiety from nicotinamide adenine dinucleotide (NAD) to specific amino acids. We recently described presence of an enzyme with this activity on cytotoxic T cells (CTL). Incubation of CTL with micromolar concentrations of NAD causes inhibition of cell proliferation and cytolytic activity. ADPRT can be released by bacterial phosphoinositol specific phospholipase C, indicating that it is a glycosylphosphatidylinositol (GPI) anchored exo-enzyme. Enzymatic release of ADPRT results in inability of NAD to modulate CTL function. Expression of ADPRT was found to be regulated, in quiescent CTL ADPRT is expressed at significant levels, however, upon TCR crosslinking it is rapidly released by an anchor hydrolyzing mechanism. This results in relative insensitivity to the inhibitory action of NAD. The question how ADPRT regulates T cell functions was investigated by incubating CTL with radioactively labeled NAD which causes modification of several proteins, pointing to potential candidates in these regulatory processes. We found that the protein tyrosine kinase p56lck but not p59tyn exists in a digitonin resistant complex with a 40 kD protein, which in its ADP-ribosylated form suppresses p56lck kinase activity. ADP-ribosylation of this protein is mediated by the arginine specific protein mono-ADPRT, presumably utilizing ecto-NAD as substrate. Release of the ADPRT by GPI-specific phospholipase C results in failure of ecto-NAD to downmodulate p56lck kinase activity. Concomitant to suppression of the kinase by ecto-NAD, CD8 mediated transmembrane signaling is found to be inhibited, whereas transmembrane signaling via CD3 is only slightly affected.

CD13/aminopeptidase N (APN) is a multifunctional ectoenzyme that is involved in anti-inflammatory reactions, control of immune reactions and differentiation of many cellular systems. In this study, we first demonstrated that CD13/APN was expressed on human gingival fibroblast (hGF). T helper (Th) 2 cell type cytokines such as IL-4 and IL-13, but not IL-2 or IL-15, preferentially increased the expression of CD13/APN, resulting in the inhibition of FMLP-mediated neutrophil activation through the degradation by CD13/APN on HGF. Our data suggested that CD13/APN expressed by hGF could contribute to the anti-inflammatory response in periodontal tissue, and may be involved in disease processes mediated by Th2 cells.

A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28,000 and pI 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H/HeN spleen cells began at 1 ng ml-1 and reached a maximal response at 100 ng ml-1 of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class 1 mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing V beta 13 T-cell receptor was observed up to 73% among the Thy 1.2+ cells in cultures stimulated with SPM, indicating expansion in a V beta-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of pI 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand V beta 8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and V beta 21+ T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.

MRL/lpr mice, which are a model of SLE and rheumatoid arthritis in humans, develop profound lymphadenopathy resulting from the accumulation of CD3 + 4 − 8 − double-negative (DN) αβT cells in peripheral lymphoid tissues. We previously indicated that these DN αβT cells preferentially proliferate in the liver and migrate to the periphery. In this study, we analyzed whether any kind of cytokine was produced by hepatic mononuclear cells (MNC) in MRL/lpr mice. The evidence obtained indicates that interleukin 6 (IL-6) was vigorously produced by hepatic MNC in diseased MRL/lpr mice under unstimulated conditions. MNC in the spleen of these mice produced small amounts of IL-6, while those in the lymph nodes did not produce any appreciable amounts of IL-6. These activities of hepatic MNC in diseased MRL/lpr mice were almost completely neutralized by anti-mouse IL-6 monoclonal antibody (mAb). On the other hand, immunohistochemical staining of light- and electron-microscopic analyses revealed that the intracellular cell adhesion molecule 1 (ICAM-1) was expressed on the hepatic sinusoidal endothelial cells of diseased MRL/lpr mice. Moreover, ICAM-1 was newly induced in the hepatic sinusoids of control C3H/He mice by an intravenous injection of 50 units of recombinant mouse IL-6. These data suggest that ICAM-1 expressed on the hepatic sinusoidal endothelial cells in MRL/lpr mice is induced by IL-6, which is produced by hepatic MNC, and that such ICAM-1 may be responsible for the saturation of inflammatory cells and the proliferation of lymphocytes in the liver of MRL/lpr mice.

We measured the principal thermal conductivities and the principal axis angle of a 0.1-mm-thick pyrolytic graphite sheet (PGS), by the integrated multi-temperature probe method. To determine its principal thermal conductivities, a sample is heated using a point heat source places on the sample surface and the transient temperature changes of nine points with radii of r1 = 2.5, r2 = 5.0, and r3 = 7.5 mm on three axes with β= 120° are measured by the integrated multi-temperature probe method. The two measured principal thermal conductivities are λp1 = 746 W/(mK) and λp2 = 925 W/(mK). The geometrical mean of the two measured principal thermal conductivities of PGS is λ= (λp1λp2)1/2 = 831 W/(mK). The relative errors in this measurement are 7.1% for λp1 and 8.7% for λp2. The calculated anisotropic factor ξ, which is the ratio of λp2 to λp1, is 1.24. The measured data of PGS showed a strong anisotropy of thermal conductivity. This suggests that there is a possibility for PGS to become a standard two-dimensional anisotropic material.

Background The production of chemokines by tissue resident cells during inflammation is considered one of the main mechanisms involved in the formation of inflammatory infiltrates. Fibroblasts are the main resident cell type in gingival and periodontal ligament tissues, and their ability to produce chemokine stromal cell-derived factor 1 alpha (SDF-1α) and its receptor CXCR4 under stimulation by gram negative bacteria, Porphyromonas gingivalis, commonly found in periodontal infections was investigated. Methods Western blots were used to assess SDF-1α and CXCR4 protein expression levels in human gingival fibroblast cells (HGF-1) induced by Lipopolysaccharide (LPS) from P. gingivalis in the presence or absence of LY294002, a highly selective inhibitor of PI-3K/Akt. RT-PCR and quantitative Real-time PCR was performed using gingival mRNAs from periodontitis patients. Immunohistochemistry was performed to analyze the expression and subcellular localization of SDF-1α and CXCR4, together with NF-kβ phosphorylation, in specimens from patients with periodontitis and in an experimental rat periodontitis model. Results We found that P. gingivalis LPS up-regulated SDF-1α and CXCR4 protein levels and elevated phosphorylation of the SDF-1α-responsive NF-kβ and Akt at 24 h in HGF-1 cells. SDF-1α and CXCR4 mRNA and protein expression levels were high in all patients with periodontitis. In the P. gingivalis-induced rat experimental periodontitis model, SDF-1α and CXCR4 immunoreactivity was higher in gingival and periodontal ligament tissues compared to the control. Conclusion Our data showed that PI-3K/Akt is an upstream participant in the P. gingivalis LPS-mediated induction of SDF-1α. Taken together, these results suggest that the chemokine SDF-1α and its receptor CXCR4 contribute to P. gingivalis-induced periodontal inflammation.