Language：English   Publishing type：Research paper (scientific journal)   Publisher：Scientific Reports  

Alveolar bone loss caused by periodontal disease eventually leads to tooth loss. Periodontal ligament stem cells (PDLSCs) are the tissue-specific cells for maintaining and repairing the periodontal ligament, cementum, and alveolar bone. Here, we investigated the role of erythropoietin receptor (EPOR), which regulates the microenvironment-modulating function of mesenchymal stem cells, in PDLSC-based periodontal therapy. We isolated PDLSCs from patients with chronic periodontal disease and healthy donors, referred to as PD-PDLSCs and Cont-PDLSCs, respectively. PD-PDLSCs exhibited reduced potency of periodontal tissue regeneration and lower expression of EPOR compared to Cont-PDLSCs. EPOR-silencing suppressed the potency of Cont-PDLSCs mimicking PD-PDLSCs, whereas EPO-mediated EPOR activation rejuvenated the reduced potency of PD-PDLSCs. Furthermore, we locally transplanted EPOR-silenced and EPOR-activated PDLSCs into the gingiva around the teeth of ligament-induced periodontitis model mice and demonstrated that EPOR in PDLSCs participated in the regeneration of the periodontal ligament, cementum, and alveolar bone in the ligated teeth. The EPOR-mediated paracrine function of PDLSCs maintains periodontal immune suppression and bone metabolic balance via osteoclasts and osteoblasts in the periodontitis model mice. Taken together, these results suggest that EPOR signaling is crucial for PDLSC-based periodontal regeneration and paves the way for the development of novel options for periodontal therapy.

DOI： 10.1038/s41598-024-57361-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemistry and Biophysics Reports  

Introduction & objectives: Stem cell therapy for regenerative medicine has been sincerely investigated, but not still popular although some clinical trials show hopeful results. This therapy is suggested to be a representative candidate such as bone defect due to the accident, iatrogenic resection oncological tumor, congenital disease, and severe periodontitis in oral region. Recently, the Bio-3D printer “Regenova®” has been introduced as an innovative three-dimensional culture system, equipped scaffold-free bio-assembling techniques without any biomaterials. Therefore, we expected a mount of bone defect could be repaired by the structure established from this Bio-3D printer using osteogenic potential stem cells. Material & methods: The gingival tissue (1x1 mm) was removed from the distal part of the lower wisdom tooth of the patients who agreed our study. Human Gingival Mesenchymal Stem Cells (hGMSCs) were isolated from this tissue and cultured, since we confirmed the characteristics such as facile isolation and accelerated proliferation, further, strong potential of osteogenic-differentiation. Spheroids were formed using hGMSC in 96-well plates designed for low cell adhesion. The size of the spheroids was measured, and fluorescent immunostaining was employed to verify the expression of stem cell and apoptosis marker, and extracellular matrix. Following four weeks of bone differentiation, μCT imaging was performed. Calcification was confirmed by alizarin red and von Kossa staining. Fluorescent immunostaining was utilized to assess the expression of markers indicative of advanced bone differentiation. Results: We have established and confirmed the spheroids (∼600 μm in diameter) constructed from human GMSCs (hGMSCs) still maintain stem cell potentials and osteogenic differentiation abilities from the results that CD73 and not CD34 were expressed as stem cell positive and negative marker, respectively. These spheroids were pilled up like cylindal shape to the “Kenzan” platform of Bio-3D printer and cultured for 7days. The cylindal structure originated from compound spheroids were tried to differentiate into bone four weeks with osteogenic induction medium. The calcification of bio-3D printed bone-like structures was confirmed by alizarin red and Von Kossa staining. In addition, μCT analysis revealed that the HU (Hounsfield Unit) of the calcified structures was almost identical to that of trabecular bone. Immunofluorescent staining detected osteocalcin expression, a late-stage bone differentiation marker. Conclusion: For the first time, we have achieved the construction of a scaffold-free, bone-like luminal structure through the assembly of spheroids comprised of this hGMSCs. This success is sure to be close to the induction of clinical application against regenerative medicine especially for bone defect disease.

DOI： 10.1016/j.bbrep.2024.101656

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Language：English   Publisher：Hindawi  

Aim. The present study evaluated the therapeutic effects of luteolin in alleviating pulpitis of dental pulp- (DP-) derived microvesicles (MVs) via the inhibition of protein kinase R- (PKR-) mediated inflammation. Methodology. Proteomic analysis of immortalized human dental pulp (DP-1) cell-derived MVs was performed to identify PKR-associated molecules. The effect of luteolin on PKR phosphorylation in DP-1 cells and the expression of tumor necrosis factor-α (TNF-α) in THP-1 macrophage-like cells were validated. The effect of luteolin on cell proliferation was compared with that of chemical PKR inhibitors (C16 and 2-AP) and the unique commercially available sedative guaiacol-parachlorophenol. In the dog experimental pulpitis model, the pulps were treated with (1) saline, (2) guaiacol-parachlorophenol, and (3) luteolin. Sixteen teeth from four dogs were extracted, and the pulp tissues were analyzed using hematoxylin and eosin staining. Immunohistochemical staining was performed to analyze the expression of phosphorylated PKR (pPKR), myeloperoxidase (MPO), and CD68. Experimental endodontic-periodontal complex lesions were established in mouse molar through a silk ligature and simultaneous MV injection. MVs were prepared from DP-1 cells with or without pretreatment with 2-AP or luteolin. A three-dimensional microcomputed tomography analysis was performed on day 7 (n=6). Periodontal bone resorption volumes were calculated for each group (nonligated–ligated), and the ratio of bone volume to tissue volume was measured. Results. Proteomic analysis identified an endogenous PKR activator, and a protein activator of interferon-induced PKR, also known as PACT, was included in MVs. Luteolin inhibited the expressions of pPKR in DP-1 cells and TNF-α in THP-1 cells with the lowest suppression of cell proliferation. In the dog model of experimental pulpitis, luteolin treatment suppressed the expression of pPKR-, MPO-, and CD68-positive cells in pulp tissues, whereas guaiacol-parachlorophenol treatment caused coagulative necrosis and disruption. In a mouse model of endodontic-periodontal complex lesions, luteolin treatment significantly decreased MV-induced alveolar bone resorption. Conclusion. Luteolin is an effective and safe compound that inhibits PKR activation in DP-derived MVs, enabling pulp preservation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioMed Research International  

AIM: The present study evaluated the therapeutic effects of luteolin in alleviating pulpitis of dental pulp- (DP-) derived microvesicles (MVs) via the inhibition of protein kinase R- (PKR-) mediated inflammation. Methodology. Proteomic analysis of immortalized human dental pulp (DP-1) cell-derived MVs was performed to identify PKR-associated molecules. The effect of luteolin on PKR phosphorylation in DP-1 cells and the expression of tumor necrosis factor-α (TNF-α) in THP-1 macrophage-like cells were validated. The effect of luteolin on cell proliferation was compared with that of chemical PKR inhibitors (C16 and 2-AP) and the unique commercially available sedative guaiacol-parachlorophenol. In the dog experimental pulpitis model, the pulps were treated with (1) saline, (2) guaiacol-parachlorophenol, and (3) luteolin. Sixteen teeth from four dogs were extracted, and the pulp tissues were analyzed using hematoxylin and eosin staining. Immunohistochemical staining was performed to analyze the expression of phosphorylated PKR (pPKR), myeloperoxidase (MPO), and CD68. Experimental endodontic-periodontal complex lesions were established in mouse molar through a silk ligature and simultaneous MV injection. MVs were prepared from DP-1 cells with or without pretreatment with 2-AP or luteolin. A three-dimensional microcomputed tomography analysis was performed on day 7 (n = 6). Periodontal bone resorption volumes were calculated for each group (nonligated-ligated), and the ratio of bone volume to tissue volume was measured. RESULTS: Proteomic analysis identified an endogenous PKR activator, and a protein activator of interferon-induced PKR, also known as PACT, was included in MVs. Luteolin inhibited the expressions of pPKR in DP-1 cells and TNF-α in THP-1 cells with the lowest suppression of cell proliferation. In the dog model of experimental pulpitis, luteolin treatment suppressed the expression of pPKR-, MPO-, and CD68-positive cells in pulp tissues, whereas guaiacol-parachlorophenol treatment caused coagulative necrosis and disruption. In a mouse model of endodontic-periodontal complex lesions, luteolin treatment significantly decreased MV-induced alveolar bone resorption. CONCLUSION: Luteolin is an effective and safe compound that inhibits PKR activation in DP-derived MVs, enabling pulp preservation.

DOI： 10.1155/2024/8864513

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Publisher：Frontiers Media SA  

Drug-induced gingival overgrowth (DIGO), induced by certain immunosuppressive drugs, antihypertensive agents, and antiepileptic drugs, may contribute to the formation of deeper periodontal pockets and intractableness in periodontitis. To date, multiple factors such as enhanced matrix production, inflammation, and reduced matrix degradation might be involved in the pathogenesis of DIGO. We have previously reported that SPOCK-1, a heparan sulfate proteoglycan, could affect gingival thickening by promoting epithelial-to-mesenchymal transition (EMT) in gingival keratinocytes. However, few studies have investigated whether a combination of these factors enhances the DIGO phenotype in animal models. Therefore, we investigated whether SPOCK-1, periodontal inflammation, and cyclosporin-A (CsA) could cooperatively promote gingival overgrowth. We first confirmed that Spock-1 overexpressing (Spock1-Tg) mice showed significantly thicker gingiva and greater alveolar bone loss than WT mice in response to ligature-induced experimental periodontitis. DIGO was induced by the combination of CsA administration and experimental periodontitis was significantly enhanced in Spock1-Tg mice compared to that in WT mice. Ligature-induced alveolar bone loss in CsA-treated Spock1-Tg mice was also significantly greater than that in CsA-treated WT mice, while being accompanied by an increase in Rankl and Col1a1 levels and a reduction in matrix metalloprotease expression. Lastly, SPOCK-1 promoted RANKL-induced osteoclast differentiation in both human peripheral blood mononuclear cells and murine macrophages, while peritoneal macrophages from Spock1-Tg mice showed less TNFα and IL-1β secretion than WT mice in response to Escherichia coli lipopolysaccharide. These results suggest that EMT, periodontal inflammation, and subsequent enhanced collagen production and reduced proteinase production contribute to CsA-induced DIGO pathogenesis.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers in Physiology  

Drug-induced gingival overgrowth (DIGO), induced by certain immunosuppressive drugs, antihypertensive agents, and antiepileptic drugs, may contribute to the formation of deeper periodontal pockets and intractableness in periodontitis. To date, multiple factors such as enhanced matrix production, inflammation, and reduced matrix degradation might be involved in the pathogenesis of DIGO. We have previously reported that SPOCK-1, a heparan sulfate proteoglycan, could affect gingival thickening by promoting epithelial-to-mesenchymal transition (EMT) in gingival keratinocytes. However, few studies have investigated whether a combination of these factors enhances the DIGO phenotype in animal models. Therefore, we investigated whether SPOCK-1, periodontal inflammation, and cyclosporin-A (CsA) could cooperatively promote gingival overgrowth. We first confirmed that Spock-1 overexpressing (Spock1-Tg) mice showed significantly thicker gingiva and greater alveolar bone loss than WT mice in response to ligature-induced experimental periodontitis. DIGO was induced by the combination of CsA administration and experimental periodontitis was significantly enhanced in Spock1-Tg mice compared to that in WT mice. Ligature-induced alveolar bone loss in CsA-treated Spock1-Tg mice was also significantly greater than that in CsA-treated WT mice, while being accompanied by an increase in Rankl and Col1a1 levels and a reduction in matrix metalloprotease expression. Lastly, SPOCK-1 promoted RANKL-induced osteoclast differentiation in both human peripheral blood mononuclear cells and murine macrophages, while peritoneal macrophages from Spock1-Tg mice showed less TNFα and IL-1β secretion than WT mice in response to Escherichia coli lipopolysaccharide. These results suggest that EMT, periodontal inflammation, and subsequent enhanced collagen production and reduced proteinase production contribute to CsA-induced DIGO pathogenesis.

DOI： 10.3389/fphys.2023.1298813

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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DOI： 10.1177/00220345231181539

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DOI： 10.1177/00220345231181539

Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Periodontal disease is an infectious disease that affects many people worldwide. Disease progression destroys the alveolar bone and causes tooth loss. We have previously shown that alymphoplasia (aly/aly) mice harboring a loss-of-function mutation in the map3k14 gene, which is involved in p100 to p52 processing of the alternative NF-κB pathway, exhibited mild osteopetrosis due to decreased number of osteoclasts, suggesting the alternative NF-κB pathway as a potential drug target for the amelioration of bone disease. In the present study, wild-type (WT) and aly/aly mice were subjected to silk ligation to establish a periodontitis model. Alveolar bone resorption was suppressed in aly/aly mice by decreased numbers of osteoclasts in the alveolar bone in comparison to WT mice. Furthermore, the expression of receptor activator of NF-κB ligand (RANKL) and TNFα (cytokines involved in osteoclast induction in periligative gingival tissue) was decreased. When primary osteoblasts (POBs) and bone marrow cells (BMCs) derived from WT and aly/aly mice were prepared and co-cultured, osteoclasts were induced from WT-derived BMCs, regardless of the origin of the POBs, but hardly formed from aly/aly mouse-derived BMCs. Furthermore, the local administration of an NIK inhibitor, Cpd33, inhibited osteoclast formation and thereby inhibited alveolar bone resorption in the periodontitis model. Therefore, the NIK-mediated NF-κB alternative pathway can be a therapeutic target for periodontal disease.

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Language：English   Publisher：Frontiers in Immunology  

Periodontal disease is an infectious disease that affects many people worldwide. Disease progression destroys the alveolar bone and causes tooth loss. We have previously shown that alymphoplasia (aly/aly) mice harboring a loss-of-function mutation in the map3k14 gene, which is involved in p100 to p52 processing of the alternative NF-κB pathway, exhibited mild osteopetrosis due to decreased number of osteoclasts, suggesting the alternative NF-κB pathway as a potential drug target for the amelioration of bone disease. In the present study, wild-type (WT) and aly/aly mice were subjected to silk ligation to establish a periodontitis model. Alveolar bone resorption was suppressed in aly/aly mice by decreased numbers of osteoclasts in the alveolar bone in comparison to WT mice. Furthermore, the expression of receptor activator of NF-κB ligand (RANKL) and TNFα (cytokines involved in osteoclast induction in periligative gingival tissue) was decreased. When primary osteoblasts (POBs) and bone marrow cells (BMCs) derived from WT and aly/aly mice were prepared and co-cultured, osteoclasts were induced from WT-derived BMCs, regardless of the origin of the POBs, but hardly formed from aly/aly mouse-derived BMCs. Furthermore, the local administration of an NIK inhibitor, Cpd33, inhibited osteoclast formation and thereby inhibited alveolar bone resorption in the periodontitis model. Therefore, the NIK-mediated NF-κB alternative pathway can be a therapeutic target for periodontal disease.

DOI： 10.3389/fimmu.2023.1179007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Diabetes  

Insulin resistance and hyperglycemia are risk factors for periodontitis and poor wound healing in diabetes, which have been associated with selective loss of insulin- activation of the PI3K/Akt pathway in the gingiva. This study showed that insulin resistance in the mouse gingiva due to selective deletion of smooth muscle and fibroblast insulin receptor (SMIRKO mice) or systemic metabolic changes induced by high fat diet (HFD) in HFD-fed mice exacerbated periodontitis-induced alveolar bone loss, preceded by delayed neutrophil and monocyte recruitment and impaired bacterial clearance compared to their respective controls. The immunocytokines, CXCL1, CXCL2, MCP-1, TNFα, IL-1β and IL-17A exhibited delayed maximal expression in the gingiva of male SMIRKO and HFD-fed mice compared to controls. Targeted overexpression of CXCL1 in the gingiva by adenovirus normalized neutrophil and monocyte recruitment and prevented bone loss in both mouse models of insulin resistance. Mechanistically, insulin enhanced bacterial lipopolysaccharide-induced CXCL1 production in mouse and human gingival fibroblasts (GFs), via Akt pathway and NF-κB activation, which were reduced in GFs from SMIRKO and HFD-fed mice. These results provided the first report that insulin signaling can enhance endotoxin induced CXCL1 expression to modulate neutrophil recruitment, suggesting CXCL1 as a new therapeutic direction for periodontitis or wound healing in diabetes.

DOI： 10.2337/db22-1014

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：The Japanese Academy of Clinical Periodonorogy  

DOI： 10.57303/tjacp.40.2_43

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Archives of Biochemistry and Biophysics  

A well-tuned inflammatory response is crucial for an effective immune process. Nuclear factor-kappa B (NF-κB) is a key mediator of inflammatory and innate immunity responses, and its dysregulation is closely associated with immune-related diseases. MicroRNAs (miRNAs) are important inflammation modulators. However, miRNA-regulated mechanisms that implicate NF-κB activity are not fully understood. This study aimed to identify a potential miRNA that could modulate the dysregulated NF-κB signaling during inflammation. We identified miR-582-5p that was significantly downregulated in inflamed murine adipose tissues and RAW264.7 cells. S-phase kinase-associated protein 1 (SKP1), a core component of an E3 ubiquitin ligase that regulates the NF-κB pathway, was proposed as a biological target of miR-582-5p by using TargetScan. The binding of miR-582-5p to a 3'-untranslated region site on Skp1 was confirmed using a dual-luciferase reporter assay; in addition, transfection with a miR-582-5p mimic suppressed SKP1 expression in RAW264.7 cells. Importantly, exogenous miR-582-5p attenuated the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 through suppressing the degradation of the NF-κB inhibitor alpha, followed by the nuclear translocation of NF-κB. Therefore, exogenously applied miR-582-5p can attenuate the NF-κB signaling pathway via targeting Skp1; this provides a prospective therapeutic strategy for treating inflammatory and immune diseases.

DOI： 10.1016/j.abb.2022.109501

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DOI： 10.3389/icell.2022.1061216

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The expression profiles of exosomal microRNAs (miRNAs) are regulated by the microenvironment, and appropriate priming with mesenchymal stem cells (MSCs) is one of the strategies to enhance the paracrine potency of MSCs. Our previous work demonstrated that exosomes from tumor necrosis factor (TNF)-α-primed human gingiva-derived MSCs (GMSCs) could be a therapeutic tool against periodontitis, and that TNFα-inducible exosomal miR-1260b is essential for the inhibition of alveolar bone loss. However, the precise molecular mechanism underlying miR-1260b-mediated inhibition of osteoclastogenesis is not yet fully understood. Here, we found that the activating transcription factor (ATF)-6β, a novel miR-1260b-targeting gene, is critical for the regulation of osteoclastogenesis under endoplasmic reticulum (ER) stress. An experimental periodontal mouse model demonstrated that induction of ER stress was accompanied by enhanced ATF6β expression, and local administration of miR-1260b and ATF6β siRNA using polyethylenimine nanoparticles (PEI-NPs) significantly suppressed the periodontal bone resorption. In periodontal ligament (PDL) cells, the ER stress inducer, tunicamycin, enhanced the expression of the receptor activator of NF-κB ligand (RANKL), while miR-1260b-mediated downregulation of ATF6β caused RANKL inhibition. Furthermore, the secretome from miR-1260b/ATF6β-axis-activated PDL cells inhibited osteoclastogenesis in human CD14^+ peripheral blood-derived monocytes. These results indicate that the miR-1260b/ATF6β axis mediates the regulation of ER stress, which may be used as a novel therapeutic strategy to treat periodontal disease.

DOI： 10.3389/fcell.2022.1061216

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Aims

Pancreatic β-cell apoptosis may be involved in the onset and progression of type 2 diabetes mellitus, although its mechanism remains unclear. We previously demonstrated that macrophage-derived interferon (IFN) β induced X-linked inhibitor of apoptosis–associated factor 1 (XAF1) expression in β-cells and accelerated β-cell apoptosis in vitro. Here, we explored the effects of XAF1 on β-cell function and progression of diabetes in vivo.

Methods

Pancreatic β-cell-selective XAF1 overexpressing (Xaf1 Tg) mice were generated. Xaf1 Tg mice and their wild-type (WT) littermates were fed either a normal diet or a 40% or 60% high-fat diet (HFD). The effects of β-cell XAF1 on β-cell apoptosis and exacerbation of diabetes were investigated.

Results

Palmitic acid induced IFNβ expression in macrophages, and HFD intake promoted macrophage infiltration in pancreatic islets, both of which cooperatively upregulated XAF1 expression in mouse islets. Furthermore, HFD-fed Xaf1 Tg mice demonstrated increased β-cell apoptosis, lowered insulin expression, and impaired glucose tolerance compared with WT mice fed the same diet. These effects were more pronounced in the 60%HFD group than in the 40%HFD group.

Conclusions

Pancreatic β-cell XAF1 expression was enhanced via HFD-induced, macrophage-derived IFNβ, which promoted β-cell apoptosis and led to a reduction in insulin secretion and progression of diabetes. To our knowledge, this is the first report to demonstrate an association between pancreatic β-cell XAF1 overexpression and exacerbation of diabetes, thus providing insight into the mechanism of β-cell mass reduction in diabetes.

DOI： 10.1007/s00592-022-01930-y

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Other Link： https://link.springer.com/article/10.1007/s00592-022-01930-y/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

Immunoregulatory properties of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are promising. Gingival tissue-derived MSCs (GMSCs) have unique immunoregulatory capacity and secrete large amounts of EVs. Recent findings suggest that priming MSCs with inflammatory stimuli is an effective strategy for cell-free therapy. However, the precise mechanism by which the contents of EVs are customized has not been fully elucidated. Here, we show that EVs derived from GMSCs primed with a combination of two pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α), synergistically promote anti-inflammatory M2 macrophage polarization by increasing the expression of cluster of differentiation 73 (CD73) and CD5 molecule-like (CD5L). Expression of CD73 by TNF-α/IFN-α stimulation was transcriptionally upregulated by the activation of mammalian target of rapamycin signaling and nuclear translocation of hypoxia-inducible factor 1α in GMSCs. TNF-α/IFN-α treatment also significantly increased the expression of CD5L mRNA via the transcription factor DNA-binding protein inhibitor ID3 and liver X receptor. Interestingly, exosomal CD5L is a prerequisite for the synergistic effect of EVs-mediated M2 macrophage polarization. These results indicate that combined pre-licensing with TNF-α and IFN-α in GMSCs is ideal for enhancing the anti-inflammatory function of EVs, which contributes to the establishment of a therapeutic tool.

DOI： 10.1038/s41598-022-17692-0

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Introduction: Dental caries and apical periodontitis are ones of the most prevalent chronic diseases and involve infection by cariogenic and endodontic bacteria. It can be said that the most required to cure is disinfection. We hypothesized that NB-UVB could be used for intraoral disinfection without affecting cells, and that it could be used in combination with TiO2 to disinfect complex root canals. The objectives were to investigate the effects of UV on dental pulp cells and oral bacteria and to evaluate the enhancement effect of a photocatalyst on bactericidal effects of UV irradiation. Methods: UV irradiation devices of UVB (310, 285 nm) and UVC (265 nm) were prepared. Cell proliferation and cytotoxicity assays after UV irradiation were performed using human dental pulp cells. The antibacterial activity of UV irradiation was investigated in Streptococcus mutans, Staphylococcus aureus, Enterococcus faecalis, Actinomyces naeslundii, Porphyromonas gingivalis, and Fusobacterium nucleatum. A curved simulated root canal with plastic training block was used to evaluate the effect of combined UV and TiO2 treatment. Data were analyzed by one-way ANOVA (p < 0.05) followed by Tukey’s post hoc tests (p < 0.05). Results: Human dental pulp cell proliferation was decreased by 265 nm, 285 nm, and 310 nm UV irradiation, although 310 nm UV irradiation did not show cytotoxic effects on these cells. Oral bacterial growth was suppressed following UV irradiation at 285 nm and 265 nm. Viability of all bacteria significantly decreased with UV irradiation. In the curved simulated root canal, viability of E. faecalis in UV irradiation at 285 nm with long taper fibers was significantly decreased in the 300 s irradiation group. E. faecalis proliferation was inhibited by combined UV irradiation and TiO2 application with long taper fibers in the curved simulated root canal. Conclusions: The wavelength of UVB and UVC showed bactericidal effects on oral bacteria including caries-related bacteria and apical periodontitis–related bacteria while NB-UVB did not. UVB with longer fibers was more effective in disinfection on E. faecalis in curved simulated root canal, and the combined use of photocatalyst further improved the disinfection effect.

DOI： 10.1007/s41547-021-00145-8

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Palmoplantar pustulosis (PPP) is a recurrent pustular dermatosis located on the palms and soles. Focal infection may exacerbate the symptoms of PPP, but the etiology is not fully clear. A 56-year-old woman with PPP was diagnosed with severe chronic periodontitis. Initial treatment for periodontitis combined with topical application of antibiotics and surgical treatment was performed. In this case, attention was paid to the relevance of systemic inflammation caused by periodontitis with the clinical symptoms of PPP. With periodontal treatment, the symptoms of PPP and periodontitis, high-sensitivity C-reactive protein (hs-CRP) level, and periodontal inflamed surface area (PISA) improved. This case highlights the importance of comprehensive dental examinations, including those for oral infections, such as periodontitis and other unrecognized sources of infection, and dental treatment in the overall management of PPP.

DOI： 10.1155/2021/5548760

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Amelogenin directly binds to glucose-regulated protein 78 (Grp78). Cell migration activity is expected to increase when human periodontal ligament cells (hPDLCs) overexpressing Grp78 are treated with amelogenin. Geranylgeranylacetone (GGA) is a drug that induces the expression of heat shock protein and is routinely used to treat gastric ulcers. Here, we investigated the changes in the properties and behavior of hPDLCs in response to treatment with GGA and the synergistic effects of amelogenin stimulation in hPDLCs pretreated with GGA for the establishment of a novel periodontal tissue regenerative therapy. We observed that GGA treatment increased Grp78 protein expression in hPDLCs and enhanced cell migration. Microarray analysis demonstrated that increased Grp78 expression triggered the production of angiopoietin-like 4 and amphiregulin, which are involved in the enhancement of angiogenesis and subsequent wound healing via the activation of hypoxia-inducible factor 1α and peroxisome proliferator-activated receptors as well as the phosphorylation of cAMP response element-binding protein and protein kinase A. Moreover, the addition of recombinant murine amelogenin (rM180) further accelerated hPDLC migration and tube formation of human umbilical vein endothelial cells due to the upregulation of interleukin-8 (IL-8), monocyte chemotactic protein 1, and IL-6, which are also known as angiogenesis-inducing factors. These findings suggest that the application of GGA to gingival tissue and alveolar bone damaged by periodontal disease would facilitate the wound healing process by inducing periodontal ligament cells to migrate to the root surface and release cytokines involved in tissue repair. Additionally, supplementation with amelogenin synergistically enhanced the migratory capacity of these cells while actively promoting angiogenesis. Therefore, the combined application of GGA and amelogenin may establish a suitable environment for periodontal wound healing and further drive the development of novel therapeutics for periodontal tissue regeneration.

DOI： 10.1002/jcb.29903

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Introduction Enlarged adipose tissue is characterized by infiltration of activated immune cells and increased expression of chemokines recruiting these cells including C-C motif ligand 19 (CCL19), although the role of adipose CCL19 is still inconclusive. Research design and methods Adipocyte-specific Ccl19 knock-in (KI) mice were generated, and the mice were fed either a normal diet or 40% or 60% fat diet (FD) to investigate the effects of CCL19 on the induction of inflammation and lipid metabolism. Results Ccl19KI mice exhibited increased inflammatory signs in adipose tissue and enlarged subcutaneous white and brown adipose tissue than those of wild-type (WT) mice. The adipose tissue of Ccl19KI mice was characterized by increased extracellular signal-regulated kinase 1/2 and decreased AMP-activated protein kinase α phosphorylation. The protein expression of peroxisome proliferator-activated receptor γcoactivator 1α and uncoupling protein 1 was significantly reduced in brown adipose tissue of Ccl19KI mice compared with that in WT mice. The most remarkable changes between genotypes were observed in mice fed a 40% FD. Conclusion A 40% FD enhanced the effects of CCL19 overexpression, and these mice could be a suitable model to study metabolic disorders in overweight Asians.

DOI： 10.1136/bmjdrc-2020-001871

Language：English   Publishing type：Research paper (scientific journal)  

Periodontal disease is a chronic inflammatory disease of the periodontal tissue. The periodontal inflamed surface area (PISA) is a proposed index for quantifying the inflammatory burden resulting from periodontitis lesions. This study aimed to investigate longitudinal changes in the periodontal status as evaluated by the PISA following the active periodontal treatment. To elucidate the prognostic factors of PISA, mixed-effect modeling was performed for clinical parameters, tooth-type, and levels of periodontal pathogens as independent variables. One-hundred-twenty-five patients with chronic periodontitis who completed the active periodontal treatment were followed-up for 24 months, with evaluations conducted at 6-month intervals. Five-times repeated measures of mean PISA values were 130+/-173, 161+/-276, 184+/-320, 175+/-417, and 209+/-469 mm2. Changes in clinical parameters and salivary and subgingival periodontal pathogens were analyzed by mixed-effect modeling. Plaque index, clinical attachment level, and salivary levels of Porphyromonas gingivalis were associated with changes in PISA at the patient- and tooth-level. Subgingival levels of P. gingivalis and Prevotella intermedia were associated with changes in PISA at the sample site. For most patients, changes in PISA were within 10% of baseline during the 24-month follow-up. However, an increase in the number of bleeding sites in a tooth with a deep periodontal pocket increased the PISA value exponentially.

DOI： 10.3390/jcm10061165

Language：Others   Publishing type：Research paper (scientific journal)  

Mesenchymal stem cell (MSC)–derived exosome plays a central role in the cell-free therapeutics involving MSCs and the contents can be customized under disease-associated microenvironments. However, optimal MSC-preconditioning to enhance its therapeutic potential is largely unknown. Here, we show that preconditioning of gingival tissue-derived MSCs (GMSCs) with tumor necrosis factor-alpha (TNF-α) is ideal for the treatment of periodontitis. TNF-α stimulation not only increased the amount of exosome secreted from GMSCs, but also enhanced the exosomal expression of CD73, thereby inducing anti-inflammatory M2 macrophage polarization. The effect of GMSC-derived exosomes on inflammatory bone loss were examined by ligature-induced periodontitis model in mice. Local injection of GMSC-derived exosomes significantly reduced periodontal bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and these effects were further enhanced by preconditioning of GMSCs with TNF-α. Thus, GMSC-derived exosomes also exhibited anti-osteoclastogenic activity. Receptor activator of NF-κB ligand (RANKL) expression was regulated by Wnt5a in periodontal ligament cells (PDLCs), and exosomal miR-1260b was found to target Wnt5a-mediated RANKL pathway and inhibit its osteoclastogenic activity. These results indicate that significant ability of the TNF-α-preconditioned GMSC-derived exosomes to regulate inflammation and osteoclastogenesis paves the way for establishment of a therapeutic approach for periodontitis.

DOI： 10.1016/j.actbio.2020.12.046

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Innate lymphoid cells (ILCs) are a family of developmentally related leukocytes that rapidly secrete polarized sets of cytokines to combat infection and promote tissue repair at mucosal barriers. Among them, group 3 ILCs (ILC3s) play an important role in maintenance of the gut homeostasis by producing IL-22, and their development and function critically depend on the transcription factor RORγt. Although recent evidence indicates that RORγt+ ILC3s are reduced in the gut in the absence of the Cdc42 activator DOCK8 (dedicator of cytokinesis 8), the underlying mechanism remains unclear. We found that genetic deletion of Dock8 in RORγt+-lineage cells markedly reduced ILC3s in the lamina propria of the small intestine. By analyzing BrdU incorporation, it was revealed that DOCK8 deficiency did not affect the cell proliferation. Furthermore, when lineage marker-negative (Lin-) α4β7+ CD127+ RORγt- fetal liver cells were cultured with OP9 stromal cells in the presence of stem cell factor (SCF) and IL-7 in vitro, RORγt+ ILC3s normally developed irrespective of DOCK8 expression. However, DOCK8-deficient ILC3s exhibited a severe defect in survival of ILC3s under the condition with or without IL-7. Similar defects were observed when we analyzed Dock8VAGR mice having mutations in the catalytic center of DOCK8, thereby failing to activate Cdc42. Thus, DOCK8 acts in cell-autonomous manner to control survival of ILC3s in the gut through Cdc42 activation.

DOI： 10.1093/intimm/dxaa066

Language：English   Publishing type：Research paper (scientific journal)  

The periodontal inflamed surface area (PISA) is a useful index for clinical and epidemiological assessments, since it can represent the inflammation status of patients in one contentious variable. However, calculation of the PISA is difficult, requiring six point probing depth measurements with or without bleeding on probing on 28 teeth, followed by data input in a calculation program. More simple methods are essential for screening periodontal disease or in epidemiological studies. In this study, we tried to establish a convenient partial examination method to estimate PISA. Cross-sectional data of 254 subjects who completed active periodontal therapy were analyzed. Teeth that represent the PISA value were selected by an item response theory approach. The maxillary second molar, first premolar, and lateral incisor and the mandibular second molar and lateral incisor were selected. The sum of the PISAs of these teeth was significantly correlated with the patient's PISA (R2 = 0.938). More simply, the sum of the maximum values of probing pocket depth with bleeding for these teeth were also significantly correlated with the patient's PISA (R2 = 0.6457). The simple model presented in this study may be useful to estimate PISA.

DOI： 10.3390/jcm10040723

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Japan Diabetes Complication and Prevention prospective (JDCP) study was conducted to examine the association between glycemic control and oral conditions in a large database of Japanese patients with diabetes. It included a total of 6099 patients with diabetes (range, 40–75 years) who had been treated as outpatients between 2007 and 2009. The mean number of present teeth at baseline was 19.8 and women with type 2 diabetes had fewer teeth than men with type 2 diabetes. Within the previous year, 17% of all patients had lost teeth. At baseline, 32% had experienced gingival swelling, 69% had brushed more than twice a day, 37% had used interdental cleaning aids, and 43% had undergone regular dental checkups. Multiple logistic regression analysis indicated that type 1 patients with HbA1c ≥ 7.0% were at higher risk of having fewer than 20 teeth (odds ratio [OR] 2.38; 95% confidence interval [CI] 1.25–4.78), and type 2 patients with HbA1c ≥ 8.0% also were at high risk of having fewer than 20 teeth (OR 1.16; 95% CI 1.00–1.34), after adjustment for nine possible confounding factors. In conclusion, patients with diabetes were found to be at high risk of tooth loss, and the poorer the glycemic control, the higher the risk of tooth loss in these patients.

DOI： 10.1007/s13340-020-00465-3

Language：English   Publishing type：Research paper (scientific journal)  

Few studies have investigated the role of extracellular-matrix proteoglycans in the pathogenesis of drug-induced gingival overgrowth (DIGO). SPOCK1 is an extracellular proteoglycan that induces epithelial to mesenchymal transition (EMT) in several cancer cell lines and exhibits protease-inhibitory activity. However, the role of SPOCK1 in non-cancerous diseases such as DIGO has not been well-addressed. We demonstrated that the expression of SPOCK1, TGF-β1, and MMP-9 in calcium channel blocker-induced gingival overgrowth is higher than that in non-overgrowth tissues. Transgenic mice overexpressing Spock1 developed obvious gingival-overgrowth and fibrosis phenotypes, and positively correlated with EMT-like changes. Furthermore, in vitro data indicated a tri-directional interaction between SPOCK1, TGF-β1, and MMP-9 that led to gingival overgrowth. Our study shows that SPOCK1 up-regulation in a noncancerous disease and SPOCK1-induced EMT in gingival overgrowth occurs via cooperation and crosstalk between several potential signaling pathways. Therefore, SPOCK1 is a novel therapeutic target for gingival overgrowth and its expression is a potential risk of EMT induction in cancerous lesions.

DOI： 10.1038/s41598-020-66660-z

Language：English   Publishing type：Research paper (scientific journal)  

Periodontal examination data have a complex structure. For epidemiological studies, mass screenings, and public health use, a simple index that represents the periodontal condition is necessary. Periodontal indices for partial examination of selected teeth have been developed. However, the selected teeth vary between indices, and a justification for the selection of examination teeth has not been presented. We applied a graded response model based on the item response theory to select optimal examination teeth and sites that represent periodontal conditions. Data were obtained from 254 patients who participated in a multicenter follow-up study. Baseline data were obtained from initial follow-up. Optimal examination sites were selected using item information calculated by graded response modeling. Twelve sites-maxillary 2nd premolar (palatal-medial), 1st premolar (palatal-distal), canine (palatal-medial), lateral incisor (palatal-central), central incisor (palatal-distal) and mandibular 1st premolar (lingual, medial)-were selected. Mean values for clinical attachment level, probing pocket depth, and bleeding on probing by full mouth examinations were used for objective variables. Measuring the clinical parameters of these sites can predict the results of full mouth examination. For calculating the periodontal index by partial oral examination, a justification for the selection of examination sites is essential. This study presents an evidence-based partial examination methodology and its modeling.

DOI： 10.3390/jcm9113754

Language：English   Publishing type：Research paper (scientific journal)  

MicroRNA (miRNA) plays an important role in diverse cellular biological processes such as inflammatory response, differentiation and proliferation, and carcinogenesis. miR-146a has been suggested as a negative regulator of the inflammatory reaction. Although, it has been reported as expressed in inflamed adipose and periodontal tissues, however, miR-146a's inhibitory effects against inflammatory response in both the tissues, are not well understood. Therefore, in this study, the inhibitory effects of miR-146a on both adipose and periodontal inflammation, was investigated. In vitro study has revealed that miR-146a transfection into either adipocytes or gingival fibroblasts, has resulted in a reduced cytokine gene expression, observed on co-culturing the cells with macrophages in the presence of lipopolysaccharides (LPS), in comparison to the control miRNA transfected. Similarly, miR-146a transfection into macrophages resulted in a reduced expression of TNF-α gene and protein in response to LPS stimulation. In vivo study revealed that a continuous intravenous miR-146a administration into mice via tail vein, protected the mice from developing high-fat diet-induced obesity and the inflammatory cytokine gene expression was down-regulated in both adipose and periodontal tissues. miR-146a appeared to be induced by macrophage-derived inflammatory signals such as TNF-α by negative feed-back mechanism, and it suppressed inflammatory reaction in both adipose and periodontal tissues. Therefore, miR-146a could be suggested as a potential therapeutic molecule and as a common inflammatory regulator for both obesity-induced diabetes and related periodontal diseases.

DOI： 10.1016/j.bbrep.2020.100757

Language：English   Publishing type：Research paper (scientific journal)  

Enamel matrix derivatives (EMDs)-based periodontal tissue regenerative therapy is known to promote healing with minimal inflammatory response after periodontal surgery, i. e., it promotes wound healing with reduced pain and swelling. It has also been reported that macrophages stimulated with amelogenin, a major component of EMD, produce various anti-inflammatory cytokines and growth factors. We previously found that stimulation of monocytes with murine recombinant M180 (rM180) amelogenin suppresses major histocompatibility complex class II (MHC II) gene expression using microarray analysis. However, the detailed molecular mechanisms for this process remain unclear. In the present study, we demonstrated that rM180 amelogenin selectively downmodulates the interferon gamma (IFNγ)-induced cell surface expression of MHC II molecules in macrophages and this mechanism mediated by rM180 appeared to be widely conserved across species. Furthermore, rM180 accumulated in the nucleus of macrophages at 15 min after stimulation and inhibited the protein expression of class II transactivator (CIITA) which controls the transcription of MHC II by IFNγ. In addition, reduced MHC II expression on macrophages pretreated with rM180 impaired the expression of T cell activation markers CD25 and CD69, T cell proliferation ability, and IL-2 production by allogenic CD4⁺ T lymphocytes in mixed lymphocyte reaction assay. The chromatin immunoprecipitation assay showed that IFNγ stimulation increased the acetylation of histone H3 lysine 27, which is important for conversion to euchromatin, as well as the trimethylation of histone H3 lysine 4 levels in the CIITA promoter IV (p-IV) region, but both were suppressed in the group stimulated with IFNγ after rM180 treatment. In conclusion, the present study shows that amelogenin suppresses MHC II expression by altering chromatin structure and inhibiting CIITA p-IV transcription activity, and attenuates subsequent T cell activation. Clinically observed acceleration of wound healing after periodontal surgery by amelogenin may be partially mediated by the mechanism elucidated in this study. In addition, the use of recombinant amelogenin is safe because it is biologically derived protein. Therefore, amelogenin may also be used in future as an immunosuppressant with minimal side effects for organ transplantation or MHC II-linked autoimmune diseases such as type I diabetes, multiple sclerosis, and rheumatoid arthritis, among others.

DOI： 10.3389/fimmu.2020.00709

Language：English   Publishing type：Research paper (scientific journal)  

SPOCK1 is a calcium-binding matricellular proteoglycan that has been extensively studied in several cancer cells. Previously, we generated a mouse line overexpressing SPOCK1 (Spock1-Tg mouse) and showed that SPOCK1 might play an important role in drug-induced gingival overgrowth, indicating that it possesses physiological functions in non-cancer diseases as well. Although SPOCK1 was reported to be secreted from human adipocytes, its role in adipocyte physiology has not been addressed yet. In this study, SPOCK1 protein expression was confirmed in pancreas, adipose tissues, spleen, and liver of normal diet (ND)-fed mice. Interestingly, SPOCK1 was up-regulated in the pancreas and adipose tissues of the high-fat diet (HFD)-fed mice. Spock1-Tg mice fed with ND showed increased maturation in epididymal and inguinal adipose tissues. In addition, Spock1 overexpression strongly decreased expression of UCP-1 in adipose tissues, suggesting that SPOCK1 might regulate thermogenic function through suppression of UCP-1 expression. Finally, exogenous SPOCK1 treatment directly accelerated the differentiation of 3T3-L1 adipocytes, accompanied by the up-regulation of adipocyte differentiation-related gene expression. In conclusion, we demonstrated for the first time that SPOCK1 induced adipocyte differentiation via the up-regulation of adipogenesis-related genes.

DOI： 10.1016/j.bbrc.2020.09.129

Language：English   Publishing type：Research paper (scientific journal)  

Differentiation-inducing factor-1 (DIF-1) has been reported to inhibit the proliferation of various mammalian cells by unknown means, although some possible mechanisms of its action have been proposed, including the activation of glycogen synthase kinase-3 (GSK-3). Here, we report an alternative mechanism underlying the action of DIF-1 in human breast cancer cell line MCF-7, on which the effects of DIF-1 have not been examined previously. Intragastric administration of DIF-1 reduced the tumor growth from MCF-7 cells injected into a mammary fat pad of nude mice, without causing adverse effects. In cultured MCF-7, DIF-1 arrested the cell cycle in G₀/G₁ phase and suppressed cyclin D1 expression, consistent with our previous results obtained in other cell species. However, DIF-1 did not inhibit the phosphorylation of GSK-3. Investigating an alternative mechanism for the reduction of cyclin D1, we found that DIF-1 reduced the protein levels of signal transducer and activator of transcription 3 (STAT3). The STAT3 inhibitor S3I-201 suppressed cyclin D1 expression and cell proliferation and the overexpression of STAT3 enhanced cyclin D1 expression and accelerated proliferation. Differentiation-inducing factor-1 did not reduce STAT3 mRNA or reduce STAT3 protein in the presence of cycloheximide, suggesting that DIF-1 inhibited STAT3 protein synthesis. Seeking its mechanism, we revealed that DIF-1 inhibited the activation of 70 kDa and/or 85 kDa ribosomal protein S6 kinase (p70^S6K/p85^S6K). Inhibition of p70^S6K/p85^S6K by rapamycin also reduced the expressions of STAT3 and cyclin D1. Therefore, DIF-1 suppresses MCF-7 proliferation by inhibiting p70^S6K/p85^S6K activity and STAT3 protein synthesis followed by reduction of cyclin D1 expression.

DOI： 10.1111/cas.14204

Language：English   Publishing type：Research paper (scientific journal)  

It is well known that dental pulp tissue can evoke some of the most severe acute inflammation observed in the human body. We found that dental pulp cells secrete a factor that induces tumor necrosis factor-α production from macrophages, and designated this factor, dental pulp cell-derived powerful inducer of TNF-α (DPIT). DPIT was induced in dental pulp cells and transported to recipient cells via microvesicles. Treatment of dental pulp cells with a PKR inhibitor markedly suppressed DPIT activity, and weak interferon signals were constitutively activated inside the cells. In recipient macrophages, stimulation with DPIT-containing supernatants from pulp cells resulted in activation of both nuclear factor-κB and MAP kinases like JNK and p38. Proteomics analyses revealed that many stress granule-related proteins were present in supernatants from dental pulp cells as well as microvesicle marker proteins like GAPDH, β-actin, HSPA8, HSPB1, HSPE1, and HSPD1. Furthermore, giant molecule AHNAK and PKR were detected in microvesicles derived from dental pulp cells, and gene silencing of AHNAK in dental pulp cells led to reduced DPIT activity. Thus, it appeared that the core protein of DPIT was PKR, and that PKR was maintained in an active state in stress granule aggregates with AHNAK and transported via microvesicles. The activity of DPIT for TNF-α induction was far superior to that of gram-negative bacterial endotoxin. Therefore, we, report for the first time, that active PKR is transported via microvesicles as stress granule aggregates and induces powerful inflammatory signals in macrophages.

DOI： 10.1038/s41598-019-40046-2

Language：English   Publishing type：Research paper (scientific journal)  

Background: The chemokine receptor CCR7, expressed on various immune cells, is associated with cell migration and lympho-node homing. Mice lacking Ccr7 are protected from diet-induced obesity and subsequent insulin resistance. We evaluated the mechanism underlying these protective effects from the standpoint of energy expenditure. Methods: Wild-type and Ccr7 null mice were fed a high-fat diet, and the regulation of energy metabolism and energy metabolism-related molecules, e.g., Ucp1, Cidea, and Pgc1α, were evaluated. Results: Food intake did not differ between groups. O₂ consumption and CO₂ production were higher in Ccr7 null mice than in wild-type mice, despite a similar respiratory quotient and glucose and lipid utilization, suggesting that energy expenditure increased in Ccr7 null mice via enhanced metabolism. In white adipose tissues of Ccr7 null mice, Prdm16, Cd137, Tmem26, Th, and Tbx1 expression increased. Similarly, in brown adipose tissues of Ccr7 null mice, Dio2, Pgc1α, Cidea, Sirt1, and Adiponectin expression increased. In both white and brown adipose tissues, Ucp1 gene and protein expression levels were higher in null mice than in wild-type mice. Conclusions: In Ccr7 null mice, browning of white adipocytes as well as the activation of brown adipocytes cause enhanced energy metabolism, resulting in protection against diet-induced obesity.

DOI： 10.1186/s12986-019-0372-5

Language：English   Publishing type：Research paper (scientific journal)  

Background: Recently, clinical studies have shown the protective effects of sodium glucose co-transporter2 (SGLT2) inhibitors against progression of diabetic nephropathy, but the underlying molecular mechanisms remain unclear. Methods: Diabetic mice were prepared by injecting nicotinamide and streptozotocin, followed by high-sucrose diet feeding (NA/STZ/Suc mice). The SGLT2 inhibitor canagliflozin was administered as a 0.03% (w/w) mixture in the diet for 4 weeks. Then, various parameters and effects of canagliflozin on diabetic nephropathy were investigated. Results: Canagliflozin administration to NA/STZ/Suc mice normalized hyperglycemia as well as elevated renal mRNA of collagen 1a1, 1a2, CTGF, TNFα and MCP-1. Microscopic observation revealed reduced fibrotic deposition in the kidneys of canagliflozin-treated NA/STZ/Suc mice. Interestingly, the protein level of Pin1, reportedly involved in the inflammation and fibrosis affecting several tissues, was markedly increased in the NA/STZ/Suc mouse kidney, but this was normalized with canagliflozin treatment. The cells showing increased Pin1 expression in the kidney were mainly mesangial cells, along with podocytes, based on immunohistochemical analysis. Furthermore, it was revealed that canagliflozin induced AMP-activated kinase (AMPK) activation concentration-dependently in CRL1927 mesangial as well as THP-1 macrophage cell lines. AMPK activation was speculated to suppress mesangial cell proliferation and exert anti-inflammatory effects in hematopoietic cells. Conclusion: Therefore, we can reasonably suggest that normalized Pin1 expression and AMPK activation contribute to the molecular mechanisms underlying SGLT2 inhibitor-induced suppression of diabetic nephropathy, possibly at least in part by reducing inflammation and fibrotic change.

DOI： 10.1186/s13098-019-0454-6

Language：English   Publishing type：Research paper (scientific journal)  

Non-shivering thermogenesis in adipocytes provides defense against low temperatures and obesity development, but the underlying regulatory mechanism remains to be fully clarified. Based on both markedly increased Pin1 expression in states of excess nutrition and resistance to obesity development in Pin1 null mice, we speculated that adipocyte Pin1 may play a role in thermogenic programs. Adipose-specific Pin1 knockout (adPin1 KO) mice showed enhanced transcription of thermogenic genes and tolerance to hypothermia when exposed to cold. In addition, adPin1 KO mice were resistant to high-fat diet-induced obesity and glucose intolerance. A series of experiments revealed that Pin1 binds to PRDM16 and thereby promotes its degradation through the ubiquitin-proteasome system. Consistent with these results, Pin1 deletion in differentiated adipocytes showed enhancement of thermogenic programs in response to the β3 agonist CL316243 through the upregulation of PRDM16 proteins. These observations indicate that Pin1 is a negative regulator of non-shivering thermogenesis.

DOI： 10.1016/j.celrep.2019.02.066

Language：English   Publishing type：Research paper (scientific journal)  

We report the draft genome sequence of Porphyromonas gingivalis strain 381 Okayama (381OKJP). The strain, obtained from the Socransky collection, has been used for experimentation since 1987. This sequence allows for comparisons to other sequenced 381 strains to observe acquisition of mutations and genome rearrangements in a commonly used laboratory strain.

DOI： 10.1128/MRA.01641-18

Language：English   Publishing type：Research paper (scientific journal)  

Nitric oxide (NO) is thought to play a pivotal regulatory role in dental pulp tissues under both physiological and pathological conditions. However, little is known about the NO functions in dental pulp stem cells (DPSCs). We examined the direct actions of a spontaneous NO gas-releasing donor, NOC-18, on the odontogenic capacity of rat DPSCs (rDPSCs). In the presence of NOC-18, rDPSCs were transformed into odontoblast-like cells with long cytoplasmic processes and a polarized nucleus. NOC-18 treatment increased alkaline phosphatase activity and enhanced dentin-like mineralized tissue formation and the expression levels of several odontoblast-specific genes, such as runt related factor 2, dentin matrix protein 1 and dentin sialophosphoprotein, in rDPSCs. In contrast, carboxy-PTIO, a NO scavenger, completely suppressed the odontogenic capacity of rDPSCs. This NO-promoted odontogenic differentiation was activated by tumor necrosis factor-NF-κB axis in rDPSCs. Further in vivo study demonstrated that NOC-18-application in a tooth cavity accelerated tertiary dentin formation, which was associated with early nitrotyrosine expression in the dental pulp tissues beneath the cavity. Taken together, the present findings indicate that exogenous NO directly induces the odontogenic capacity of rDPSCs, suggesting that NO donors might offer a novel host DPSC-targeting alternative to current pulp capping agents in endodontics.

DOI： 10.1038/s41598-018-21183-6

Language：English   Publishing type：Research paper (scientific journal)  

Dental pulp cells (DPCs) are promising candidates for use as transplantable cells in regenerative medicine. However, ex vivo expansion of these cells typically requires culture media containing fetal bovine serum, which may cause infection and immunological reaction following transplantation. In addition, the proliferation and differentiation of DPCs markedly depend upon serum batches. Therefore, the present study examined whether DPCs could be expanded under serum-free conditions. DPCs obtained from four donors were identified to proliferate actively in the serum-free medium, STK2, when compared with those cells in control medium (Dulbecco's modified Eagle's medium containing 10% serum). The high proliferative potential with STK2 was maintained through multiple successive culture passages. DNA microarray analyses demonstrated that the gene expression profile of DPCs grown in STK2 was similar to that of cells grown in the control medium; however, a number of genes related to cell proliferation, including placental growth factor and inhibin-βE, were upregulated in the STK2 cultures. Following induction of osteogenesis, DPCs grown in STK2 induced alkaline phosphatase activity and calcification at higher levels compared with the control medium cultures, indicating maintenance of differentiation potential in STK2. This serum-free culture system with DPCs may have applications in further experimental studies and as a clinical strategy in regenerative medicine.

DOI： 10.3892/br.2018.1066

Language：English   Publishing type：Research paper (scientific journal)  

Metabolic endotoxemia has been implicated in the pathogenesis of type 2 diabetes. In addition to adipose tissue inflammation, inflammatory cell infiltration is also observed in islets, although its effect on islets is largely unknown. We hypothesized that macrophage infiltration into islets leads to impairment of α or β cell function, which ultimately act to exacerbate the pathophysiology of diabetes. Gene expression in a murine α cell line, αTC1, and β cell line, βTC6, was investigated by DNA microarray after co-culturing the cells with a murine macrophage cell line, RAW 264.7, in the presence or absence of bacterial endotoxin. Among the genes showing highly upregulated expression, genes specifically upregulated only in β cells were evaluated to determine the roles of the gene products on the cellular function of β cells. In both α and β cells, expression of type I interferon-responsive genes was highly upregulated upon endotoxin stimulation. Among these genes, expression of the X-linked inhibitor of apoptosis (Xiap)-associated factor 1 (Xaf1) gene, which is associated with the induction of apoptosis, was specifically enhanced in β cells by endotoxin stimulation. This upregulation appeared to be mediated by macrophage-derived interferon β (IFNβ), as endotoxin-stimulated macrophages produced higher amounts of IFNβ, and exogenous addition of IFNβ into βTC6 cultures resulted in increased Xaf1 protein production and cleaved caspase 3, which accelerated β-cell apoptosis. Macrophages activated by metabolic endotoxemia infiltrated into islets and produced IFNβ, which induced β-cell apoptosis by increasing the expression of Xaf1.

DOI： 10.1055/s-0043-121467

Language：English   Publishing type：Research paper (scientific journal)  

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.

DOI： 10.1016/j.bbrc.2017.12.136

Language：English   Publishing type：Research paper (scientific journal)  

Objectives It is well-known that the complement system plays an essential role in host immunity. Observational studies have indicated that complement system-related molecules such as complement factor B (CfB) and other components are correlated with obesity and/or insulin resistance parameters. In this study, we investigated the role of adipocyte-derived CfB in adipose tissue metabolism. Methods We investigated the expression level of complement system-related genes in adipocytes. To understand the role of CfB in adipocyte, we performed Cfb overexpression in 3T3-L1 preadipocytes and generated adipocyte-specific Cfb transgenic mice. Results Cfb expression was markedly enhanced in 3T3-L1 adipocytes co-cultured with macrophages following endotoxin stimulation. In Cfb-overexpressing cells, the expression of adipocyte differentiation/maturation-related genes encoding peroxisome proliferator-activated receptor γ (Pparγ), adipocyte Protein 2 and perilipin was significantly enhanced. Cfb transgenic mice showed a marked increase in the expression of genes encoding Pparγ, perilipin, sterol regulatory element-binding protein 1 c, and Cd36 in the subcutaneous adipose tissue. Conclusions CfB plays a crucial role in late-phase of adipocyte differentiation and subsequent lipid droplet formation.

DOI： 10.1016/j.bbrc.2017.11.069

Language：English   Publishing type：Research paper (scientific journal)  

Periodontal disease is assessed and its progression is determined via observations on a site-by-site basis. Periodontal data are complex and structured in multiple levels; thus, applying a summary statistical approach (i.e., the mean) for site-level evaluations results in loss of information. Previous studies have shown the availability of mixed effects modeling. However, clinically beneficial information on the progression of periodontal disease during the follow-up period is not available. We conducted a multicenter prospective cohort study. Using mixed effects modeling, we analyzed 18,834 sites distributed on 3,139 teeth in 124 patients, and data were collected 5 times over a 24-month follow-up period. The change in the clinical attachment level (CAL) was used as the outcome variable. The CAL at baseline was an important determinant of the CAL changes, which varied widely according to the tooth surface. The salivary levels of periodontal pathogens, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were affected by CAL progression. “Linear”- and “burst”-type patterns of CAL progression occurred simultaneously within the same patient. More than half of the teeth that presented burst-type progression sites also presented linear-type progression sites, and most of the progressions were of the linear type. Maxillary premolars and anterior teeth tended to show burst-type progression. The parameters identified in this study may guide practitioners in determining the type and extent of treatment needed at the site and patient levels. In addition, these results show that prior hypotheses concerning "burst" and "linear" theories are not valid.

DOI： 10.1371/journal.pone.0188670

Language：English   Publishing type：Research paper (scientific journal)  

Objectives Amelogenin, the major component of the enamel matrix derivative (EMD), has been suggested as a bioactive candidate for periodontal regeneration. Apart from producing a regenerative effect on periodontal tissues, amelogenin has also been reported to have an anti-inflammatory effect. However, the precise molecular mechanisms underlying these effects remain unclear. In the present study, we examined the immunomodulatory effects of amelogenin on macrophages. Design Human phorbol 12-myristate 13-acetate (PMA)-differentiated U937 macrophages and CD14+ peripheral blood-derived monocytes (PBMC)-derived macrophages were stimulated with recombinant amelogenin (rM180). After performing a detailed microarray analysis, the effects of rM180 on macrophage phenotype and signal transduction pathways were evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, confocal microscopy and flow cytometry. Results The microarray analysis demonstrated that rM180 increased the expression of anti-inflammatory genes in lipopolysaccharide (LPS)-challenged macrophages after 24 h, while it temporarily up-regulated inflammatory responses at 4 h. rM180 significantly enhanced the expression of M2 macrophage markers (CD163 and CD206). rM180-induced M2 macrophage polarisation was associated with morphological changes as well as vascular endothelial growth factor (VEGF) production. rM180 enhanced prostaglandin E2 (PGE2) expression, and the activation of the cAMP/cAMP-responsive element binding (CREB) signaling pathway was involved in amelogenin-induced M2 macrophage polarisation. Blocking of PGE2 signaling by indomethacin specifically abrogated rM180 with or without LPS-induced M2 shift in PBMC-derived macrophages. Conclusion Amelogenin could reprogram macrophages into the anti-inflammatory M2 phenotype. It could therefore contribute to the early resolution of inflammation in periodontal lesions and provide a suitable environment for remodeling-periodontal tissues.

DOI： 10.1016/j.archoralbio.2017.08.005

Language：English   Publishing type：Research paper (scientific journal)  

Tripalmitoyl-S-glyceryl-L-Cys-Ser-(Lys)₄ (Pam₃CSK₄) is a highly conserved molecular motif found in various classes of lipoproteins. The requirement for leukocyte to respond to synthetic Pam₃CSK₄ were studied. Pam₃CSK₄ primed neutrophils for a respiratory burst in a serum-dependent manner. Pam₃CSK₄ upregulated CD11b, CD14, and cytochrome b₅₅₈, and downregulated Leu-8. Treatment of neutrophils with anti-CD14 antibodies and treatment of serum with anti-LPS binding protein (LBP) antibodies resulted in the inhibition of priming for respiratory burst by Pam₃CSK₄. It should be noted that LBP could not replicate the effects of serum in priming of neutrophils for respiratory burst by Pam₃CSK₄. Serum LBP bound to immobilized Pam₃CSK₄. Pam₃CSK₄ induced the interleukin-8 (IL-8) production by leukocytes in a serum-dependent manner. Further, Pam₃CSK₄-induced priming of neutrophils for respiratory burst was not inhibited by the LPS antagonists LA-14-PP, Rhodobacter sphaeroides LPS, or E5531, and Pam₃CSK₄-induced IL-8 production by leukocytes was not affected by LPS antagonist, E5531, indicating that Pam₃CSK₄ was recognized by a different receptor than LPS. Thus, Pam₃CSK₄ and LPS had similar biological activities and similar requirement to act on leukocytes, but were recognized by different receptors. Serum in the action of Pam₃CSK₄ on leukocytes was not replicated by LBP, suggesting that Pam₃CSK₄ might be disaggregated by serum to result in the activation of leukocytes.

DOI： 10.1016/j.intimp.2017.06.006

Language：English   Publishing type：Research paper (scientific journal)  

Aims/Introduction: To explore the relationships between periodontitis and microvascular complications as well as glycemic control in type 2 diabetes patients. Materials and Methods: This multicenter, hospital-based, cross-sectional study included 620 patients with type 2 diabetes. We compared the prevalence and severity of periodontitis between patients with ≥1 microvascular complication and those without microvascular complications. We also compared the prevalence and severity of periodontitis among patients with different degrees of glycemic control. Results: After adjusting for confounding factors, multiple logistic regression analysis showed that the severity of periodontitis was significantly associated with the number of microvascular complications (odds ratio 1.3, 95% confidence interval 1.1–1.6), glycated hemoglobin ≥8.0% (64 mmol/mol; odds ratio 1.6; 95% confidence interval 1.1–2.3), and older age (≥50 years; odds ratio 1.7; 95% confidence interval 1.1–2.6). However, the prevalence of periodontitis was not significantly associated with the number of microvascular complications, but was associated with male sex, high glycated hemoglobin (≥8.0% [64 mmol/mol]), older age (≥40 years), longer duration of diabetes (≥15 years) and fewer teeth (≤25). Furthermore, propensity score matching for age, sex, diabetes duration and glycated hemoglobin showed that the incidence of severe periodontitis was significantly higher among patients with microvascular complications than among those without microvascular complications (P < 0.05). Conclusions: The number of microvascular complications is a risk factor for more severe periodontitis in patients with type 2 diabetes, whereas poor glycemic control is a risk factor for increased prevalence and severity of periodontitis.

DOI： 10.1111/jdi.12633

Language：English   Publishing type：Research paper (scientific journal)  

The effects of adherence on neutrophil superoxide anion (O₂ ⁻) generation triggered by surface, soluble ligand, or adherence were studied. Resting-neutrophils adhered to the uncoated tubes resulting in O₂ ⁻ generation, but not on plasma-, fibrinogen-, vitronectin-, fibronectin-, laminin-, collagen-, or poly HEMA-coated surfaces. Enhanced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated O₂ ⁻ generation by LPS-primed-neutrophils was induced by the incubation on plasma, fibrinogen, vitronectin, fibronectin, or laminin in the absence of Mg²⁺. In the presence of Mg²⁺, this response was observed in cells on collagen or poly HEMA. LPS-primed-neutrophils adhered to uncoated, BSA- or IgG-coated tubes and did not respond to fMLP, indicating that the fMLP-response of LPS-primed-neutrophils was suppressed by adherence. Upon incubation on plasma, fibrinogen, vitronectin, fibronectin in the presence of Mg²⁺, LPS-primed-neutrophils showed O₂ ⁻ generation. Upon incubation on collagen or poly HEMA, the primed-neutrophils neither generated O₂ ⁻ nor adhered. We found that O₂ ⁻ response of LPS-primed-neutrophils was attenuated depending on the time of exposure to plasma-coated surface. This attenuation was evident on plasma or fibrinogen, but not on collagen in the presence of Mg²⁺, indicating that O₂ ⁻ generation by LPS-primed-neutrophils was attenuated dependent on adherence but not on Mg²⁺. Thus, adhesion attenuated the O₂ ⁻ generation triggered by both soluble (fMLP) and insoluble (surface) stimuli.

DOI： 10.1016/j.imbio.2017.05.001

Language：English   Publishing type：Research paper (scientific journal)  

Introduction and Aims Several studies have reported that angiopoietin-like protein 2 (Angptl2) is expressed abundantly in adipocytes and is associated with adipose tissue inflammation. In the present study, we found that osteoblasts and mesenchymal stem cells also expressed Angptl2 at high levels. The aim of this study was to understand the role of Angptl2 in osteoblastic cell differentiation. Methods Angptl2 expression was examined during osteoblast and adipocyte differentiation. The role of Angptl2 on cell differentiation and associated signaling was analyzed by gene knockdown using Angptl2 small interfering ribonucleic acid (siRNA). Results Angptl2 was highly expressed in MC3T3-E1 cells, ST2 cells and primary osteoblasts, but not in RAW264 cells. Inhibition of Angptl2 expression using siRNA markedly inhibited alkaline phosphatase (ALP) expression and osteoblastic differentiation in MC3T3-E1, ST2 cells and primary osteoblasts. Angptl2 siRNA also inhibited adipocyte differentiation in ST2 cells. Treatment of MC3T3-E1 cells with Angptl2 siRNA led to the down-regulation of the activities of several cell signaling pathways, including extracellular signal-regulated kinase (ERK), Jun amino-terminal kinase (JNK), Akt, and nuclear factor kappa B (NF-κB) signals. It also down-regulated the expression of Osterix, but not that of runt-related transcription factor 2 (Runx2), suggesting that Angptl2 is a positive activator of Osterix and its down-stream signals. Treatment of MC3T3-E1 cells with anti-Angptl2 antibodies suppressed ALP gene expression. In addition, treatment of Angptl2 siRNA-treated cells with culture supernatants of normal MC3T3-E1 cells restored ALP gene expression, indicating that Angptl2 acts in an autocrine manner. Conclusions The results suggest that Angptl2 is an autocrine positive regulator of cell differentiation. Thus, it is suggested that Angptl2 regulates not only adipose tissue metabolism but also bone metabolism.

DOI： 10.1016/j.metabol.2017.01.006

Language：English   Publishing type：Research paper (scientific journal)  

Background and aims Epicatechin (EC) intake has been suggested to be beneficial for the prevention of cardiovascular disorders, and it is well known that adipose tissue inflammation is one of the major risk factors for coronary heart diseases. The purpose of the present study was to determine the in vitro and in vivo effects of EC on adipose tissue inflammation and obesity. Methods and results DNA microarray analysis was performed to evaluate the effects of EC on gene expression in adipocytes co-cultured with bacterial endotoxin-stimulated macrophages. To determine the in vivo effects of the catechin, C57BL/6 mice were fed either a high-fat diet (HFD) or HFD combined with EC, and metabolic changes were observed EC suppressed the expression of many inflammatory genes in the adipocytes co-cultured with endotoxin-stimulated macrophages. Specifically, EC markedly suppressed chemokine (C–C motif) ligand 19 (CCL19) expression. The target cell of EC appeared to macrophages. The in vivo study indicated that mice fed the EC-supplemented HFD were protected from diet-induced obesity and insulin resistance. Accordingly, the expression levels of genes associated with inflammation in adipose tissue and in the liver were downregulated in this group of mice. Conclusions EC exerts beneficial effects for the prevention of adipose tissue inflammation and insulin resistance. Since we previously reported that mice deficient in the CCL19 receptor were protected from diet-induced obesity and insulin resistance, it can be concluded that the beneficial effects of EC could be mediated, at least in part, by marked suppression of CCL19 expression.

DOI： 10.1016/j.numecd.2016.11.008

Language：English   Publishing type：Research paper (scientific journal)  

CD14 and Toll-like receptor 4/MD2 (TLR4/MD2) mediate the action of LPS on neutrophils. The anti-CD14 antibody and the TLR4/MD2-antagonist, synthetic lipid IVa (LA-14-PP), are known to inhibit the response of neutrophils to LPS. We studied the role of CD14 in LPS-induced priming of neutrophils for enhanced release of the superoxide anion. The anti-CD14 antibody at much higher concentrations than required to saturate CD14 was required to inhibit priming by LPS. The inhibitory effect of the anti-CD14 antibody was overcome by LPS. After washing, anti-CD14-treated neutrophils showed upregulated CD14 upon incubation at 37°C and responded to LPS with a delayed time-course. Thus, CD14-blocked neutrophils gained responsiveness to LPS through newly upregulated CD14. These results suggested that the unbound/free anti-CD14 antibody was essential to inhibit LPS-induced priming by blocking CD14 that were newly expressed during incubation at 37°C. LA-14-PP inhibited the response of neutrophils to LPS in an anti-CD14 antibody sensitive manner. When neutrophils were treated with LA-14-PP followed by treatment with the anti-CD14 antibody, CD14 was upregulated upon warming, but priming was blocked, suggesting that TLR4/MD2 was not newly expressed by warming in association with CD14 molecules. Thus, in addition to blocking CD14, the anti-CD14 antibody was found to induce the expression of new CD14.

DOI： 10.1080/08820139.2016.1238925

Language：English   Publishing type：Research paper (scientific journal)  

Fimbriae are virulence factors of Porphyromonas gingivalis (P. gingivalis). In this study, the action of fimbriae on neutrophil respiratory burst and cytokine production by mononuclear cells (MNC) were investigated. Native or denatured form of purified P. gingivalis fimbriae contained endotoxin at an equivalence of 1– 3 μg lipopolysaccharides (LPS)/mg protein. The endotoxin could be reduced to the equivalent of 1 ng-LPS/mg protein by phase separation using Triton X-114. Unfractionated fimbriae caused serum-dependent priming of neutrophils for enhanced respiratory burst, but both native and denatured forms of Triton X-114-fractionated fimbriae were not active at 100 μg/mL. Unfractionated fimbriae induced serum-dependent production of IL-1β by MNC. Triton X-114-fractionated fimbriae (10 μg/mL)-induced production of IL-1β, IL-8 or TNF-α was much lower than that induced by unfractionated fimbriae or 10 ng/mL P. gingivalis-LPS preparation. Triton X-114-fractionated fimbriae immobilized on polystyrene tubes induced adhesion-stimulated superoxide release by LPS-primed neutrophils in a β₂ integrin-dependent manner. P. gingivalis cells caused priming of neutrophils; however, Toll-like receptor (TLR) 4 antagonists did not affect this response. Thus, P. gingivalis fimbriae were ineffective in inducing innate immune response in leukocytes; however, they induced β₂ integrin-mediated response by neutrophils. Immune-stimulatory components of P. gingivalis might be recognized by receptors other than TLR4.

DOI： 10.1016/j.jim.2016.11.012

Language：English   Publishing type：Research paper (scientific journal)  

Mechanical reduction of infectious bacteria by using physical instruments is considered the principal therapeutic strategy for periodontal disease; addition of antibiotics is adjunctive. However, local antibiotic treatment, combined with conventional mechanical debridement, has recently been shown to be more effective in periodontitis subjects with type 2 diabetes. This suggests that some bacteria may invade the inflamed inner gingival epithelium, and mechanical debridement alone will be unable to reduce these bacteria completely. Therefore, we tried to establish infected organ culture models that mimic the inner gingival epithelium and aimed to see the effects of antibiotics in these established models. Mouse dorsal skin epithelia were isolated, and periodontal bacteria were injected into the epithelia. Infected epithelia were incubated with test antibiotics, and colony-forming ability was evaluated. Results indicated that effective antibiotics differed according to injected bacteria and the bacterial combinations tested. Overall, in organ culture model, the combination of amoxicillin or cefdinir and metronidazole compensate for the effects of less effective bacterial combinations on each other. This in vitro study would suggest effective periodontal treatment regimens, especially for severe periodontitis.

DOI： 10.1002/cre2.48

Language：English   Publishing type：Research paper (scientific journal)  

Background: The diagnosis of the progression of periodontitis presently depends on the use of clinical symptoms (such as attachment loss) and radiographic imaging. The aim of the multicenter study described here was to evaluate the diagnostic use of the bacterial content of subgingival plaque recovered from the deepest pockets in assessing disease progression in chronic periodontitis patients. Methods: This study consisted of a 24-month investigation of a total of 163 patients with chronic periodontitis who received trimonthly follow-up care. Subgingival plaque from the deepest pockets was recovered and assessed for bacterial content of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans using the modified Invader PLUS assay. The corresponding serum IgG titers were measured using ELISA. Changes in clinical parameters were evaluated over the course of 24 months. The sensitivity, specificity, and prediction values were calculated and used to determine cutoff points for prediction of the progression of chronic periodontitis. Results: Of the 124 individuals who completed the 24-month monitoring phase, 62 exhibited progression of periodontitis, whereas 62 demonstrated stable disease. The P. gingivalis counts of subgingival plaque from the deepest pockets was significantly associated with the progression of periodontitis (p < 0.001, positive predictive value = 0.708). Conclusions: The P. gingivalis counts of subgingival plaque from the deepest pockets may be associated with the progression of periodontitis.

DOI： 10.1186/s12903-017-0337-x

Language：English   Publishing type：Research paper (scientific journal)  

Background and Objective: A diagnosis of periodontitis progression is presently limited to clinical parameters such as attachment loss and radiographic imaging. The aim of this multicenter study was to monitor disease progression in patients with chronic periodontitis during a 24-mo follow-up program and to evaluate the amount of bacteria in saliva and corresponding IgG titers in serum for determining the diagnostic usefulness of each in indicating disease progression and stability. Material and Methods: A total of 163 patients with chronic periodontitis who received trimonthly follow-up care were observed for 24 mo. The clinical parameters and salivary content of Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were assessed using the modified Invader PLUS assay, and the corresponding serum IgG titers were measured using ELISA. The changes through 24 mo were analyzed using cut-off values calculated for each factor. One-way ANOVA or Fisher's exact test was used to perform between-group comparison for the data collected. Diagnostic values were calculated using Fisher's exact test. Results: Of the 124 individuals who completed the 24-mo monitoring phase, 62 exhibited periodontitis progression, whereas 62 demonstrated stable disease. Seven patients withdrew because of acute periodontal abscess. The ratio of P. gingivalis to total bacteria and the combination of P. gingivalis counts and IgG titers against P. gingivalis were significantly related to the progression of periodontitis. The combination of P. gingivalis ratio and P. gingivalis IgG titers was significantly associated with the progression of periodontitis (p = 0.001, sensitivity = 0.339, specificity = 0.790). Conclusions: It is suggested that the combination of P. gingivalis ratio in saliva and serum IgG titers against P. gingivalis may be associated with the progression of periodontitis.

DOI： 10.1111/jre.12353

Language：English   Publishing type：Research paper (scientific journal)  

Sphingosine-1-phosphate (S1P) is a signaling sphingolipid that also plays crucial roles in bone regeneration. Recently, we reported that the S1P receptors S1PR1 and S1PR2 were mainly expressed in osteoblast-like cells, and that the S1P/S1PR1 signaling pathway up-regulated osteoprotegerin and osteoblast differentiation. However, the involvement of S1P/S1PR2 signaling in osteoblast differentiation is not well understood. Here we investigate the role of S1P/S1PR2-mediated signaling in osteoblast differentiation and clarify the underlying signaling mechanisms. We found that an S1P/S1PR2/Gi-independent signaling pathway activated RhoA activity, leading to phosphorylation of Smad1/5/8 in mouse osteoblast-like MC3T3-E1 cells and primary osteoblasts. Furthermore, this signaling pathway promoted nuclear translocation of Smad4, and increased the amount of Smad6/7 protein in the nucleus. S1P also up-regulated runt-related transcription factor 2 (Runx2) expression through S1PR2/RhoA/ROCK/Smad1/5/8 signaling. Moreover, we found that S1P partially triggered S1PR2/RhoA/ROCK pathway leading to bone formation in vivo. These findings suggest that S1P induces RhoA activity, leading to the phosphorylation of Smad1/5/8, thereby promoting Runx2 expression and differentiation in osteoblasts. Our findings describe novel molecular mechanisms in S1P/S1PR2-mediated osteoblast differentiation that could aid future studies of bone regeneration.

DOI： 10.1016/j.bone.2016.09.003

Language：English   Publishing type：Research paper (scientific journal)  

The infiltration of macrophages into adipose tissue and their interaction with adipocytes are essential for the chronic low-grade inflammation of obese adipose tissue. In this study, we identified the serum amyloid A3 (Saa3) gene as a key adipocyte-derived factor that is affected by interaction with macrophages. We showed that the Saa3 promoter in adipocytes actually responds to activated macrophages in a co-culture system. Decreasing C/EBPβ abundance in 3T3-L1 adipocytes or point mutation of C/EBPβ elements suppressed the increased promoter activity in response to activated macrophages, suggesting an essential role of C/EBPβ in Saa3 promoter activation. Bioluminescence based on Saa3 promoter activity in Saa3-luc mice was promoted in obese adipose tissue, showing that Saa3 promoter activity is most likely related to macrophage infiltration. This study suggests that the level of expression of the Saa3 gene could be utilized for the number of infiltrated macrophages in obese adipose tissue.

DOI： 10.1038/srep38697

Language：English   Publishing type：Research paper (scientific journal)  

Mechanical reduction of infectious bacteria by using physical instruments is considered the principal therapeutic strategy for periodontal disease; addition of antibiotics is adjunctive. However, local antibiotic treatment, combined with conventional mechanical debridement, has recently been shown to be more effective in periodontitis subjects with type 2 diabetes. This suggests that some bacteria may invade the inflamed inner gingival epithelium, and mechanical debridement alone will be unable to reduce these bacteria completely. Therefore, we tried to establish infected organ culture models that mimic the inner gingival epithelium and aimed to see the effects of antibiotics in these established models. Mouse dorsal skin epithelia were isolated, and periodontal bacteria were injected into the epithelia. Infected epithelia were incubated with test antibiotics, and colony‐forming ability was evaluated. Results indicated that effective antibiotics differed according to injected bacteria and the bacterial combinations tested. Overall, in organ culture model, the combination of amoxicillin or cefdinir and metronidazole compensate for the effects of less effective bacterial combinations on each other. This in vitro study would suggest effective periodontal treatment regimens, especially for severe periodontitis.

Language：English  

Mechanical reduction of infectious bacteria by using physical instruments is considered the principal therapeutic strategy for periodontal disease; addition of antibiotics is adjunctive. However, local antibiotic treatment, combined with conventional mechanical debridement, has recently been shown to be more effective in periodontitis subjects with type 2 diabetes. This suggests that some bacteria may invade the inflamed inner gingival epithelium, and mechanical debridement alone will be unable to reduce these bacteria completely. Therefore, we tried to establish infected organ culture models that mimic the inner gingival epithelium and aimed to see the effects of antibiotics in these established models. Mouse dorsal skin epithelia were isolated, and periodontal bacteria were injected into the epithelia. Infected epithelia were incubated with test antibiotics, and colony‐forming ability was evaluated. Results indicated that effective antibiotics differed according to injected bacteria and the bacterial combinations tested. Overall, in organ culture model, the combination of amoxicillin or cefdinir and metronidazole compensate for the effects of less effective bacterial combinations on each other. This in vitro study would suggest effective periodontal treatment regimens, especially for severe periodontitis.

Language：English   Publishing type：Research paper (scientific journal)  

In this study, we investigated the involvement of Wnt signaling in sphingosine-1-phosphate (S1P)-enhanced osteogenic differentiation of C3H10T1/2 pluripotent stem cells. We found that S1P enhanced the expression of Wnt5a and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) during osteogenic differentiation. Wnt5a-neutralizing antibody inhibited S1P-enhanced expression of LRP5/6 and alkaline phosphatase, which are essential for osteogenic differentiation. Conversely, S1P did not affect endogenous canonical Wnt signaling. Taken together, S1P-enhanced Wnt5a promotes LRP5/6 expression, resulting in the trigger of osteogenic differentiation of C3H10T1/2 cells. These findings suggest a potential beneficial role for S1P in bone regeneration.

DOI： 10.1002/cbin.10652

Language：English   Publishing type：Research paper (scientific journal)  

Interleukin-17A (IL-17A) is known to induce inflammatory responses and to be involved in the pathogenesis of not only autoimmune diseases, but also several metabolic and infectious diseases. In this study, IL-17A is shown to induce IL-6 expression in 3T3-L1 mature adipocytes. Interestingly, we found that IL-17A synergistically amplified TNFα-induced secretion of IL-6 and upregulation of IL-17RA expression in 3T3-L1 adipocytes. Its synergistic effects on IL-6 production were inhibited by pre-treatment with inhibitors of IκBα and JNK. Furthermore, IL-17A cooperatively enhanced LPS-mediated IL-6 production in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. In addition, IL-17A also enhanced CCL20 production in 3T3-L1 adipocytes stimulated with TNFα or co-cultured with LPS-stimulated RAW macrophages. In high-fat diet-fed mouse epididymal adipose tissues, IL-17RA and RORγt mRNA levels were significantly increased and the serum level of CCL20 was also upregulated. Taken together, these data show that, in adipose tissues, IL-17A contributes to exacerbating insulin resistance-enhancing IL-6 production and promotes the infiltration of Th17 cells in cooperation with TNFα; these findings represent a novel hypothesis for the association between IL-17A-producing cells and type 2 diabetes.

DOI： 10.1016/j.bbrc.2016.06.049

Language：English   Publishing type：Research paper (scientific journal)  

The effect of macrolides on the superoxide (O₂ ⁻) production by neutrophils was studied. Resting neutrophils become primed by lipopolysaccharide (LPS) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), and primed neutrophils generate O₂ ⁻ in response to fMLP or adhesion, respectively. Both LPS-primed fMLP-stimulated O₂ ⁻ generation by macrolide-treated neutrophils and adhesion-stimulated O₂ ⁻ generation by macrolide-treated fMLP-primed neutrophils were inhibited. Macrolide inhibition of O₂ ⁻ generation was dependent on serum or pH. Serum could be substituted by NaHCO₃. The intensity of inhibition was azithromycin = roxithromycin > clarithromycin > erythromycin, in that order. Non-antimicrobial derivatives of erythromycin, that is, EM703 and EM900, inhibited O₂ ⁻ generation at pH 7.4. NH₄Cl abolished the activity of azithromycin (AZ) only when added to neutrophils with AZ but not after incubation with AZ, suggesting that NH₄Cl prevented the influx of AZ. AZ did not affect the expression of alkaline phosphatase, CD11b, and cytochrome b₅₅₈ in both resting and LPS-primed neutrophils. These results suggested that macrolides did not affect granule mobilization but inhibited O₂ ⁻ generation selectively.

DOI： 10.1007/s10753-016-0333-3

Language：English   Publishing type：Research paper (scientific journal)  

We studied the interaction of LPS with albumin, hemoglobin or high-density lipoprotein (HDL), and whether the interaction affected the activity of LPS on neutrophils. These proteins disaggregated LPS, depending upon temperature and LPS:protein ratio. Albumin-treated LPS was absorbed by immobilized anti-albumin antibody and was eluted with Triton X-100, indicating that LPS formed a hydrophobic complex with albumin. Rd mutant LPS was not disaggregated by the proteins, and did not form a complex with the proteins. But triethylamine-treated Rd mutant LPS formed complexes. When LPS was incubated with an equal concentration of albumin and with polymyxin B (PMXB), PMXB-LPS-protein three-way complexes were formed. After removal of PMXB, the complexes consisted of 11-15 LPS monomers bound to one albumin or hemoglobin molecule. LPS primed neutrophils for enhanced release of formyl peptide-stimulated superoxide, in a serum- and LPS-binding protein (LBP)-dependent manner. Although LPS plus LBP alone did not prime neutrophils, albumin-, hemoglobin- or HDL-treated LPS primed neutrophils when added with LBP. Triethylamine-treated Rd mutant LPS primed neutrophils only when incubated with one of the proteins and with LBP. Thus, in addition to LBP, disaggregation and complex formation of LPS with one of these proteins is required for LPS to prime neutrophils.

DOI： 10.1093/femspd/ftw003

Language：English   Publishing type：Research paper (scientific journal)  

Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose-regulated protein 78 (Grp78) as a new amelogenin-binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1-17, which possesses the most typical characteristics of PDLSCs. Confocal co-localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1-17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration-associated genes in 1-17 cells. Moreover, Grp78 overexpression enhanced rM180-induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin-induced migration in 1-17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin-induced periodontal tissue regeneration. J. Cell. Physiol. 231: 414-427, 2016.

DOI： 10.1002/jcp.25087

Language：English   Publishing type：Research paper (scientific journal)  

Periodontitis is a chronic inflammatory disorder caused by specific bacteria residing in the biofilm, particularly Porphyromonas gingivalis (Pg). Sprouty2 (Spry2) functions as a negative regulator of the fibroblast growth factor (FGF) signaling pathway. We previously demonstrated that sequestration of Spry2 induced proliferation and osteogenesis in osteoblastic cells by basic FGF (bFGF) and epidermal growth factor (EGF) stimulation in vitro, but diminished cell proliferation in gingival epithelial cells. In addition, Spry2 knockdown in combination with bFGF and EGF stimulation increases periodontal ligament cell proliferation and migration accompanied by prevention of osteoblastic differentiation. In this study, we investigated the mechanisms through which Spry2 depletion by interferon (IFN) γ and Pg lipopolysaccharide (LPS) stimulation affected the physiology of macrophages in vitro. Transfection of macrophages with Spry2 small-interfering RNA (siRNA) promoted the expression of genes characteristic of M2 alternative activated macrophages, induced interleukin (IL)-10 expression, and enhanced arginase activity, even in cells stimulated with IFNγ and Pg LPS. In addition, we found that phosphoinositide 3-kinase (PI3K) and AKT activation by Spry2 downregulation enhanced efferocytosis of apoptotic cells by increasing Rac1 activation and decreasing nuclear factor kappa B (NFkB) p65 phosphorylation but not signal transducer and activator of transcription 1 (STAT1) phosphorylation. Collectively, our results suggested that topical administration of Spry2 inhibitors may efficiently resolve inflammation in periodontal disease as macrophage-based anti-inflammatory immunotherapy and may create a suitable environment for periodontal wound healing. These in vitro findings provide a molecular basis for new therapeutic approaches in periodontal tissue regeneration.

DOI： 10.1002/iid3.99

Language：English   Publishing type：Research paper (scientific journal)  

Resistin-like molecule β (RELMβ) reportedly has multiple functions including local immune responses in the gut. In this study, we investigated the possible contribution of RELMβ to non-alcoholic steatohepatitis (NASH) development. First, RELMβ knock-out (KO) mice were shown to be resistant to methionine-choline deficient (MCD) diet-induced NASH development. Since it was newly revealed that Kupffer cells in the liver express RELMβ and that RELMβ expression levels in the colon and the numbers of RELMβ-positive Kupffer cells were both increased in this model, we carried out further experiments using radiation chimeras between wild-type and RELMβ-KO mice to distinguish between the contributions of RELMβ in these two organs. These experiments revealed the requirement of RELMβ in both organs for full manifestation of NASH, while deletion of each one alone attenuated the development of NASH with reduced serum lipopolysaccharide (LPS) levels. The higher proportion of lactic acid bacteria in the gut microbiota of RELMβ-KO than in that of wild-type mice may be one of the mechanisms underlying the lower serum LPS level the former. These data suggest the contribution of increases in RELMβ in the gut and Kupffer cells to NASH development, raising the possibility of RELMβ being a novel therapeutic target for NASH.

DOI： 10.1038/srep20157

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Clinically, irreversible pulpitis is treated by the complete removal of pulp tissue followed by replacement with artificial materials. There is considered to be a high potential for autologous transplantation of human dental pulp stem cells (DPSCs) in endodontic treatment. The usefulness of DPSCs isolated from healthy teeth is limited. However, DPSCs isolated from diseased teeth with irreversible pulpitis (IP-DPSCs) are considered to be suitable for dentin/pulp regeneration. In this study, we examined the stem cell potency of IP-DPSCs. In comparison with healthy DPSCs, IP-DPSCs expressed lower colony-forming capacity, population-doubling rate, cell proliferation, multipotency, in vivo dentin regeneration, and immunosuppressive activity, suggesting that intact IP-DPSCs may be inadequate for dentin/pulp regeneration. Therefore, we attempted to improve the impaired in vivo dentin regeneration and in vitro immunosuppressive functions of IP-DPSCs to enable dentin/pulp regeneration. Interferon gamma (IFN-γ) treatment enhanced in vivo dentin regeneration and in vitro T cell suppression of IP-DPSCs, whereas treatment with tumor necrosis factor alpha did not. Therefore, these findings suggest that IFN-γ may be a feasible modulator to improve the functions of impaired IP-DPSCs, suggesting that autologous transplantation of IFN-γ-accelerated IP-DPSCs might be a promising new therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment.

DOI： 10.1038/srep19286

Language：English   Publishing type：Research paper (scientific journal)  

Objectives: We previously demonstrated that a dominant-negative Sprouty2 (Spry2) mutation promotes osteoblast proliferation and differentiation after basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulation, whereas it diminishes proliferation of gingival epithelial cells, thereby inducing favourable conditions for periodontal tissue regeneration. In this study, we investigated how Spry2 inhibition affects the cellular physiology of periodontal ligament (PDL) cells. Methods: A total of 1-17 PDL cells (multipotent clonal human PDL cell line) were stimulated with bFGF and EGF after transfection of Spry2 siRNA. Cell proliferation, migration, ALP staining, real-time PCR, Western blot and immunofluorescence assays were performed. Results: ERK1/2 activation and proliferation of 1-17 PDL cells were significantly upregulated by the addition of Spry2 siRNA in the presence of bFGF and EGF. In addition, Spry2 siRNA reduced transcription of osteogenesis-related genes and ALP staining relative to control cells. Furthermore, it increased AKT/phosphatidylinositol 3-kinase (PI3K) phosphorylation; consequently, Rac1 but not Cdc42 was activated, thereby promoting lamellipodia formation, cell proliferation and migration after stimulation by bFGF and EGF. Conclusion: Spry2 combined with bFGF and EGF stimulation reduced PDL cell migration and proliferation with inducing osteoblastic differentiation. These in vitro findings may provide a molecular basis for novel therapeutic approaches for establishing periodontal tissue regeneration.

DOI： 10.1111/odi.12369

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2169/naika.104.1907

Language：English   Publishing type：Research paper (scientific journal)  

Objective Several chemokines play important roles in recruiting the monocyte/macrophage lineage into adipose tissues. We previously found CCL19 was highly expressed in adipocytes cocultured with macrophages stimulated by endotoxin. This study aimed to evaluate the role of CCL19-CCR7 axis on obesity and insulin resistance. Methods Serum CCL19 concentration was examined in obese model mice challenged by endotoxin. CCL19 receptor-null, Ccr7^-/-, mice and wild-type mice fed a high-fat diet or normal diet were used to investigate the role of CCL19 signals on obesity-associated inflammation. Results CCL19 protein was elevated in the sera of obese model mice challenged by endotoxin. Ccr7^-/- mice were protected from diet-induced obesity and insulin resistance. The adipose tissue and liver expression of inflammatory genes of Ccr7^-/- mice was much lower than in diet-induced obese mice. Ccr7^-/- mice were protected from fatty liver and dyslipidemia and exhibited increased thermogenesis on high-fat feeding. CCL19 attracts activated dendritic cells (DC). The expression of the DC markers, CD11b and 11c, was not observed in the adipose tissues of Ccr7^-/- mice fed a high-fat diet, which might be closely associated with the protection of these mice from obesity. Conclusions The CCL19-CCR7 pathway associates with the development of high-fat-induced obesity and insulin resistance.

DOI： 10.1002/oby.21127

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Background: DPP-4 inhibitors reportedly exert effects on both alpha and beta cells, and promote the proliferation and survival of beta cells. We investigated the effects of anagliptin on structurally-impaired islets of Langerhans in streptozotocin (STZ)-treated mice, fed either a normal or a high-fat diet. Pdx-1 expression in the pancreas and serum insulin/glucagon concentrations were also examined. Findings: Anagliptin treatment significantly up-regulated pancreatic Pdx-1 expression, with elevated serum glucagon-like peptide-1 concentrations, regardless of whether the diet was normal or high-fat. However, interestingly, the beta cell regeneration, structural normalization of islets of Langerhans including alpha cell: beta cell area ratios, and serum insulin elevation, all observed with anagliptin administration in the animals fed a normal diet, were markedly suppressed in the high-fat fed group. Conclusions: High-fat diet feeding clearly weakened the regenerative effects of anagliptin on the islets of Langerhans in STZ-treated mice. Our findings suggest the importance of normalizing lipid metabolism for full manifestation of DPP-4 inhibitor effects on the islets of Langerhans.

DOI： 10.1186/s13098-015-0047-y

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Objective Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). PP which contains tandem serine/asparatic acid rich repeats (SDrr) is known to enhance dentin mineralization. The nucleotide sequences coding SDrr are identified in the DSPP genes of toothed animals and the length variations of SDrr between intra- and inter-species have been reported. However, it remains unknown about the relationship between the length variations in SDrr and the functions of PP in matrix mineralization. Design By utilizing a mammalian expression system, we generated several recombinant PP proteins (rPP) containing SDrr of different lengths and analyzed their effects on the precipitation of calcium phosphate with an in vitro gel diffusion system. Results rPP-Δ37.6 SDrr and rPP-Δ63.5 SDrr, which possessed shortened SDrr that accounted for 62.4 and 36.5% the length of SDrr in full-length rPP (rPP-full), respectively, induced the precipitation of calcium phosphate similar to that of rPP-full at the same molar concentration, whereas rPP-ΔSDrr, in which SDrr were flipped, did not. Furthermore, rPP-Δ63.5 SDrr significantly increased the accumulation of calcium compared with rPP-full at adjusted concentrations so that the same amounts of SDrr were embedded. The results of an ELISA analysis indicated that the amounts of rPP-Δ37.6 SDrr and rPP-Δ63.5 SDrr secreted from transfected cells were 5.2- and 7.1-fold greater than that of rPP-full, respectively. Conclusions The generated rPP-Δ63.5 SDrr which can be substituted for rPP-full may be a candidate for a therapeutic molecule to facilitate hard tissue generation such as reparative dentin formation.

DOI： 10.1016/j.archoralbio.2015.05.013

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Sprouty was identified as an inhibitor of the fibroblast growth factor (FGF) receptor, and Sprouty2 (Spry2) functions as a negative regulator of receptor tyrosine kinase signaling. In this study, we investigated how inhibition of Spry2 affects osteoblasts and gingival epithelial cells in periodontal tissue regeneration in vitro. Transduction of a dominant-negative mutant of Spry2 (Y55A-Spry2) enhanced basic fibroblast growth factor (bFGF)- and epidermal growth factor (EGF)-induced ERK activation in MC3T3-E1 osteoblastic cells. In contrast, it decreased their activation in GE1 cells. Consistent with these observations, Y55A-Spry2 increased osteoblast proliferation with bFGF and EGF stimulation, whereas the proliferation of Y55A-Spry2-introduced GE1 cells was decreased via the ubiquitination and degradation of EGF receptors (EGFRs). In addition, Y55A-Spry2 caused upregulation of Runx2 expression and downregulation of Twist, a negative regulator of Runx2, with treatment of bFGF and EGF, resulting in enhanced osteoblastogenesis accompanied by alkaline phosphatase activation and osteocalcin expression in MC3T3-E1 cells. These data suggest that suppression of Spry2 expression induces proliferation and differentiation of osteoblastic cells after the addition of a bFGF and EGF cocktail but inhibits proliferation in gingival epithelial cells. These in vitro experiments may provide a molecular basis for novel therapeutic approaches in periodontal tissue regeneration. Taken together, our study proposes that combined application of an inhibitor for tyrosine 55 of Spry2, bFGF, and EGF may effectively allow alveolar bone growth and block the ingrowth of gingival epithelial cells toward bony defects, biologically mimicking a barrier effect in guided tissue regeneration, with in vivo investigation in the future. J. Cell. Biochem. 116: 628-639, 2015.

DOI： 10.1002/jcb.25014

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Several lines of evidence have suggested a role of gut microbiota in the etiology of nonalcoholic steatohepatitis (NASH). NASH subjects reportedly showed a prolonged orocecal transit time coexistent with small intestinal bacterial overgrowth. We considered the possibility that enhanced gastrointestinal motility would influence gut microbiota and thus investigated the effects of the gastroprokinetic agent mosapride citrate (MC) on gut microbiota and the development of NASH using a methionine-choline deficient (MCD) diet-fed rodent model. Mice were divided into three groups, given the normal chow diet (NCD), the MCD diet, or the MCD diet containing 10 mg·kg^–1·day^–1 of MC (MCD plus MC) for 6 wk. NASH development was evaluated based on hepatic histochemical findings, serum parameters and various mRNA and/or protein expression levels. MC treatment suppressed MCD diet-induced NASH development, with reduced serum lipopoly-saccharide and increased plasma glucagon-like peptide-1 (GLP-1) concentrations. Calculation of the relative abundance of each strain based on gut microbiota analyses indicated lactic acid bacteria specifically, such as Bifidobacterium and Lactobacillus, in feces to be decreased in the MCD, compared with the NCD group. Interestingly, the reduction in lactic acid bacteria in the MCD diet group was reversed in the MCD plus MC group. In addition, colon inflammation observed in the MCD diet group was reduced in the MCD plus MC group. Therefore, MC showed a protective effect against MCD diet-induced NASH development in our rodent model, with possible involvements of increased fecal lactic acid bacteria, protection against colon inflammation and elevated plasma GLP-1.

DOI： 10.1152/ajpgi.00198.2014

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It remains unclear whether serum antibody titer against Porphyromonas gingivalis (Pg) and inflammatory components lead to periodontal deterioration in each gender, as periodontal and systemic status is influenced by gender. The present study investigates the gender-specific probable effects of titer against Pg and inflammatory markers on periodontal health status in a longitudinal study. A retrospective study design was used. At two time points over an 8-year period (in 2003 and 2011), 411 individuals (295 males with a mean age of 57.6 ± 11.2 years and 116 females with a mean age of 59.2 ± 10.3 years) were surveyed. Periodontal status, serum antibody titer against Pg, and high-sensitive C-reactive protein (hsCRP) were evaluated. Poisson regression analyses revealed that the elevated titer against Pg and hsCRP significantly predicted the persistence of periodontal disease 8 years later in females with periodontal disease in 2003. Elevated hsCRP was significantly associated with the incidence of periodontal disease 8 years later in females who were periodontally healthy in 2003. Males had a weaker association among titer against Pg, inflammatory markers, and periodontal disease. These findings suggest that immune response to Pg infection in addition to inflammatory components affects periodontal deterioration in females.

DOI： 10.1155/2015/897971

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Dipeptidyl peptidase IV (DPPIV) expression in visceral adipose tissue is reportedly increased in obese patients, suggesting an association of DPP-IV with inflammation. In this study, first, lipopolysaccharide (LPS)- or palmitateinduced elevations of inflammatory cytokine mRNA expressions in RAW264.7 macrophages were shown to be significantly suppressed by coincubation with a DPP-IV inhibitor, anagliptin (10 μM), despite low DPP-IV expression in the RAW264.7 cells. Regarding the molecular mechanism, LPS-induced degradation of IκBα and phosphorylations of p65, JNK, and p38, as well as NF- κB and AP-1 promoter activities, were revealed to be suppressed by incubation with anagliptin, indicating suppressive effects of anagliptin on both NF- κB and AP-1 signaling pathways. Anagliptin also acted on 3T3-L1 adipocytes, weakly suppressing the inflammatory cytokine expressions induced by LPS and TNFα. When 3T3-L1 and RAW cells were cocultured and stimulated with LPS, the effects of anagliptin on the suppression of cytokine expressions in 3T3-L1 adipocytes were more marked and became evident at the 10 μM concentration. Anti-inflammatory effects of anagliptin were also observed in vivo on the elevated hepatic and adipose expressions and serum concentrations of inflammatory cytokines in association with the suppression of hepatic NF- κB transcriptional activity in LPS-infused mice. Taking these observations together, the anti-inflammatory properties of anagliptin may be beneficial in terms of preventing exacerbation of diabetes and cardiovascular events.

DOI： 10.1152/ajpendo.00553.2014

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Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). Gene duplications in the ancestor dentin matrix protein-1 (DMP-1) genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Many SIBLING members have been shown to evoke various cell responses through the integrinbinding Arg-Gly-Asp (RGD) domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-⁴⁶³SDESDTNSESANESGSRGDA⁴⁸²-OH) was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-⁴⁷⁸SRGDASYTSDESSDDDNDSDSH⁴⁹⁹-OH). This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala⁴⁸². Furthermore, various point mutations in Ala⁴⁸² and/or Ser⁴⁸³ converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala⁴⁸²-Ser⁴⁸³ flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PPRGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin-mediated signaling molecule.

DOI： 10.1371/journal.pone.0112490

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DOI： 10.1007/s00520-014-2432-8

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DOI： 10.1016/j.jebdp.2014.04.017

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Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca²⁺ utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca²⁺, the additive effects of Ca²⁺-promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca²⁺-promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A. actinomycetemcomitans cells grown in the presence of 1 mm CaCl₂ exhibited enhanced cell aggregation and increased levels of cell-associated Ca²⁺. Biofilm-derived cells grown in the presence of Ca²⁺ exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca²⁺-promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A. actinomycetemcomitans to adapt to its natural environment. The Ca²⁺-promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.

DOI： 10.1111/omi.12046

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BackgroundChronic kidney disease (CKD) is an important risk factor for coronary heart disease, and previous studies indicated the involvement of low-grade inflammation in the pathogenesis of CKD.MethodsThe study was designed to (i) identify and confirm genes and their products upregulated in mesangial cells cocultured with endotoxin-stimulated macrophages and (ii) determine the clinical relevance of genes and proteins upregulated in mesangial cells under inflammatory conditions by an epidemiological approach.ResultsDNA microarray analysis revealed upregulated expression of many genes and their products including several cytokines and chemokines, as well as the inflammatory marker, lipocalin 2 gene. The gene expression and protein upregulation of lipocalin 2 were synergistically affected by endotoxin and tumor necrosis factor (TNF)-α stimulation. In human studies, lipocalin 2 level was significantly associated with creatinine (r = 0.419, P < 0.001) and negatively associated with eGFR (r = -0.365, P < 0.001). Multiple logistic regression analysis revealed a significant association between lipocalin 2 and soluble tumor necrosis factor receptor 2 (sTNF-R2), eGFR and uric acid in general subjects attending regular annual medical check-up (n = 420). When subjects with diabetes were excluded from the analysis, lipocalin 2 remained associated with sTNF-R2, eGFR and uric acid.ConclusionsSince an activated TNF system, as demonstrated by elevated sTNF-R2, and elevated uric acid were recently implicated in an elevated CKD risk, we conclude that inflammation could play an important role in the pathogenesis of CKD, and that lipocalin 2 is a potential universal marker for impaired kidney function. Furthermore, the results obtained by the current microarray analysis could improve the understanding of gene profiles associated with the pathophysiology of CKD under inflammatory conditions.

DOI： 10.1093/ndt/gft449

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Mesenchymal stem cells (MSCs) can differentiate into a number of cell types, including adipocytes and osteoblasts. MSC differentiation into adipocytes inhibits osteogenic differentiation and vice versa. Therefore, understanding the mechanisms of MSC differentiation at the signaling level can lead to the development of novel therapeutic strategies toward tissue regeneration. Sphingosine-1-phosphate (S1P) is a signaling molecule that regulates many cellular responses, including cellular differentiation. However, the effects of S1P on MSC differentiation are largely unknown. The purpose of study was to investigate whether S1P drives MSCs toward either adipogenic or osteogenic differentiation, and if so, to clarify the underlying signaling mechanisms for such differentiation. We found that S1P inhibited adipogenic differentiation of C3H10T1/2 multipotent stem cells, while promoting their osteogenic differentiation. During adipogenic differentiation, S1P suppressed the cAMP accumulation in a Gi-protein-dependent manner. The Gi-dependent S1P signaling suppressed C/EBPβ expression, which is essential for adipogenic differentiation. Furthermore, S1P did not affect cAMP-independent adipogenic differentiation. These findings suggest that S1P suppresses cAMP accumulation, leading to inhibition of C/EBPβ expression, thereby resulting in decreased adipogenic differentiation of C3H10T1/2 cells. Thus, our findings provide novel molecular mechanisms as regards how S1P inhibits adipogenic differentiation of C3H10T1/2 cells, indicating a potential beneficial role for regeneration and repair of tissues.

DOI： 10.1007/s11010-014-2290-1

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Background: Azithromycin is an antibiotic belonging to the macrolides. Previous case reports showed that azithromycin has a regenerative effect on periodontal tissue in addition to improving periodontal gingival inflammation. Recently, we experienced three periodontitis cases, all of which showed severe bone loss. However, their gingival inflammatory signs differed greatly. The present case reports evaluated the regenerative effects of azithromycin on periodontitis sites with different clinical signs of gingival inflammation. Methods: In Case 1, generalized chronic periodontitis with severe gingival inflammation was treated with azithromycin before periodontal treatment. In contrast, Case 2 presented with few clinical signs of gingival inflammation, but was treated with azithromycin prescribed within a day of scaling and root planing. In Case 3, teeth with moderate gingival inflammation were treated with azithromycin after a series of scaling and root planing. Results: Remarkable alveolar bone growth, regardless of baseline gingival inflammation, was noted in all three cases. Conclusions: The use of adjunctive azithromycin in scaling and root planing may be effective for periodontal tissue regeneration. This property may be independent of the degree of baseline gingival inflammation.

DOI： 10.1111/adj.12177

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Summary: Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are major periodontal pathogens that cause several types of periodontal disease. Our previous study suggested that P. gingivalis gingipains secreted in the subgingival environment are related to the detachment of A.actinomycetemcomitans biofilms. However, it remains unclear whether arginine-specific cysteine proteinase (Rgp) and lysine-specific proteinase (Kgp) play different roles in the detachment of A. actinomycetemcomitans biofilm. The aim of this study was to investigate possible disruptive roles of Kgp and Rgp in the aggregation and attachment of A. actinomycetemcomitans. While P. gingivalis ATCC33277 culture supernatant has an ability to decrease autoaggregation and coaggregation of A. actinomycetemcomitans cells, neither the boiled culture supernatant of ATCC33277 nor the culture supernatant of KDP136 showed this ability. The addition of KYT-1 and KYT-36, specific inhibitors of Rgp and Kgp, respectively, showed no influence on the ability of P. gingivalis culture supernatant. The result of gelatin zymography suggested that other proteases processed by gingipains mediated the decrease of A. actinomycetemcomitans aggregations. We also examined the biofilm-destructive effect of gingipains by assessing the detachment of A. actinomycetemcomitans from polystyrene surfaces. Scanning electron microscope analysis indicated that A. actinomycetemcomitans cells were detached by P. gingivalis Kgp. The quantity of A. actinomycetemcomitans in biofilm was decreased in co-culture with P. gingivalis. However, this was not found after the addition of KYT-36. These findings suggest that Kgp is a critical component for the detachment and decrease of A. actinomycetemcomitans biofilms.

DOI： 10.1111/omi.12051

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Macrophage infiltration in the adipose tissue, and the interaction with adipocytes, is well documented to be involved in fat inflammation and obesity-associated complications. In this study, we isolated IκB kinase ε (IKKε) as a key adipocyte factor that is potentially affected by interaction with macrophages in adipose tissue in vivo. We showed that IKKε mRNA expression levels in white adipose tissue were increased in both genetic and diet-induced obese mouse. Furthermore, IKKε mRNA expression was decreased by the administration of vitamin B6, an anti-inflammatory vitamin, and that IKKε expression levels in adipose tissue were closely correlated with the numbers of infiltrating macrophages. In a co-culture system, we showed that IKKaε expression in adipocytes was upregulated by interaction with activated macrophages. This study provides novel insight into IKKε, which is involved in adipose tissue inflammation during the development of obesity.

DOI： 10.1080/09168451.2014.925776

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Aim: Chronic kidney disease (CKD) is associated with cardiovascular events. Tumor necrosis factor (TNF) and/or its receptors have been postulated to be involved in renal pathophysiology. It is unclear whether an increased TNF system activity is present before the development of apparent CKD. Methods: Four hundred and twenty non-diabetic Japanese subjects with an estimated GFR (eGFR) greater than 60 ml/min/1.73 m² were recruited for measurement of the HbA1c, insulin, TNF system activity (TNF-α, soluble TNF receptor 1 (sTNF-R1) and sTNF-R2) levels and various parameters, including the lipid, high-sensitivity C-reactive protein (hsCRP), high-molecular-weight (HMW) adiponectin and leptin levels. The subjects were stratified according to the eGFR: the G1 level (eGFR ≥90 ml/min/1.73 m²) and the G2 level (90 <eGFR ≥60 ml/min/1.73 m²). Results: Whereas no significant differences were observed in gender, body mass index (BMI), blood pressure, insulin, TNF-α, hsCRP, HMW adiponectin or leptin between the two groups, the values for age, HbA1c, triglycerides, sTNF-R1 and sTNF-R2 were significantly higher in the subjects with a G2 level of eGFR than in those with a G1 level. In contrast, the HDL cholesterol levels were significantly lower in the subjects with a G2 level than in those with a G1 level. Linear negative correlations were also observed between eGFR and age, BMI, HbA1c, triglycerides, sTNF-R1 and sTNFR2, respectively. A multiple logistic regression analysis revealed that only sTNF-R2 was associated with the presence of a G2 level of eGFR (Odds ratio 1.092, 95% CI 1.013-1.177, P= 0.021). Conclusions: The circulating sTNF-R2 level is closely associated with the kidney function in nondiabetic Japanese subjects.

DOI： 10.5551/jat.21055

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Gut microbiota alterations are associated with various disorders. In this study, gut microbiota changes were investigated in a methionine-choline-deficient (MCD) dietinduced nonalcoholic steatohepatitis (NASH) rodent model, and the effects of administering Lactobacillus casei strain Shirota (LcS) on the development of NASH were also investigated. Mice were divided into three groups, given the normal chow diet (NCD), MCD diet, or the MCD diet plus daily oral administration of LcS for 6 wk. Gut microbiota analyses for the three groups revealed that lactic acid bacteria such as Bifidobacterium and Lactobacillus in feces were markedly reduced by the MCD diet. Interestingly, oral administration of LcS to MCD diet-fed mice increased not only the L. casei subgroup but also other lactic acid bacteria. Subsequently, NASH development was evaluated based on hepatic histochemical findings, serum parameters, and various mRNA and/or protein expression levels. LcS intervention markedly suppressed MCD-diet-induced NASH development, with reduced serum lipopolysaccharide concentrations, suppression of inflammation and fibrosis in the liver, and reduced colon inflammation. Therefore, reduced populations of lactic acid bacteria in the colon may be involved in the pathogenesis of MCD diet-induced NASH, suggesting normalization of gut microbiota to be effective for treating NASH.

DOI： 10.1152/ajpgi.00225.2013

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Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology. It is used in periodontal surgery to regenerate cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying periodontal regeneration are still unclear. In this study, we investigated the proteins bound to amelogenin, which are suggested to play a pivotal role in promoting periodontal tissue regeneration. To identify new molecules that interact with amelogenin and are involved in osteoblast activation, we employed coupling affinity chromatography with proteomic analysis in fractionated SaOS-2 osteoblastic cell lysate. In SaOS-2 cells, many of the amelogenin-interacting proteins in the cytoplasm were mainly cytoskeletal proteins and several chaperone molecules of heat shock protein 70 (HSP70) family. On the other hand, the proteomic profiles of amelogenin-interacting proteins in the membrane fraction of the cell extracts were quite different from those of the cytosolic-fraction. They were mainly endoplasmic reticulum (ER)-associated proteins, with lesser quantities of mitochondrial proteins and nucleoprotein. Among the identified amelogenin-interacting proteins, we validated the biological interaction of amelogenin with glucose-regulated protein 78 (Grp78/Bip), which was identified in both cytosolic and membrane-enriched fractions. Confocal co-localization experiment strongly suggested that Grp78/Bip could be an amelogenin receptor candidate. Further biological evaluations were examined by Grp78/Bip knockdown analysis with and without amelogenin. Within the limits of the present study, the interaction of amelogenin with Grp78/Bip contributed to cell proliferation, rather than correlate with the osteogenic differentiation in SaOS-2 cells. Although the biological significance of other interactions are not yet explored, these findings suggest that the differential effects of amelogenin-derived osteoblast activation could be of potential clinical significance for understanding the cellular and molecular bases of amelogenin-induced periodontal tissue regeneration.

DOI： 10.1371/journal.pone.0078129

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Background: Platelet-activating factor (PAF) has been implicated in the pathology of neuropathic pain. Previous studies reported that PAF receptor (PAF-R) antagonists have varied anti-allodynia effects by route of administration and nerve injury models in rats. Methods: The present study elucidated the effectiveness of PAF antagonists against neuropathic pain in four different models of peripheral nerve injury and provided insights into the mode of anti-allodynia action. Results: PAF antagonists, TCV-309, BN 50739 and WEB 2086 by intravenous (i.v.) and oral administration have potent and long-lasting anti-allodynia action in mice neuropathic pain models. Treatment with PAF antagonists before surgery delayed the initiation of allodynia until the effects of these treatments were abolished. Intrathecal (i.t.) injection of the PAF antagonists and siRNA against PAF receptor ameliorated allodynia. I.t. injection of the glycine receptor (GlyR)α3 siRNA reduced the anti-allodynia effect of PAF antagonists. This evidence suggests that the anti-allodynia effect of PAF antagonists is at least in part mediated by spinal relief of PAF-induced dysfunction of GlyRα3. An analysis of the mode of anti-allodynia action of TCV-309 in vivo revealed a competitive action against PAF shortly after the injection of TCV-309, converting to a non-competitive action later. Conclusions: The present results revealed the effectiveness in anti-allodynia of PAF antagonists in different nerve injury models, and the unique mode of action; long-lasting anti-allodynia effects mediated by spinal GlyRa3 with a competitive manner at the initial stage and the following non-competitive manner of inhibition.

DOI： 10.1002/j.1532-2149.2013.00289.x

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OBJECTIVE - : Resistin-like molecule (RELM) β is a secretory protein homologous to resistin and reportedly contributes to local immune response regulation in gut and bronchial epithelial cells. However, we found that activated macrophages also express RELMβ and thus investigated the role of RELMβ in the development of atherosclerosis. APPROACH AND RESULTS - : It was demonstrated that foam cells in atherosclerotic lesions of the human coronary artery abundantly express RELMβ. RELMβ knockout ( ^-/-) and wild-type mice were mated with apolipoprotein E-deficient background mice. RELMβ^-/- apolipoprotein E-deficient mice exhibited less lipid accumulation in the aortic root and wall than RELMβ^+/+ apolipoprotein E-deficient mice, without significant changes in serum lipid parameters. In vitro, RELMβ^-/- primary cultured peritoneal macrophages (PCPMs) exhibited weaker lipopolysaccharide- induced nuclear factor-κB classical pathway activation and inflammatory cytokine secretion than RELMβ^+/+, whereas stimulation with RELMβ upregulated inflammatory cytokine expressions and increased expressions of many lipid transporters and scavenger receptors in PCPMs. Flow cytometric analysis revealed inflammatory stimulation-induced RELMβ in F4/80(+) CD11c(+) PCPMs. In contrast, the expressions of CD11c and tumor necrosis factor were lower in RELMβ^-/- PCPMs, but both were restored by stimulation with recombinant RELMβ. CONCLUSIONS - : RELMβ is abundantly expressed in foam cells within plaques and contributes to atherosclerosis development via lipid accumulation and inflammatory facilitation.

DOI： 10.1161/ATVBAHA.113.301546

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Pin1 and Par14 are parvulin-type peptidyl-prolyl cis/trans isomerases. Although numerous proteins have been identified as Pin1 substrates, the target proteins of Par14 remain largely unknown. Par14 expression levels are increased in the livers and embryonic fibroblasts of Pin1KOmice, suggesting a compensatory relationship between the functions of Pin1 and Par14. In this study, the association of Par14 with insulin receptor substrate 1 (IRS-1) was demonstrated in HepG2 cells overexpressing both as well as endogenously in the mouse liver. The analysis using deletion-mutated Par14 and IRS-1 constructs revealed the N-terminal portion containing the basic domain of Par14 and the two relatively C-terminal portions of IRS-1 to be involved in these associations, in contrast to the WW domain of Pin1 and the SAIN domain of IRS-1. Par14 overexpression in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events, PI3K binding with IRS-1 and Akt phosphorylation. In contrast, treating HepG2 cells with Par14 siRNA suppressed these events. In addition, overexpression of Par14 in the insulin-resistant ob/ob mouse liver by adenoviral transfer significantly improved hyperglycemia with normalization of hepatic PEPCK and G6Pase mRNA levels, and gene suppression of Par14 using shRNA adenovirus significantly exacerbated the glucose intolerance in Pin1 KO mice. Therefore, although Pin1 and Par14 associate with different portions of IRS-1, the prolyl cis/trans isomerization in multiple sites of IRS-1 by these isomerases appears to be critical for efficient insulin receptor-induced IRS-1 phosphorylation. This process is likely to be one of the major mechanisms regulating insulin sensitivity and also constitutes a potential therapeutic target for novel insulin-sensitizing agents.

DOI： 10.1074/jbc.M113.485730

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The dynamic process of adipose differentiation involves stepwise expressions of transcription factors and proteins specific to the mature fat cell phenotype. In this study, it was revealed that expression levels of IntS6 and IntS11, subunits of the Integrator complex, were increased in 3T3-L1 cells in the period when the cells reached confluence and differentiated into adipocytes, while being reduced to basal levels after the completion of differentiation. Suppression of IntS6 or IntS11 expression using siRNAs in 3T3-L1 preadipocytes markedly inhibited differentiation into mature adipocytes, based on morphological findings as well as mRNA analysis of adipocyte-specific genes such as Glut4, perilipin and Fabp4. Although Pparγ2 protein expression was suppressed in IntS6 or IntS11-siRNA treated cells, adenoviral forced expression of Pparγ2 failed to restore the capacity for differentiation into mature adipocytes. Taken together, these findings demonstrate that increased expression of Integrator complex subunits is an indispensable event in adipose differentiation. Although further study is necessary to elucidate the underlying mechanism, the processing of U1, U2 small nuclear RNAs may be involved in cell differentiation steps.

DOI： 10.1016/j.bbrc.2013.03.029

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Aims: Periodontal treatment reduces glycated hemoglobin (HbA1) in subjects with type 2 diabetes, although effective strategy for different severities of periodontitis remains unclear. We hypothesized that resolution of periodontitis-induced inflammation by the therapy combined with antibiotics may have beneficial effects on the glycemic control of diabetes. Methods: A total of 523 subjects with type 2 diabetes were screened for periodontal disease. Of these, 160 subjects who visited dentists were divided into two groups according to high-sensitivity c-reactive protein (hsCRP) level: >500. ng/ml and <500. ng/ml. The group with hsCRP over 500. ng/ml was further sub-divided into two groups according to treatment strategy: topical application of antibiotics combined with conventional mechanical debridement (group A), and debridement alone (B). Subjects with hsCRP below 500. ng/ml were sub-divided similarly (C: combination therapy; D: debridement alone). hsCRP was measured after 1 month and changes of HbA1c after 3 months. These parameters were also measured in control subjects (N= 118) who did not visit dentists (E: initial hsCRP > 500. ng/ml; F: hsCRP < 500. ng/ml). Results: A multiple comparison by ANOVA revealed that only group A showed a significant reduction in HbA1c over time (P< 0.001). Multivariable analyses revealed elevated hsCRP and the combination treatment with antibiotics were two independent variables influencing the decrease of HbA1c over the study (P< 0.01 and P< 0.001, respectively). Conclusions: In subjects with type 2 diabetes and periodontitis-induced mild inflammation (hsCRP. > 500. ng/ml), treatment to reduce hsCRP using antibiotics is recommended.

DOI： 10.1016/j.diabres.2013.01.028

Language：English   Publishing type：Research paper (scientific journal)  

Macrophage infiltration into adipose tissue is associated with obesity and the crosstalk between adipocytes and infiltrated macrophages has been investigated as an important pathological phenomenon during adipose tissue inflammation. Here, we sought to identify adipocyte mRNAs that are regulated by interaction with infiltrated macrophages in vivo. An anti-inflammatory vitamin, vitamin B6, suppressed macrophage infiltration into white adipose tissue and altered mRNA expression. We identified >3500 genes whose expression is significantly altered during the development of obesity in db/db mice, and compared them to the adipose tissue mRNA expression profile of mice supplemented with vitamin B6. We identified PTX3 and MMP3 as candidate genes regulated by macrophage infiltration. PTX3 and MMP3 mRNA expression in 3T3-L1 adipocytes was up-regulated by activated RAW264.7 cells and these mRNA levels were positively correlated with macrophage number in adipose tissue in vivo. Next, we screened adipose genes down-regulated by the interaction with macrophages, and isolated RASSF6 (Ras association domain family 6). RASSF6 mRNA in adipocytes was decreased by culture medium conditioned by activated RAW264.7 cells, and RASSF6 mRNA level was negatively correlated with macrophage number in adipose tissue, suggesting that adipocyte RASSF6 mRNA expression is down-regulated by infiltrated macrophages in vivo. Finally, this study also showed that decreased RASSF6 expression up-regulates mRNA expression of several genes, such as CD44 and high mobility group protein HMGA2. These data provide novel insights into the biological significance of interactions between adipocytes and macrophages in adipose tissue during the development of obesity.

DOI： 10.1371/journal.pone.0061931

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Aims: Chronic low-grade inflammation and/or obesity are suggested to induce chronic kidney disease (CKD) in patients with type 2 diabetes. This cross-sectional study was performed to investigate the relationship between inflammatory biomarkers and CKD in non-obese patients with type 2 diabetes. Methods: 106 non-obese Japanese patients with type 2 diabetes were recruited for the measurement of GFR, TNF, HMW adiponectin, leptin, hsCRP and some variables including urinary albumin. BMI, serum creatinine, and urinary albumin levels were 22.2±0.2kg/m² (17.1-24.9kg/m²), 0.76±0.02mg/dl (0.39-1.38mg/dl), 40.4±4.3mg/gCr (1.6-195.0mg/gCr), respectively. They were stratified into two groups based on the value of eGFR: low eGFR (eGFR<60ml/min/1.73m²) and normal eGFR (eGFR>60ml/min/1.73m²). Logistic regression analysis was used for statistical analysis. Results: Whereas univariate logistic regression analysis showed that gender, diabetes duration, triglyceride, HDL cholesterol, uric acid, urinary albumin, and soluble TNF receptors (sTNF-R1, sTNF-R2) are associated with the development of stage 3 CKD, multivariate logistic regression analysis revealed that sTNF-R2 (Odds ratio 1.003, 95% confidence interval 1.000 to 1.005, P= 0.030) showed significant associations with the development of stage 3 CKD. Conclusions: Circulating TNF receptor 2 is an independent risk factor for CKD in non-obese Japanese patients with type 2 diabetes.

DOI： 10.1016/j.diabres.2012.11.002

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Aim: To find possible reagents to minimize inflammatory responses by using an established pulpitis models for the purpose of developing new pulp-capping materials, and to test the possible use of phosphorylated pullulan as a carrier for such an anti-inflammatory reagent. Methodology: Co-culturing was performed using transwell systems. Inflammatory responses were evaluated by measuring cytokines produced by the cells. The effects of two flavonoids, luteolin and quercetin, as anti-inflammatory reagents, and phosphorylated pullulan, which potentially achieves a sufficient marginal sealing to hydroxyapatite and slowly releases luteolin, as a carrier for flavonoids, were tested. Results: Flavonols, particularly luteolin, dramatically attenuated inflammatory cytokine production, which was augmented by co-cultures. Luteolin was successfully enclosed by phosphorylated pullulan. Finally, it was confirmed that luteolin released from phosphorylated pullulan was effective in reducing cytokine production by co-cultures. Conclusions: Combination of phosphorylated pullulan and luteolin could be potentially used in the treatment of dental pulp inflammation.

DOI： 10.1111/j.1365-2591.2012.02095.x

Language：English   Publishing type：Research paper (scientific journal)  

Inflammation involving adipose tissue is regarded as one of the major molecular mechanisms underlying obesity-related insulin resistance. Recent studies have suggested a series of angiotensin II receptor blockers (ARBs) to improve insulin resistance or protect against the development of diabetes mellitus. We previously demonstrated that valsartan suppresses the inflammatory response of macrophages. Interestingly, however, this effect did not occur via peroxisome proliferator-activated receptor (PPAR) γ or the AT1a receptor. This suppression appears to secondarily lead to amelioration of insulin resistance and reductions in abnormal gene expressions in adipocytes. In addition to these in vitro findings, we herein demonstrate the in vivo effects of valsartan, using mice constitutively infused with lipopolysaccharide (LPS) for 4 weeks. Oral administration of valsartan to LPS-infused mice normalized the increased expressions of inflammatory cytokines in adipose and liver tissues. These results raise the possibility that valsartan not only contributes to normalization of obesity-related insulin resistance, but is also beneficial for the treatment of other diseases with inflammation related to the metabolic syndrome such as atherosclerosis and non-alcoholic steatohepatitis. Further study is necessary to clarify these issues.

DOI： 10.4161/adip.21837

Language：English   Publishing type：Research paper (scientific journal)  

Inflammation involving adipose tissue is regarded as one of the major molecular mechanisms underlying obesity-related insulin resistance. Recent studies have suggested a series of angiotensin II receptor blockers (ARBs) to improve insulin resistance or protect against the development of diabetes mellitus. We previously demonstrated that valsartan suppresses the inflammatory response of macrophages. Interestingly, however, this effect did not occur via peroxisome proliferator-activated receptor (PPAR) γ or the AT1a receptor. This suppression appears to secondarily lead to amelioration of insulin resistance and reductions in abnormal gene expressions in adipocytes. In addition to these in γ vitro findings, we herein demonstrate the in vivo effects of valsartan, using mice constitutively infused with lipopolysaccharide (LPS) for 4 weeks. Oral administration of valsartan to LPS-infused mice normalized the increased expressions of inflammatory cytokines in adipose and liver tissues. These results raise the possibility that valsartan not only contributes to normalization of obesity-related insulin resistance, but is also beneficial for the treatment of other diseases with inflammation related to the metabolic syndrome such as atherosclerosis and non-alcoholic steatohepatitis. Further study is necessary to clarify these issues.

DOI： 10.4161/adip.21837

Language：English   Publishing type：Research paper (scientific journal)  

Aim To establish an ex vivo pulpitis model by co-culturing dental pulp cells with macrophages. Methodology As dental pulp cells, immortalized human dental pulp cells, named DP-1, were used, whilst as macrophage cell lines, the differentiated human monocytic cell line, THP-1, was used. In some experiments, primary dental pulp cells were isolated and used to confirm the results obtained in the experiments using immortalized cells. Co-culturing was performed using transwell systems. Inflammatory responses were evaluated by measuring cytokines produced by the cells. Results Co-culturing both cell types markedly up-regulated inflammatory cytokine production as compared with the cells cultured independently, suggesting that both cell types interact with each other to synergistically produce higher amounts of inflammatory cytokines. Interestingly, both DP-1 and primary dental pulp cells appeared to produce molecules stimulating macrophages to produce tumour necrosis factor-α- Conclusion Co-culturing immortalized dental pulp cells and macrophages may be a new ex vivo model for studying the pathophysiology of reversible pulpitis.

DOI： 10.1111/j.1365-2591.2012.02074.x

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Nonalcoholic steatohepatitis (NASH) is a disorder characterized by simultaneous fat accumulation and chronic inflammation in the liver. In this study, Pin1 expression was revealed to be markedly increased in the livers of mice with methionine choline-deficient (MCD) diet-induced NASH, a rodent model of NASH. In addition, Pin1 KO mice were highly resistant to MCD induced NASH, based on a series of data showing simultaneous fat accumulation, chronic inflammation, and fibrosis in the liver. In terms of Pin1-induced fat accumulation, it was revealed that the expression levels of peroxisome proliferator-activated receptor α and its target genes were higher in the livers of Pin1 KO mice than in controls. Thus, resistance of Pin1 KO mice to hepatic steatosis is partially attributable to the lack of Pin1-induced down-regulation of peroxisome proliferator-activated receptor α, although multiple other mechanisms are apparently involved. Another mechanism involves the enhancing effect of hematopoietic Pin1 on the expressions of inflammatory cytokines such as tumor necrosis factor and monocyte chemoattractant protein 1 through NF-κB activation, eventually leading to hepatic fibrosis. Finally, to distinguish the roles of hematopoietic or nonhematopoietic Pin1 in NASH development, mice lacking Pin1 in either nonhematopoietic or hematopoietic cells were produced by bone marrow transplantation between wildtype and Pin1 KO mice. The mice having nonhematopoietic Pin1 exhibited fat accumulation without liver fibrosis on the MCD diet. Thus, hepatic Pin1 appears to be directly involved in the fat accumulation in hepatocytes, whereas Pin1 in hematopoietic cells contributes to inflammation and fibrosis. In summary, this is the first study to demonstrate that Pin1 plays critical roles in NASH development. This report also raises the possibility that hepatic Pin1 inhibition to the appropriate level might provide a novel therapeutic strategy for NASH.

DOI： 10.1074/jbc.M112.397133

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Macrophages in adipose tissue reportedly play a major role in the development of insulin resistance and chronic inflammation associated with obesity. On the other hand, several clinical trials have revealed angiotensin II receptor blockers (ARBs) to improve insulin resistance. In this study, we analyzed the gene expression profile of 3T3-L1 adipocytes co-cultured with LPS-treated RAW264.7 macrophages in the presence or the absence of the angiotensin receptor 1 blocker valsartan, for 4, 8, 12 and 24 h. The genes of which expressions were affected by LPS-treated RAW macrophages but normalized by co-addition of valsartan were analyzed using KeyMolnet Lite. They included many NF-κB, thyroid receptor and AP-1 target transcripts. In addition, the expression patterns of caspases, integrins, matrix metallopeptidases and adipogenic genes, altered by co-culture with LPS-treated RAW cells, were generally normalized by valsartan treatment. In light of these data, it is reasonable to consider valsartan to normalize altered gene expression patterns in adipose tissue infiltrated by macrophages, and to ameliorate inflammation, apoptosis and fibrotic changes of adipose tissue. Although there may be multiple mechanisms by which ARBs ameliorate insulin resistance, for example, through effects on muscle or other tissues via the circulatory system, this is the first report demonstrating that a favorable effect of valsartan involves normalization of the interaction between adipocytes and macrophages. This mechanism of valsartan action holds promise for developing treatments for obesity-related insulin resistance.

DOI： 10.1016/j.orcp.2012.05.005

Language：English   Publishing type：Research paper (scientific journal)  

Dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) are highly phosphorylated proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and are essential for proper development of hard tissues such as teeth and bones. In order to understand how they contribute to tissue organization, DSPP and DMP-1 have been analyzed for over a decade using both in vivo and in vitro techniques. Among the five SIBLINGs, the DSPP and DMP-1 genes are located next to each other and their gene and protein structures are most similar. In this review we examine the phenotypes of the genetically engineered mouse models of DSPP and DMP-1 and also introduce complementary in vitro studies into the molecular mechanisms underlying these phenotypes. DSPP affects the mineralization of dentin more profoundly than DMP-1. In contrast, DMP-1 significantly affects bone mineralization and importantly controls serum phosphate levels by regulating serum FGF-23 levels, whereas DSPP does not show any systemic effects. DMP-1 activates integrin signalling and is endocytosed into the cytoplasm whereupon it is translocated to the nucleus. In contrast, DSPP only activates integrin-dependent signalling. Thus it is now clear that both DSPP and DMP-1 contribute to hard tissue mineralization and the tissues affected by each are different presumably as a result of their different expression levels. In fact, in comparison with DMP-1, the functional analysis of cell signalling by DSPP remains relatively unexplored.

DOI： 10.1016/j.archoralbio.2012.03.005

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Chronic low-grade infection has been suggested to be associated with metabolic disorder such as diabetes. However, the molecular mechanism underlying this important association is largely unknown. The only clue established so far is that many subjects exhibit elevated levels of C-reactive protein as measured by highly sensitive assay. Here, we hypothesized that adipocyte-macrophage interaction plays a key role in amplifying such low grade infection to the level of influencing metabolic disorders. The presence of macrophages in abdominal adipose tissues was investigated by immunohistochemistry. To see whether molecules associated with acute phase protein, LPS signaling, and persistent recruitment of monocytes, are produced at higher amounts in adipocytes co-cultured with macrophages stimulated with low concentration of LPS (1 ng/ml), we measured serum amyloid A (SAA), LPS binding protein (LBP), soluble CD14 (sCD14), and RANTES levels in culture supernatant of co-cultures. Lastly, we investigated in vivo effect of low-grade LPS infusion on the production of these molecules using obese model mice. The macrophages were certainly identified in abdominal adipose tissues. Investigated molecules, especially LBP, SAA, and RANTES were produced at higher amounts in co-cultures stimulated with LPS compared with the cells without LPS. The ob/ob, and high-fat diet-induced obesity mice produced higher amounts of LBP, SAA, and RANTES one day after LPS infusion (1 ng/ml/g body weight) compared with ob/- and normal-fat fed control mice. Thus, adipocytes and infiltrated macrophages, and their interaction with low endotoxin stimulation appear to play an important role in amplifying and maintaining LPS-induced low-grade inflammation.

DOI： 10.1177/1753425910393370

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Objective-: Hyperuricemia is common in patients with metabolic syndrome. We investigated the role of xanthine oxidoreductase (XOR) in atherosclerosis development, and the effects of the XOR inhibitor allopurinol on this process. Methods and Results-: Oral administration of allopurinol to ApoE knockout mice markedly ameliorated lipid accumulation and calcification in the aorta and aortic root. In addition, allopurinol treatment or siRNA-mediated gene knockdown of XOR suppressed transformation of J774.1 murine macrophage cells, treated with acetylated LDL or very low density lipoprotein (VLDL) into foam cells. This inhibitory effect of allopurinol was also observed in primary cultured human macrophages. In contrast, overexpression of XOR promoted transformation of J774.1 cells into foam cells. Interestingly, SR-A1, SR-B1, SR-B II, and VLDL receptors in J774.1 cells were reduced by XOR knockdown, and increased by XOR overexpression. Conversely, expressions of ABCA1 and ABCG1 were increased by XOR knockdown and suppressed by XOR overexpression. Finally, productions of inflammatory cytokines accompanied by foam cell formation were also reduced by allopurinol administration. Conclusion-: These results strongly suggest XOR activity and/or its expression level to contribute to macrophage foam cell formation. Thus, XOR inhibitors may be useful for preventing atherosclerosis.

DOI： 10.1161/ATVBAHA.111.234559

Language：English   Publishing type：Research paper (scientific journal)  

Macrophages are integrated into adipose tissues and interact with adipocytes in obese subjects, thereby exacerbating adipose insulin resistance. This study aimed to elucidate the molecular mechanism underlying the insulin-sensitizing effect of the angiotensin II receptor blocker (ARB) valsartan, as demonstrated in clinical studies. Insulin signaling, i.e., insulin receptor substrate-1 and Akt phosphorylations, in 3T3-L1 adipocytes was impaired markedly by treatment with tumor necrosis factor-α (TNFα) or in the culture medium of lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages, and valsartan had no effects on these impairments. However, in contrast, when cocultured with RAW 264.7 cells using a transwell system, the LPS-induced insulin signaling impairment in 3T3-L1 adipocytes showed almost complete normalization with coaddition of valsartan. Furthermore, valsartan strongly suppressed LPS-induced productions of cytokines such as interleukin (IL)-1β, IL-6, and TNFα with nuclear factor-κB activation and c-Jun NH ₂-terminal kinase phosphorylation in RAW 264.7 and primary murine macrophages. Very interestingly, this effect of valsartan was also observed in THP-1 cells treated with angiotensin II type 1 (AT1) siRNA or a peroxisome proliferator-activated receptor-γ (PPARγ) antagonist as well as macrophages from AT1a receptor-knockout mice. We conclude that valsartan suppresses the inflammatory response of macrophages, albeit not via PPARγ or the AT1a receptor. This suppression appears to secondarily improve adipose insulin resistance.

DOI： 10.1152/ajpendo.00324.2011

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Background: The effect of smoking on leptin regulation is controversial. Smoking may induce low-grade inflammation. Recent series of studies indicated the critical role of macrophage migration in the establishment of adipose tissue inflammation. In this study, we aimed to see the effects of smoking and inflammation on leptin regulation both at cellular and epidemiological levels. Methods. We compared the concentration of inflammatory markers and serum leptin levels among Japanese male subjects. Additionally, leptin and intercellular adhesion molecule (ICAM) -1 gene expression was assessed in adipocytes co-cultured with or without macrophages in the presence or absence of nicotine and/or lipopolysaccharide (LPS). Results: In subjects with BMI below 25 kg/m ², both WBC counts and soluble-ICAM-1 levels are significantly higher in smokers than in non-smokers. However, leptin concentration did not differ according to smoking status. However, in subjects with BMI over 25 kg/m ², smokers exhibited significantly lower serum leptin level as well as higher WBC counts and s-ICAM-1 concentration as compared with non-smokers. Leptin gene expression was markedly suppressed in adipocytes co-cultured with macrophages than in adipocyte culture alone. Furthermore, nicotine further suppressed leptin gene expression. ICAM-1 gene expression was markedly up-regulated in adipocytes co-cultured with macrophages when stimulated with LPS. Conclusions: Adipose tissue inflammation appears to down-regulate leptin expression in adipose tissues. Nicotine further suppresses leptin expression. Thus, both smoking and inflammation may diminish leptin effect in obese subjects. Therefore, obese, but not normal weight, smokers might be more resistant to weight loss than non-smokers.

DOI： 10.1186/1617-9625-10-3

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Background: Cigarette smokers have increased white blood cell (WBC) counts and the activation of tumor necrosis factor (TNF). The effect of smoking on WBC counts and TNF system activity, however, has not been separately investigated yet. Subjects and Methods. One hundred and forty-two Japanese male subjects with normal glucose tolerance were recruited. They were stratified into two groups based on the questionnaire for smoking: one with current smokers (n = 48) and the other with current non-smokers (n = 94). Whereas no significant differences were observed in age, BMI, high molecular weight (HMW) adiponectin, and TNF- between the two groups, current smokers had significantly higher soluble TNF receptor 1 (sTNF-R1) (1203 30 vs. 1116 21 pg/ml, p = 0.010) and increased WBC counts (7165 242 vs. 5590 163/l, p < 0.001) and lower HDL cholesterol (55 2 vs. 60 1 mg/dl, p = 0.031) as compared to current non-smokers. Next, we classified 48 current smokers into two subpopulations: one with heavy smoking (Brinkman index 600) and the other with light smoking (Brinkman index < 600). Results: Whereas no significant difference was observed in age, BMI, HMW adiponectin, WBC counts and TNF-, sTNF-R1 and sTNF-R2 were significantly higher in heavy smoking group (1307 44 vs. 1099 30 pg/ml, p < 0.001; 2166 86 vs. 827 62 pg/ml, p = 0.005) than in light smoking group, whose sTNF-R1 and sTNF-R2 were similar to non-smokers (sTNF-R1: 1116 15 pg/ml, p = 0.718, sTNF-R2; 1901 32 pg/ml, p = 0.437). In contrast, WBC counts were significantly increased in heavy (7500 324/l, p < 0.001) or light (6829 352/l, p = 0.001) smoking group as compared to non-smokers (5590 178/l). There was no significant difference in WBC counts between heavy and light smoking group (p = 0.158). Conclusion: We can hypothesize that light smoking is associated with an increase in WBC counts, while heavy smoking is responsible for TNF activation in Japanese male subjects with normal glucose tolerance.

DOI： 10.1186/1617-9625-9-12

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Purpose Coagulase-negative staphylococci (CoNS) are frequently isolated from blood cultures of hematopoietic cell transplantation (HCT) patients. Generally, the use of central venous catheters is recognized as a significant risk factor for CoNS infection, while the impact of CoNS infection from oral ulcerative mucositis, which occurs frequently in HCT, may be underestimated. Here, we examined the bacteria on the buccal mucosa after HCT. Methods Sixty-one patients were examined for bacteria on the buccal mucosa routinely once a week from 1 week before to 3 weeks after allogeneic HCT. Subjects were divided into groups with short and long periods of antibiotic use, and differences in bacterial substitution were evaluated. The relationships between type of HCT (conventional HCT or RIST) and bacterial substitution were also evaluated. Results The changes in detection frequencies of CoNS and α-streptococci from before to 3 weeks after HCT were significant (P<0.05, χ2 test): 14.5-53.3% and 92.7-53.1%, respectively. Significant bacterial substitution of CoNS for streptococci was observed in the long-term antibiotic use group (P<0.05, χ2 test), but also occurred in cases with short-term or no antibiotic use. No relationships between type of HCT (conventional HCT or RIST) were observed. Conclusion Bacterial substitution of CoNS for streptococci occurred frequently on the buccal mucosa after HCT. In addition to antibiotic use, environmental factors may be involved in bacterial substitution. It is important to consider the presence of oral mucositis in CoNS infection after HCT.

DOI： 10.1007/s00520-010-0923-9

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Multiple risk factor syndrome is a clustering of cardiovascular risk factors, such as diabetes, dyslipidemia, hypertension, and obesity associated epidemiologically with insulin resistance. This report describes the clinical course of a patient suffering from severe periodontitis with multiple risk factor syndrome, and discusses the association between periodontal infection and systemic health. The patient had a history of type 2 diabetes, dyslipidemia, and hypertension for over 10 years. At baseline, her hemoglobin A1 c was 8.1%. However, she had no diabetic complications except periodontitis. The IgG antibody titers against Porphyromonas gingivalis FDC 381 and SU63 were elevated above the mean of healthy subjects +2 standard deviations. Intensive periodontal treatment, including periodontal surgery, was performed to reduce periodontal infection and bacteremia. Her systemic and periodontal conditions were evaluated longitudinally for 10 years. Following periodontal treatment, antibody titers against Porphyromonas gingivalis and hemoglobin A1c values were significantly improved. The other clinical data and medication for her systemic condition also remained stable during supportive periodontal therapy. However, she developed myocardial infarction, and showed continuous deterioration of hemoglobin A1 c level and periodontitis. The long-term clustering of risk factors, such as diabetes, dyslipidemia, hypertension, and periodontitis, are associated with the development of myocardial infarction. Treatment of systemic conditions in combination with comprehensive periodontal treatment is important in management of patients with multiple risk factor syndrome.

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Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and pointmutated Pin1 and IRS-1 constructs revealed the WW domain located in theNterminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Aktphosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.

DOI： 10.1074/jbc.M110.206904

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Although periodontal disease may be associated with increased risk for atherosclerosis, the mechanism by which the disease causes atherosclerosis is still unknown. The candidates contributing to atherosclerosis in periodontal disease include low-grade inflammation such as C-reactive protein (CRP) and insulin resistance. A previous study demonstrated that periodontal therapy leads to an improvement in CRP as well as insulin resistance, indicating the relationship between periodontal disease and low-grade inflammation or insulin resistance. On the other hand, we previously demonstrated that serum triglyceride (TG) per se is independently associated with CRP or insulin resistance in Japanese populations with a body mass index (BMI) of 21.5 to 27.0 (midrange BMI). To the best of our knowledge, however, the relationship between periodontal disease and serum TG is not fully clarified. The first aim of the present study is to investigate whether periodontal disease is associated with serum TG in Japanese subjects with midrange BMI. If so, another aim of the study is to determine which mechanism is responsible for the association between periodontal disease and serum TG in these subjects. We have performed a periodontal examination in the Ogaki metabolic syndrome medical examination. One hundred sixty-two participants from 40 to 74 years old (56 men and 106 women; mean age, 66.43 ± 6.25 years) were enrolled in the study. Besides medical examination, oral panoramic radiograph was taken for all participants. Average bone score was also calculated. Periodontal bone destruction increased according to the age of the participants (r = 0.227, P < .004, Spearman correlation coefficient). Periodontal bone destruction was also associated with serum TG levels (r = 0.299, P = .000). This association was more evident in subjects with midrange BMI (r = 0.332, P < .001). In subjects with midrange BMI, TG was not correlated with BMI or waste circumstances. Furthermore, TG was not associated with age itself in the midrange BMI group. We then investigated the lipolytic activity of endotoxin in cocultures of adipocytes and macrophages. Low-dose lipopolysaccharide dose-dependently increased lipolytic activity in cocultures, and this activity was neutralized by anti-tumor necrosis factor α neutralizing antibodies. These results suggest that periodontal infection, especially bacterial endotoxinemia, is associated with enhanced lipolysis and subsequent up-regulation of circulating TG in Japanese with midrange BMI.

DOI： 10.1016/j.metabol.2010.07.034

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Glucose transporter 1 (GLUT1) is widely distributed throughout various tissues and contributes to insulin- independent basal glucose uptake. Using a split-ubiquitin membrane yeast two-hybrid system, we newly identified 4F2 heavy chain (4F2hc) as a membrane protein interacting with GLUT1. Though 4F2hc reportedly forms heterodimeric complexes between amino acid transporters, such as LAT1 and LAT2, and regulates amino acid uptake, we investigated the effects of 4F2hc on GLUT1 expression and the associated glucose uptake. First, FLAG-tagged 4F2hc and hemagglutinin-tagged GLUT1 were overexpressed in human embryonic kidney 293 cells and their association was confirmed by coimmunoprecipitation. The green fluorescent protein-tagged 4F2hc and DsRed-tagged GLUT1 showed significant, but incomplete, colocalization at the plasma membrane. In addition, an endogenous association between GLUT1 and 4F2hc was demonstrated using mouse brain tissue and HeLa cells. Interestingly, overexpression of 4F2hc increased the amount of GLUT1 protein in HeLa and HepG2 cells with increased glucose uptake. In contrast, small interfering RNA (siRNA)-mediated 4F2hc gene suppression markedly reduced GLUT1 protein in both cell types, with reduced glucose uptake. While GLUT1 mRNA levels were not affected by overexpression or gene silencing of 4F2hc, GLUT1 degradation after the addition of cycloheximide was significantly suppressed by 4F2hc overexpression and increased by 4F2hc siRNA treatment. Taken together, these observations indicate that 4F2hc is likely to be involved in GLUT1 stabilization and to contribute to the regulation of not only amino acid but also glucose metabolism.

DOI： 10.1152/ajpcell.00416.2010

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Periodontitis is a chronic inflammatory disease caused by the infection of periodontopathic bacteria in dental plaque. However, an individual's susceptibility to this disease appears to be associated with multiple genetic factors, as seen in the case of leprosy. In order to gain a better understanding of the pathophysiology of periodontal disease in subjects with leprosy, we investigated the clinical features of periodontitis and the immunological responses against periodontopathic bacteria in 382 subjects with a history of leprosy and 451 age-matched control subjects. The prevalence of periodontitis and the degree of periodontal pocket depth were found to be significantly higher in leprosy patients than in age-matched controls. Furthermore, a comparison of the clinical parameters of lepromatous leprosy (L-lep) and tuberculoid leprosy (T-lep) patients showed that the probing pocket depth of L-lep patients with periodontal disease was significantly higher than that for T-lep patients. In contrast, serum IgG titers against Porphyromonas gingivalis in L-lep patients were significantly lower than in T-lep patients. These results imply that L-lep patients show more severe periodontal disease than T-lep patients or agematched control subjects, and that low humoral immunity against P. gingivalis might be one of the genetic factors determining periodontal disease susceptibility in leprosy patients.

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Fibroblasts act as important immune regulatory cells via their ability to cross-talk with T cells accumulating in lesions. Our previous study showed that fibroblasts produce several cytokines and chemokines by crosslinking HLA class II (HLA-II) molecules with monoclonal antibodies or by making T-cell receptor-peptide-HLA complexes. It is thus conceivable that the interaction of T cells and fibroblasts via HLA-II affects fibroblast responses to stimuli. This study used human gingival fibroblasts (HGF) to investigate possible effects of these fibroblast-derived soluble factors on the differentiation of nave T cells and on the subsequent fibroblast responses. After mixed lymphocyte reaction culture between nave T cells and allogeneic dendritic cells in the presence of culture supernatant from HGF stimulated via HLA-DQ molecules (DQ-sup), but not via DR, T cells exhibited a Th2-shifted phenotype, thereby producing quantitatively more IL-13 and IL-5 compared with interferon-γ. Astonishingly, analyses to identify possible factors affecting the Th2 polarization secreted from HLA-II-stimulated HGF, prostaglandin E₂, was detected only in DQ-sup. The Th2 polarization of nave T cells was blocked in the presence of supernatants from indomethacin-treated HGF with HLA-DQ stimulation. In addition, we found that the culture supernatants of Th cells activated following mixed lymphocyte reaction culture in the presence of DQ-sup had the potential to induce gene expression of type I and III collagens in HGF. These results suggested that fibroblasts stimulated via HLA-DQ molecules promote Th2 polarization in Th-cell responses and showed the counter activation of collagen synthesis, implicating orchestrated responses among these cells in the fibrosis of chronic inflammatory lesions.

DOI： 10.1038/labinvest.2010.128

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Pin1 is a unique regulator, which catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified CRTC2 as a Pin1-binding protein by overexpressing Pin1 with Myc and FLAG tags in mouse livers and subsequent purification of the complex containing Pin1. The association between Pin1 and CRTC2 was observed not only in overexpression experiments but also endogenously in the mouse liver. Interestingly, Ser ¹³⁶ in the nuclear localization signal of CRTC2 was shown to be involved in the association with Pin1. Pin1 overexpression in HepG2 cells attenuated forskolin-induced nuclear localization of CRTC2 and cAMP-responsive element (CRE) transcriptional activity, whereas gene knockdown of Pin1 by siRNA enhanced both. Pin1 also associated with CRTC1, leading to their cytosol localization, essentially similar to the action of CRTC2. Furthermore, it was shown that CRTC2 associated with Pin1 did not bind to CREB. Taken together, these observations indicate the association of Pin1 with CRTC2 to decrease the nuclear CBP·CRTC·CREB complex. Indeed, adenoviral gene transfer of Pin1 into diabetic mice improved hyperglycemia in conjunction with normalizing phosphoenolpyruvate carboxykinase mRNA expression levels, which is regulated by CRE transcriptional activity. In conclusion, Pin1 regulates CRE transcriptional activity, by associating with CRTC1 or CRTC2.

DOI： 10.1074/jbc.M110.137836

Language：English   Publishing type：Research paper (scientific journal)  

Purpose: The commercial saliva substitute Oralbalance® has been reported to alleviate symptoms of postradiotherapy xerostomia in head and neck cancer patients. Oralbalance® may also be effective for xerostomia in patients undergoing hematopoietic cell transplantation (HCT) with high-dose chemotherapy and total-body irradiation. However, HCT patients are in a severely compromised condition, and saliva substitute must not promote infection. We reported previously that Oralbalance® has antimicrobial effects against microbial species detected during HCT in vitro. This study was performed to determine the in vivo effects of Oralbalance® on oral mucosal total bacterial counts in patients undergoing HCT. Methods: A total of 18 neutropenic patients undergoing HCT were enrolled in this study. Before and after 1 week of Oralbalance® use, bacterial samples were obtained from patients by wiping an area of ω1 cm on the buccal mucosa with sterilized cotton swabs. Total bacterial counts of the obtained samples were examined by quantitative polymerase chain reaction amplification of the bacterial 16S ribosomal RNA gene. As controls, bacterial samples were also obtained from ten healthy subjects, and total bacterial counts were examined. Results: No significant increase in bacterial count was observed with use of Oralbalance®. None of the patients showed bacterial counts above the range found in healthy controls after using Oralbalance®. Conclusions: In neutropenic patients undergoing HCT, Oralbalance® did not increase the total counts of oral mucosal bacteria beyond the range found in healthy controls. Oral care using Oralbalance® may alleviate the symptoms induced by hyposalivation without promoting infection.

DOI： 10.1007/s00520-009-0789-x

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Aims: Energy sensing systems including AMPK and SIRT1 play important roles in the regulation of hepatic gluconeogenesis and fatty acid oxidation. In this study, we investigated how hepatic LKB1-AMPK signaling and SIRT1 expression are altered after 2 or 8 weeks of HFD feeding. Methods: The livers of male mice fed a HFD or a standard diet for 2 or 8 weeks were removed. The expression and phosphorylation levels of LKB1, AMPK, ACC and TORC2, and SIRT1 expression levels were examined by immunoblotting. Results: In mice fed a HFD for 2 weeks, the phosphorylations of AMPKα and ACC were decreased without significant alterations in LKB1 phosphorylation or SIRT1 protein levels, while TORC2 protein levels were increased. In mice fed a HFD for 8 weeks, marked reductions in LKB1 phosphorylation and SIRT1 protein amount were observed in addition to the decreased phosphorylations of AMPKα and ACC. Conclusions: The mechanisms underlying impaired energy sensing signaling differ with the duration of HFD feeding. In the early phase of HFD feeding, LKB1 and SIRT1 were not impaired, while in the later phase of HFD feeding, decreased SIRT1 expression and LKB1 phosphorylation may be involved in the development of severe glucose and lipid intolerance.

DOI： 10.1016/j.orcp.2010.02.002

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The differentiation of macrophages into cytokine-secreting foam cells plays a critical role in the development of diabetic angiopathy. J774.1, a murine macrophage cell line, reportedly differentiates into foam cells when incubated with oxidized LDL, ApoE-rich VLDL or WHHLMI (myocardial infarction-prone Watanabe heritable hyperlipidemic) rabbit serum. In this study, serum samples from Type 2 diabetic patients were added to the medium with J774.1 cells and the degree of foam cell induction was quantified by measuring lipid accumulation. These values were calculated relative to the activities of normal and WHHLMI rabbit sera as 0% and 100%, respectively, and termed the MMI (Macrophage Maturation Index). These MMI values reflected intracellular lipids, including cholesteryl ester assayed by GC/MS. Statistical analysis revealed MMI to correlate positively and independently with serum triglycerides, the state of diabetic retinopathy, nephropathy and obesity, but negatively with administration of α-glucosidase inhibitors or thiazolidinediones. Taken together, our results suggest that this novel assay may be applicable to the identification of patients at risk for rapidly progressive angiopathic disorders.

DOI： 10.1016/j.diabres.2009.10.011

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1902/jop.2009.090461

Language：English   Publishing type：Research paper (scientific journal)  

Interleukin (IL)-23 is an essential cytokine involved in expansion of the Th17 lineage, which is associated with many immune-related destructive tissue diseases. We hypothesized that the IL-23-induced Th17 pathway plays a role in periodontal pathology and examined the expression of cytokines, and related molecules, in periodontal lesions and control sites. IL-23 and IL-12 were expressed at significantly higher levels in periodontal lesions than in control sites. However, the relative expression of the IL-23 receptor compared with the IL-12 receptor β2 was significantly higher in periodontal lesions. Moreover, IL-17 expression was significantly higher in periodontal lesions, especially in the tissue adjacent to bone destruction, than in control sites. There was no significant difference in the expression levels of IFN-γ, an important cytokine inhibiting differentiation toward the Th17 pathway, between periodontal lesions and control sites. Together, these results suggest that the IL-23-induced Th17 pathway is stimulated in inflammatory periodontal lesions.

DOI： 10.1177/0022034509339889

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Goals of work: Oral and systemic infections arising from the oral cavity are significant problems in clinical management of patients undergoing leukemia treatment. However, there is significant disparity in the reported incidences of development of periodontal infections. Evidence is limited to those showing the systemic influence of periodontal infection in neutropenic patients. This study indicated an association between febrile neutropenia (FN) and periodontitis in a case in which periodontal treatment in the intervals between chemotherapy cycles reduced FN in subsequent courses of chemotherapy and hematopoietic transplantation (HCT). Materials and methods: Periodontal treatment was performed in a 61-year-old man with advanced periodontitis, who received HCT following three cycles of chemotherapy. After recovery from neutropenia induced by initial chemotherapy, periodontal treatment was performed in each chemotherapy interval period. Following extraction of teeth with severe advanced periodontitis, all teeth were subjected to periodontal pocket curettage and root planning, which are common periodontal treatments to reduce periodontal pockets harboring anaerobic periodontal bacteria, before HCT. Main results: Periodontal treatment successfully reduced periodontal pockets from 4.1∈±∈1.5 mm to 3.0∈±∈0.6 mm, which was almost within the healthy range (<3.0 mm), before HCT. The frequency of FN decreased significantly with increasing cycles of chemotherapy, and decreases in FN corresponded to progress of periodontal treatment. Blood cultures obtained a total of 12 times throughout leukemia treatment were all negative. Conclusions: The observations reported here indicate the importance of periodontal treatment in clinical management of patients undergoing leukemia treatment to prevent FN, although all blood cultures were negative.

DOI： 10.1007/s00520-008-0532-z

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Background and Objective: The role of human leukocyte histocompatibility antigen (HLA) class II molecules on non-antigen-presenting cells has been a matter of controversy. We previously reported that HLA-II molecules on human gingival fibroblasts (GF) do not present antigens, but transduce signals into the cells, resulting in the expression of several cytokines, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T-cell expressed and secreted (RANTES) and IL-8. However, the exact role of these cytokines, as well as other cytokines which are potentially secreted from GF, in the pathogenesis of chronic periodontal inflammation is not fully understood. The aim of this study was to observe the effects of HLA-II-induced cytokines on the proliferation of human umbilical vein endothelial cells (HUVEC). Material and Methods: Antibody-based cytokine-microarray analyses were performed to detect potential cytokines associated with angiogenesis. Next, cytokine productivity was confirmed by quantitative methods. Then, cell proliferation assay was performed to see whether these cytokines promoted the proliferation of HUVEC. Results: Besides IL-6, MCP-1, RANTES and IL-8, growth-related gene product (GRO) was newly identified as an HLA-II-induced cytokine released from GF. This was confirmed by a quantitative method. Cell culture supernatant from HLA-II-stimulated GF cultures promoted the growth of HUVEC. Addition of anti-IL-8 neutralizing antibody, anti-CXC receptor (CXCR)1 antibody and anti-MCP-1 antibody inhibited the growth of HUVEC in a dose-dependent manner, while addition of anti-GROα antibody did not. Conclusion: The HLA-II-induced IL-8, via CXCR1, as well as MCP-1 from GF, promotes endothelial cell proliferation, which is possibly associated with enhanced angiogenesis in chronic periodontal lesions.

DOI： 10.1111/j.1600-0765.2008.01097.x

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Pyogenic liver abscess (PLA) formation is thought to originate from the transmission of infection via three major routes including the biliary tract, portal vein and hepatic artery. However, about 50% of PLA cases are considered to be cryptogenic. Here we report an unusual autopsy case of PLA associated with periodontopathic bacterial infection. A 59-year-old female suddenly developed cardiopulmonary arrest and died. Despite macroscopic and microscopic examinations, the infectious routes and source of infection were unidentified, and the case appeared to be cryptogenic. Since this patient had suffered severe periodontitis for a long period of time, we investigated the involvement of periodontal infection in PLA formation by performing immunohistochemical analyses. We identified several periodontopathic bacterial species in the PLA of this patient, including Fusobacterium nucleatum, Treponema denticola, Prevotella intermedia and Porphyromonas gingivalis. Thus, we demonstrate here that periodontal infection is a potential source of infection in the formation of PLA.

Language：English   Publishing type：Research paper (scientific journal)  

Recent studies have suggested that macrophages were integrated into adipose tissues to interact with adipocytes, thereby exacerbating inflammatory responses. Furthermore, both adipocytes and macrophages appear to express toll-like receptor-4 (TLR-4), and free fatty acids may stimulate cells through TLR-4. Herein, we analyzed genes differentially expressed in adipocytes when co-cultured with macrophages in the presence of a ligand for TLR-4, bacterial lipopolysaccharide (LPS). RAW264.7, a murine macrophage cell line and differentiated 3T3-L1 adipocytes were co-cultured using a transwell system. Genes differentially expressed in adipocytes were analyzed by the DNA microarray method following 4, 8, 12 and 24 h stimulation with 1 ng ml^-1 of Escherichia coli LPS. Randomly selected genes with high expressions were confirmed by quantitative methods at both the gene and the protein level. Co-culture of macrophages and adipocytes with a low LPS concentration (1 ng ml^-1) markedly upregulated gene expressions associated with inflammation and/or angiogenesis, such as those of interleukin-6 (IL-6), MCP-1, RANTES and CXCL1/KC, in adipocytes. Furthermore, several genes associated with insulin resistance were differentially expressed. Upregulations of genes encoding MCP-1, RANTES and CXC/KC were confirmed by quantitative methods. These results suggest that ligands for TLR-4 stimulate both adipocytes and macrophages to upregulate the expressions of many genes associated with inflammation and/or angiogenesis.

DOI： 10.1038/ijo.2008.153

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Interleukin (IL)-6 has an important role in inflammatory diseases. Lysosomal enzymes cathepsins are widely expressed as cysteine proteases regulating inflammatory process. Caveolin-1 (Cav-1) is a scaffolding/regulatory membrane protein that interacts with signaling molecules. In this study, we investigated the role of Cav-1 on (1) the productivity, and (2) the enzymatic activity of cathepsin B and L in human gingival fibroblasts (HGFs) treated with IL-6 in the presence of soluble form of IL-6 receptor (sIL-6R). At first, we established the siRNA-mediated Cav-1 down-regulating in vitro systems by transient transfection of Cav-1 siRNA. The siRNA-mediated Cav-1 down-regulated cells were treated with IL-6/sIL-6R for indicated times. Then, cell lysates were collected, and examined the IL-6-induced signaling pathway, cathepsin B and L production, and measurement of cathepsins activity. To investigate the cathepsin L activity, cathepsin-(B + L) activity was measured after pretreatment with CA-074Me, a specific inhibitor for cathepsin B. We found that IL-6/sIL-6R enhanced significantly both production and activity of cathepsin B and L in HGFs. Interestingly, IL-6-mediated phosphorylation of both p44/42 MAPK and JNK was dramatically suppressed in Cav-1 down-regulated HGFs treated with IL-6/sIL-6R. In addition, both production and activity of cathepsin B and L were also significantly suppressed. Importantly, we demonstrated that JNK inhibition, but not p44/42 MAPK inhibition, significantly diminished IL-6/sIL-6R-induced cathepsin B and L production. Taken together, we concluded that IL-6/sIL-6R enhances cathepsin B and L production via IL-6/sIL-6R-mediated Cav-1-JNK-AP-1 pathway in HGFs. Our findings indicate that Cav-1 might be a therapeutic target for IL-6-mediated tissue degradation in periodontitis.

DOI： 10.1002/jcp.21517

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Goals: Hematopoietic cell transplantation (HCT) may lead to the development of xerostomia. However, there have been few reports of xerostomia in HCT patients based on objective data. We investigated moisture in the oral mucosa in patients undergoing HCT by the capacitance method using a convenient device, Moisture Checker for Mucus® (MCM; Life Co., Ltd., Saitama, Japan). Subjects and methods: Thirty-six patients undergoing HCT at Okayama University Hospital of Medicine and Dentistry (Male=22, Female=14; age=41.6±16.2 years old) were enrolled in this study. Moisture in the oral mucosa was measured by MCM in accordance with the manufacturer's instructions. The results were obtained as MCM values (%), which are the weight percentage of water content in the oral mucosal epithelium. As controls, moisture of the oral mucosa was also examined in healthy volunteers (Male=27, Female=35; age=43.0±14.6 years old). Main results: Throughout the examination period, MCM values were significantly lower in patients who underwent HCT than in controls. The degree of mucosal moisture in HCT patients showed wide interindividual differences. Conclusion: The degree of mucosal moisture in HCT patients was low and showed wide interindividual differences. Evaluation of xerostomia using such a device may contribute to appropriate oral care with saliva substitute.

DOI： 10.1007/s00520-008-0470-9

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Single nucleotide polymorphisms (SNPs) in the 5′ flanking region of IL12RB2 are frequently detected in lepromatous leprosy patients, and may be possible immunogenetic factors that reduce transcriptional activity of the IL-12Rβ2 gene in Jurkat T cells. This study determined the functional effects of these SNPs on NK-cell activity, including IFN-γ production and IL-12Rβ2 gene expression. Reporter gene assays using the NK cell line NK3.3 revealed that transcriptional activities of the variant haplotypes were significantly higher in the NK cell line, in contrast to our previous results in Jurkat T cells. IFN-γ production in activated T cells from donors was significantly lower than in cells from donors without the variant SNPs, while NK cells with these SNPs produced significantly higher amounts of IFN-γ. These results suggest that these SNPs in IL12RB2 have differential effects on cellular activation of T cells and NK cells.

DOI： 10.1089/jir.2008.0133

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Loop-mediated isothermal amplification (LAMP) was applied to develop a rapid and simple detection system for eight periodontal pathogens: Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola and Tannerella forsythia. Primers were designed from the 16S ribosomal RNA gene for each pathogen, and the LAMP amplified the targets specifically and efficiently under isothermal condition at 64°C. To simplify the manipulation of LAMP examination, boiled cells and intact cells suspended in phosphate-buffered saline (PBS) were tested as templates besides extracted DNA template. The detection limits were 1-10 cells per tube using extracted DNA template. However, LAMP methods using boiled cells and intact cells required 10-100 and 100-1000 cells per tube, respectively. LAMPs for A. actinomycetemcomitans, P. gingivalis and P. intermedia were then applied to clinical plaque samples, and the method demonstrated equal or higher sensitivity compared with the conventional real-time PCR method. These findings suggest the usefulness of the LAMP method for the rapid and simple microbiological diagnosis of periodontitis, and the possibility of LAMP examination without the DNA extraction step.

DOI： 10.1111/j.1574-695X.2008.00417.x

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Goals: The commercially available saliva substitute Oralbalance® has been reported to alleviate symptoms of post-radiotherapy xerostomia in head and neck cancer patients. Oralbalance® may also be effective for xerostomia in patients undergoing hematopoietic cell transplantation (HCT) with high-dose chemotherapy and total-body irradiation. However, HCT patients are severely compromised, and saliva substitute must therefore not promote infection. This study was performed to determine the effects of Oralbalance® on microbial species identified during HCT. Patients and methods: Microbial identification of oral mucosa was performed in 28 patients undergoing HCT. The antimicrobial effects of Oralbalance® against bacteria and fungi detected in the HCT period were examined in vitro. Briefly, bacteria and fungi were spread on agar plates, and 0.1g of Oralbalance® gel was applied (about φ1cm). After incubation at 37°C for 24h, the presence of a transparent zone of inhibition around Oralbalance® was observed. Main results: Not only bacterial species constituting normal flora of the oral mucosa but also those not usually constituting normal flora, e.g., coagulase-negative Staphylococcus, were detected. A transparent zone was observed around Oralbalance® in all bacterial species examined. No transparent zone was observed for Candida albicans, but growth was inhibited in the area where Oralbalance® was applied. Conclusions: Oralbalance® does not facilitate increases in microorganisms in the HCT period. Oral care with Oralbalance® does not promote infection in patients undergoing HCT.

DOI： 10.1007/s00520-007-0391-z

Language：English   Publishing type：Research paper (scientific journal)  

Objectives: The expression of interleukin (IL)-12Rβ2 molecule is a crucial regulatory factor in the T-helper type (Th) 1 differentiation of T cells. To elucidate the role of the cell-mediated immune (CMI) response in the pathogenesis of periodontitis, Japanese periodontal patients were subjected to single nucleotide polymorphism (SNP) analyses of the 5′ flanking region of IL12RB2, whose variants are frequently detected in lepromatous leprosy patients, in which the very weak cellular immune response is caused by low expression of IL-12Rβ2. Material and Methods: The gene polymorphisms of the 5′ flanking region of IL12RB2 were examined in subjects with several types of periodontal disease and in healthy controls. Serum immunoglobulin (Ig) G antibody titres against periodontopathic bacteria were measured and compared in periodontal patients with and without variant alleles of IL12RB2. Results: The frequencies of variant alleles of IL12RB2 were significantly higher in aggressive periodontitis patients as compared with healthy controls or chronic periodontitis patients. Serum IgG titres against all periodontal bacteria examined in subjects carrying variant alleles were higher than those in subjects without variant alleles. Conclusion: IL-12Rβ2 SNPs could be useful as genetic markers to access the susceptibility of the general population to periodontal disease. Low CMI responses or high humoral responses are associated with the pathogenesis of inflammatory periodontal diseases.

DOI： 10.1111/j.1600-051X.2008.01208.x

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Background: Dentists generally recognize the importance of periodontal treatment in patients with leukemia, with the most attention paid to preventing the development of odontogenic infection. For physicians, the worst type of infection is one caused by multidrug-resistant bacteria. Here, we report a patient with an abnormal increase in multidrug-resistant opportunistic bacteria in the gingiva during hematopoietic cell transplantation (HCT). Methods: A 53-year-old woman receiving HCT for leukemia had an insufficient blood cell count for invasive periodontal treatment before HCT. Even brushing caused difficulties with hemostasis. Therefore, frequent pocket irrigation and local minocycline administration were performed. Results: The multidrug-resistant opportunistic bacterium Stenotrophomonas maltophilia was detected first in phlegm 2 days before HCT, and it was detected in a gingival smear and a blood sample 7 and 11 days after HCT, respectively. The patient developed sepsis on day 11 and died 14 days after HCT. Frequent irrigation and local antibiotic application were ineffective against S. maltophilia on the gingiva. Inflammatory gingiva without scaling and root planing showed bleeding tendency, and this interfered with the eradication of this bacterium. Conclusions: The gingiva in patients undergoing leukemia treatment acts as sites of proliferation and reservoirs for multidrug-resistant opportunistic bacteria. Severe systemic infection by multidrug-resistant bacteria in such patients with leukemia also may involve the gingiva. To prevent abnormal increases in such bacteria on the gingiva, scaling and/or root planing before chemotherapy, which reduces bleeding on brushing during the neutropenic period caused by chemotherapy, may contribute to infection control in such patients, although it was impossible in this case.

DOI： 10.1902/jop.2008.070205

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Background and Objective: The role of human leukocyte antigen class II molecules on nonantigen-presenting cells has been a matter of controversy. We previously reported that human leukocyte antigen class II molecules on human gingival fibroblasts do not present antigens, but transduce signals into the cells by making a complex with antigenic peptide T-cell receptor or by stimulating cell surface human leukocyte antigen-DR molecules with human leukocyte antigen-DR antibody (L243), which mimics the formation of the human leukocyte antigen class II-antigenic peptide T-cell receptor complex, resulting in the expression of several cytokines. The aim of this study was to detect human leukocyte antigen class II-associated molecules mediating human leukocyte antigen class II-induced signals into the cells. Antibody-based protein-microarray analysis was performed to detect activated signaling molecules in gingival fibroblasts stimulated via human leukocyte antigen class II molecules. Then, we examined if these molecules structurally associate with human leukocyte antigen class II and actually transduce signals into the cells. Stimulation of human leukocyte antigen class II on gingival fibroblasts by L243 resulted in enhanced phosphorylation of focal adhesion kinase. Focal adhesion kinase was co-immunoprecipitated with human leukocyte antigen-DR by L243. Stimulation of gingival fibroblasts with L243 induced phosphorylation of focal adhesion kinase. Luteolin, a putative focal adhesion kinase inhibitor, suppressed phosphorylation of focal adhesion kinase and dose dependently inhibited human leukocyte antigen class II-induced cytokine production. Focal adhesion kinase is structurally associated with human leukocyte antigen-DR and mediates human leukocyte antigen class II-induced signals in gingival fibroblasts.

DOI： 10.1111/j.1600-0765.2007.00985.x

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Objective: Recent studies suggested macrophages were integrated in adipose tissues, interacting with adipocytes, thereby exacerbating inflammatory responses. Persistent low-grade infection by gram-negative bacteria appears to promote atherogenesis. We hypothesized a ligand for toll-like receptor 4 (TLR4), bacterial lipopolysaccharide (LPS), would further exaggerate macrophage-adipocyte interaction. Research Methods and Procedures: RAW264.7 macrophage cell line and differentiated 3T3-L1 preadipocytes were co-cultured using transwell system. As a control, each cell was cultured independently. After incubation of the cells with or without Escherichia coli LPS, tumor necrosis factor (TNF)-α and interleukin (IL)-6 production was evaluated. Results: Co-culture of macrophages and adipocytes with low concentration of Escherichia coli LPS (1 ng/mL) markedly up-regulated IL-6 production (nearly 100-fold higher than that of adipocyte culture alone, p < 0.01), whereas TNF-α production was not significantly influenced. This increase was partially inhibited by anti-TNF-α neutralizing antibody. Recombinant TNF-α and LPS synergistically up-regulated IL-6 production in adipocytes. However, this increase did not reach the level of production observed in co-cultures stimulated with LPS. Discussion: A ligand for TLR-4 stimulates macrophages to produce TNF-α. TNF-α, thus produced, cooperatively up-regulates IL-6 production with other soluble factors secreted either from adipocytes or macrophages in these cells. Markedly up-regulated IL-6 would greatly influence the pathophysiology of diabetes and its vascular complications.

DOI： 10.1038/oby.2007.305

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Several reports have demonstrated a possible association of periodontal infections with coronary heart disease (CHD) by elevated antibody titre to periodontopathic bacteria in CHD patients compared with non-diseased controls. Although each periodontopathic bacterium may vary in virulence for periodontitis and atherosclerosis, antibody response to multiple bacteria in CHD patients has not been understood fully. Therefore, serum levels of antibody to 12 periodontopathic bacteria together with other atherosclerotic risk markers were compared among 51 patients with CHD, 55 patients with moderate to severe chronic periodontitis and 37 healthy individuals. The antibody response was the most prevalent for Porphyromonas gingivalis, a major causative organism, in CHD as well as periodontitis patients. However, antibody positivity was different between CHD and periodontitis if the response was analysed for two different strains of P. gingivalis, namely FDC381 and Su63. While periodontitis patients were positive for both P. gingivalis FDC381 and Su63, a high frequency of antibody positivity for P. gingivalis Su63 but not for FDC381 was observed in CHD patients. The results indicate that the presence of particular periodontopathic bacteria with high virulence may affect atherogenesis. Identifying the virulence factors of P. gingivalis Su63 may gain insight into the new therapeutic modality for infection-induced deterioration of atherosclerosis.

DOI： 10.1111/j.1365-2249.2007.03450.x

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Diabetic subjects are susceptible to atherosclerosis. It has been postulated that inflammation plays a crucial role in atherogenesis. Since previous studies suggested persistent low-grade infection by Gram-negative bacteria such as Chlamydia spp. and/or periodontal infection is associated with increased atherogenesis among diabetic subjects, we hypothesized that macrophages under hyperglycemia respond to lipopolysaccharide (LPS) challenge in a more exaggerated manner than under normal glucose conditions. Therefore, we examined cytokine productivity and associated signal transduction molecules in LPS-stimulated the monocytic cell line THP-1, under conditions of hyperglycemia. Differentiated THP-1 cells were cultured under normal and high glucose conditions without fetal bovine serum, and were stimulated with Escherichia coli LPS in the presence of LPS binding protein. Following stimulation, activated signal transduction molecules were detected by protein microarray and confirmed thereafter. Results indicated that c-jun N-terminal kinase (JNK) was highly-phosphorylated at high glucose concentrations, and this was confirmed by Western-immunoblotting. Tumor necrosis factor-α and monocyte chemo-attractant protein-1 production were significantly enhanced under these conditions. SP600125, a selective inhibitor of JNK, dose-dependently suppressed the production of these cytokine. Therefore, we suggest that this may be one of the mechanisms by which sub-clinical infection by Gram-negative bacteria promotes atherosclerosis in diabetic subjects.

DOI： 10.1177/0968051907082608

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Mechanical stress results in differential gene expression that is critical to convert the stimulus into biochemical signals. Under physiological stress such as occlusal force, human periodontal ligament fibroblasts (HPLF) are associated with homeostasis of periodontal tissues however the changes in response to mechanotransduction remain uncharacterized. We hypothesized that cyclic tension-responsive (CT) genes may be used to identify a set of fundamental pathways of mechanotransduction. Our goal was to catalogue CT genes in cultured HPLF. HPLF were subjected to cyclic tension up to 16 h, and total RNA was isolated from both tension-loaded and static HPLF. The oligonucleotide arrays analysis revealed significant changes of mRNA accumulation for 122 CT genes, and their kinetics were assigned by the K-means clustering methods. Ingenuity Pathway Analysis was completed for HPLF mechanotransduction using 50 CT genes. This analysis revealed that cyclic tension immediately down-regulated all nuclear transcription factors except v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) reacting as an early responsive gene. In turn, transcription factors such as tumor protein p53 binding protein 2 (TP53BP2), and extra-nuclear molecules such as adrenergic receptor β2 (ADRB2) were up-regulated after 1-2 h, which may result in fundamental HPLF functions to adapt to cyclic tension. Subsequent inhibition assays using Y27632, a pharmacologic inhibitor of Rho-associated kinase (ROCK), suggested that HPLF has both ROCK-dependent and ROCK-independent CT genes. Mechanical stress was found to effect the expression of numerous genes, in particular, expression of an early responsive gene; FOS initiates alteration of HPLF behaviors to control homeostasis of the periodontal ligament.

DOI： 10.1016/j.biocel.2007.01.015

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DOI： 10.1111/j.1600-0757.2006.00171.x

Language：English   Publishing type：Research paper (scientific journal)  

A 38-year-old woman with pustulosis palmaris et plantaris (PPP) was referred to the dentist with a suspicion of metal allergy (Fig. 1A). She had noticed the symptoms 3 years previously on her palms and legs. As her symptoms did not improve spontaneously, she visited her dermatologist. There, she was recorded as follows: (1) pustules localized only on the palms and soles; (2) no lesions with eczema or psoriasis on any other parts of the body; and (3) no family history of pustulosis. On the basis of these observations, she was diagnosed with PPP. She was treated with topical application of corticosteroids; however, her symptoms did not improve. Her dermatologist then suspected metal allergy and performed a patch test. Her skin reacted positively with chromium sulfate, zinc chloride, and mercury bichloride. Therefore, she was referred to our dental hospital for the investigation of the possible association of PPP with oral restorative materials. Her medical records were unremarkable, except for extremely severe periodontitis, which she was aware of more than 10 years previously; however, she had not received any comprehensive periodontal treatment. On presentation, we first analyzed the restorative materials of the teeth to determine whether they contained any positive elements detected by the patch test. Unfortunately, we could not detect any positive elements in the oral cavity. Her blood chemistry did not indicate any abnormalities. The white blood cell count was 5600/mm³, with differential counts of 49.3% neutrophils, 28.0% lymphocytes, 7.6% monocytes, 14.4% eosinophils, and 0.7% basophils. C-reactive protein was not elevated, as measured by the conventional method. Immunoglobulin G (IgG), IgA, and IgM concentrations were all within the normal range (IgG, 1243.0 mg/dL; IgA, 131.6 mg/dL; IgM, 149.6 mg/dL). Because of these laboratory data, we first treated her periodontitis to determine whether there was a possible relationship of her PPP to severe periodontitis. Her periodontitis was very severe (Fig. 2A,B). The extraction of several teeth, as well as the correction of the shape of the inflamed alveolar bone (the bone supporting the teeth) to a physiological shape, was necessary. We first treated her periodontitis with the topical application of antibiotics (minocycline-HCl ointment) to determine the effect of anti-infectious periodontal treatment on PPP. We administered the antibiotics in every periodontal pocket once a week for a period of 1 month. One month after treatment, her symptoms appeared to improve slightly (Fig. 1B). We next performed surgical treatment, including teeth extractions and correction of the bone shape. Two to three days after surgery, her PPP symptoms worsened suddenly (Fig. 1C), and continued for up to 1 month (Fig. 1D). From 1 month after surgery, however, her symptoms gradually improved and complete remission was observed (Fig. 1E). In addition, although more than 2 years have passed since complete remission, no recurrence has been observed (Fig. 1F).

DOI： 10.1111/j.1365-4632.2006.02900.x

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1055/s-2006-954582

Language：English   Publishing type：Research paper (scientific journal)  

Recent studies have suggested that the periodontal disease, chronic sub-clinical inflammation, is associated with atherosclerosis, although "cause or effect" relationship is still unclear. The aim of this study was to assess the association between the degree of periodontal infection and lipid profiles in diabetic subjects. Additionally, the association of such sub-clinical inflammation with HMG-CoA reductase gene expression was evaluated. One hundred and thirty-one non-obese relatively well-controlled Japanese type 2 diabetic patients were enrolled for the study. Although no significant association was observed between serum triglycerides, HLD-cholesterol and antibody titer to Porphyromonas gingivalis (Pg), the most predominant periodontal pathogen in adults, LDL-cholesterol was significantly associated with antibody titer to Pg. Concomitantly, the same works out to be true for total cholesterol. To understand the possible mechanisms underlying this association, we evaluated 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase gene expression in cultured HepG2 cells stimulated by either bacterial lipopolysaccharide (LPS) or inflammatory cytokines. Although Pg and E. coli LPS had no effect on HMG-CoA reductase gene expression, both tumor necrosis factor-α and interleukin-6 (IL-6), especially IL-6 at low concentration, markedly up-regulated HMG-CoA reductase gene expression. It can be concluded that Pg infection is associated with increased LDL-cholesterol in diabetic subjects, which may be accompanied by increased cholesterol synthesis by inflammatory cytokines.

DOI： 10.1055/s-2006-949525

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We introduced a debate-style tutorial exercise into the third-year tutorial classes with the purpose of developing the students' logic, broadening their vision and encouraging them to express their opinions in public, before an audience. The issues for debate included medical (dental) and non-medical topics. Two separate debate exercises were performed and each session concluded with an open debate. The groups' performance was evaluated by the audience, which included students and tutors. The groups received high scores; their understanding of the subjects was superior and they provided logical arguments using good presentation skills. The program appeared to have had the desired results. Thus, it was suggested that the introduction of debates into the curriculums of lower classes would be effective in teaching students new ways of thinking about problems. This would prepare them suitably for future education.

Language：English   Publishing type：Research paper (scientific journal)  

The aim of the present study was to investigate the relationships between interleukin 6 (IL-6) and insulin resistance, serum leptin, serum adiponectin, or serum lipids including triglycerides in 98 nonobese Japanese type 2 diabetic patients. Insulin resistance was estimated by the insulin resistance index of homeostasis model assessment (HOMA-IR). Serum IL-6 concentration was negatively correlated to high-density lipoprotein cholesterol (r = -0.295, P = .004), but was not associated with HOMA-IR (r = 0.016, P = .871), body mass index (BMI) (r = 0.090, P = .375), systolic (r = 0.169, P = .116) and diastolic (r = -0.061, P = .570) blood pressures, leptin (r = 0.062, P = .544), and adiponectin (r = -0.020, P = .841) in these patients. In contrast, serum leptin level was positively correlated to HOMA-IR (r = 0.291, P = .004), BMI (r = 0.338, P < .001), and systolic blood pressure (r = 0.241, P = .025). Serum adiponectin level was negatively correlated to HOMA-IR (r = -0.288, P = .005), BMI (r = -0.308, P = .002), diastolic blood pressure (r = -0.269, P = .012), and triglycerides (r = -0.338, P < .001), and positively correlated to high-density lipoprotein cholesterol (r = 0.300, P = .003) in our patients. From these results, it can be suggested that fasting serum IL-6 is not a major factor responsible for the evolution of insulin resistance in nonobese Japanese type 2 diabetic patients.

DOI： 10.1016/j.metabol.2005.08.020

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α2 integrin on fibroblasts is reported to play an important role in the induction of drug-induced gingival overgrowth, which is characterized by excessive accumulation of type I collagen in gingival connective tissue. Silent polymorphism 807 T/C within the α2 integrin gene is associated with high/low α2 integrin expression. The aim of this study was to test the hypothesis that expression of α2 integrin 807 T/C polymorphism correlates with drug-induced gingival overgrowth. A case-control study comparing 136 subjects taking calcium channel blockers (72 with vs. 64 without drug-induced gingival overgrowth) demonstrated that the frequency of the +807 C allele was significantly higher in the case group than in the controls (odds ratio, 3.61; 95% confidence interval, 2.14 - 6.10; P < 0.05). The present findings suggest that the α2 +807 C allele is one of the genetic risk factors for drug-induced gingival overgrowth.

DOI： 10.1177/154405910508401217

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Background: Obesity is an important risk factor for diabetes, cardiovascular disease, and periodontal disease. Adipocytes appear to secrete proinflammatory cytokines which may be the molecules linking the pathogenesis of these diseases. We evaluated the relationship between obesity, periodontal disease, and diabetes mellitus insulin resistance as well as the plasma levels of tumor necrosis factor alpha (TNFα) and its soluble receptors (sTNFα) to assess the relationship of inflammation to obesity, diabetes, and periodontal infections. Methods: The relationship between periodontal disease, obesity, and insulin resistance was examined in the Third National Health and Nutrition Examination Survey (NHANES III). In a population of 12,367 non-diabetic subjects, the variable body mass index (BMI) was used as an assessment of obesity and periodontal disease was assessed by mean clinical attachment loss. The plasma levels of TNFα and sTNFα were assessed in subsets of 1,221 adults from Erie County, New York, who represented the highest and lowest quartile of BMI. These subjects had extensive periodontal and medical evaluations. Results: In the NHANES III portion of the study, BMI was positively related to severity of periodontal attachment loss (P<0.001). Weighted multiple logistic regressions showed that this relationship is likely mediated by insulin resistance, since overweight individuals (with BMI ≥27 kg/m²) with high levels of insulin resistance (IR) exhibited an odds ratio of 1.48 (95% confidence interval 1.13 - 1.93) for severe periodontal disease as compared to overweight subjects with low IR. In the Erie County adult population, the highest levels of TNFα and sTNFα receptors were found in those individuals in the highest quartile of BMI. A positive correlation of TNFα levels with periodontal disease was found only in those in the lowest quartile of BMI. Conclusions: Obesity is a significant predictor of periodontal disease and insulin resistance appears to mediate this relationship. Furthermore, obesity is associated with high plasma levels of TNFα and its soluble receptors, which in turn may lead to a hyperinflammatory state increasing the risk for periodontal disease and also accounting in part for insulin resistance. Further studies of the molecular basis of insulin resistance and its relationship to diabetes, periodontal disease, and obesity are necessary to fully test the hypothesis that adipocyte production of proinflammatory cytokines is a pathogenic factor linking obesity to diabetes and periodontal infections.

DOI： 10.1902/jop.2005.76.11-S.2075

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Pulpal wound healing followed by cavity preparation may involve reactionary or reparative dentinogenesis in relation to the cavity position; however, little is known about the molecular responses. We aimed to isolate and analyze genes induced or suppressed in the wounded pulp to identify molecular processes involved in the pulp responses to injury. Twenty-three cDNAs were isolated by cDNA subtraction between healthy and wounded pulp of rats. By library screening, we identified rat 14.7K-interacting protein (rFIP)-2A and B genes homologous to human FIP-2, being involved in regulating membrane trafficking and cellular morphogenesis. RT-PCR analysis showed induction for only rFIP-2B in the wounded pulp. In situ hybridization analysis revealed that both rFIP-2s were expressed strongly in condensing mesenchymal cells of the palatal process and surrounding Meckel's cartilage, but not in intramembranous chondrogenic cells. Thus, up-regulated rFIP-2B expression may play a role in regulating membrane trafficking or cellular morphogenesis of these embryonic and wounded pulpal cells.

DOI： 10.1177/154405910508400912

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Background: Aplastic anemia (AA) is a rare hematologic disease characterized by hypo-cellular bone marrow. The clinical features include fatigue, increased bruising, and gingival bleeding caused by anemia, leukopenia, and thrombocytopenia. A patient with AA is at high risk for infection because of leukopenia. The risk of systemic infection is especially high in AA patients with severe local infections, including periodontitis. Accordingly, periodontal treatment should include antibiotic prophylaxis to reduce the risk of systemic infection. However, treatment of periodontitis in the AA patient is significantly complicated by the bleeding disorder. We present a case report of the successful periodontal treatment of an AA patient with spontaneous gingival bleeding. Methods: The patient was closely monitored for platelet and neutrophil counts before every treatment. The patient's platelet count was always under 10,000/μl. Therefore, it was necessary to increase platelet counts to over 25,000/μl by transfusion, after which subgingival scaling with anesthesia was performed. When the neutrophil count was less than 2,000/μl, local minocycline chemotherapy was applied to the pockets. Periodontal infection was monitored by detection of bacterial DNA and measurement of serum immunoglobulin (Ig) G titer against periodontal bacteria. Results: Following the physical and chemical treatment, the gingival appearance improved dramatically and the spontaneous gingival bleeding disappeared. Moreover, the IgG titer against periodontal bacteria decreased to normal range and specific periodontal pathogens were no longer detectable in the tested pockets. Conclusion: We believe that the treatment strategy in the present report provides new sight into treatment planning for severely medically compromised patients.

DOI： 10.1902/jop.2005.76.7.1211

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Human β-defensin-2 (hBD-2) is an antimicrobial peptide with a broad spectrum of antimicrobial activity against bacteria, yeast and fungi. Here, we analyzed the transcriptional regulation of hBD-2 in cultured human cervical carcinoma (HeLa) cells with or without lipopolysaccharide (LPS). DNA from position -329 to -39 in the hBD-2 promoter region contained the consensus binding sites for transcription factors, one site for nuclear factor for IL-6 expression (NF-IL6) and two sites for nuclear factor-κB (NF-κB). Reporter gene assays for promoter activity revealed that the region had the highest level of responsiveness to LPS. Furthermore, mutations in both of the NF-κB binding sites caused a significant reduction of the responsiveness to LPS, whereas mutation in the NF-IL6 binding site resulted in an elevation of the basal promoter activity. Electrophoretic mobility shift assays demonstrated that LPS induced the binding of HeLa nuclear factors to 60-bp probe containing the two NF-κB binding sites, suggesting that the sites were essential for the binding. Our results suggest that the two NF-κB binding sites contribute to LPS-mediated hBD-2 transcription while the NF-IL6 binding site represses LPS-independent hBD-2 transcription in the HeLa cells.

DOI： 10.1016/j.femsim.2005.01.008

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Background: Individual differences in T cell responsiveness to interleukin 12 (IL-12), resulting from inherited factors, may be responsible for differences in the intensity of cell mediated immune (CMI) responses in patients with leprosy, a disease with a wide clinical spectrum. Aim: Polymorphisms in the 5′ flanking region of the IL12RB2 gene were analysed to determine potential immunogenetic factors affecting CMI responses, using leprosy as a model. Methods: Polymorphisms in the 5′ flanking region of IL12RB2 were examined using direct sequencing techniques, and allele frequencies between patients with lepromatous leprosy and patients with tuberculoid leprosy were compared. The effect of these single nucleotide polymorphisms (SNPs) on IL12RB2 expression was estimated using the dual luciferase reporter gene assay in Jurkat T cells. Results: Several SNPs, including -1035A>G, -1023A>G, -650delG, and -465A>G, were detected within the 5′ flanking region of IL12RB2. The frequency of haplotype 1 (-1035A, -1023A, -650G, -464A) was high in the general Japanese population, but was significantly lower in lepromatous patients compared with tuberculoid patients and healthy controls. Reporter gene assays using Jurkat T cells revealed that all haplotypes carrying one or more SNP exhibited a lower transcriptional activity compared with haplotype 1. Conclusion: SNPs within the 5′ flanking region of IL12RB2 affect the degree of expression of this gene and may be implicated in individual differences in CMI responsiveness to mycobacterial antigens, leading to lepromatous or tuberculoid leprosy.

DOI： 10.1136/jcp.2004.023903

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Outer membrane protein with a 53-kDa molecular weight (Ag53) isolated from Porphyromonas gingivalis evokes strong humoral immune responses in many periodontitis patients. To examine the effects of cytokines produced by Ag53-specific Th cells on the IgG production against Ag53, we established Ag53-specific Th-cell lines from patients with early onset periodontitis and from healthy volunteers. We then developed a mixed lymphocyte culture system between Ag53-specific Th cells and auto- or alloderived T-cell-depleted leukocytes produced from the subjects whose HLA class II haplotypes were completely matched. Interferon-γ production was observed in all Th cell lines from patients and healthy subjects. As for Th2 type cytokines, interleukin (IL)-4, IL-5, IL-6 and IL-10 production varied greatly in Th cells regardless of the periodontal condition of the donor. Only Th cell lines with a high Th2/Th1 ratio induced Ag53-specific IgG production when cocultured with T-cell-depleted leukocytes. Thus, the difference in Th2/Th1 balance may regulate the Ag53-specific IgG production.

DOI： 10.1111/j.1399-302X.2004.00203.x

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A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64°C with a detection limit of 20 cells of P. gingivalis. Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. Detection limits of these naked-eye inspections were 20 cells and 200 cells, respectively. Although false-positive DNA amplification was observed from more than 10⁷ cells of Porphyromonas endodontalis, no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real-time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10²-10⁶ cells. The real-time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real-time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis, especially in its rapidity and simplicity.

DOI： 10.1016/j.femsim.2004.08.005

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Periodontal disease has been considered as a complication of diabetes mellitus. A recent epidemiological study revealed that obesity is an independent risk factor for periodontal disease. Obesity and type 2 diabetes mellitus are associated with many metabolic disorders including insulin resistance, dyslipidemia, hypertension and atherosclerosis. Chronic sub-clinical inflammation, although often for the most part in a healthy reference range, has recently been declared part of the insulin resistance syndrome, as such inflammatory responses appear to participate in the progression of metabolic disorders, including type 2 diabetes and atherosclerosis. We hypothesized that periodontal disease is one such sub-clinical inflammation. Here, we summarize current knowledge supporting this concept primarily based on data obtained from our own studies and propose a new concept that periodontal disease should be considered as part of the insulin resistance syndrome.

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An elevated level of C-reactive protein (CRP) predicts the future development of coronary heart disease. Periodontitis appears to up-regulate CRP. CRP is produced by hepatocytes in response to interleukin-6 (IL-6). A major source of IL-6 in obese subjects is adipocytes. We hypothesized that lipopolysaccharide (LPS) from periodontal pathogens stimulated adipocytes to produce IL-6, and that the production was suppressed by the drugs targeted against insulin resistance, thiazolidinedione (pioglitazone), since this agent potentially showed an anti-inflammatory effect. Mouse 3T3-L1 adipocytes were stimulated with E. coli, P. gingivalis, and F. nucleatum LPS. The IL-6 concentration in culture supernatants was measured. All LPS stimulated adipocytes to produce IL-6. Although pioglitazone changed adipocyte appearance from large to small, and completely suppressed P. gingivalis and F. nucleatum LPS-induced IL-6 production, E. coli LPS-induced IL-6 production was not efficiently blocked. Thus, pioglitazone completely blocked periodontal-bacteria- derived LPS-induced IL-6 production in adipocytes, a major inducer of CRP.

DOI： 10.1177/154405910508400306

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Background: Gingival overgrowth is a common side-effect following administration of cyclosporin A. We reported previously that lysosomal protease cathepsin-L activity, but not cathepsin-B, was significantly suppressed by short-term cyclosporin A exposure in human gingival fibroblasts. Although this suppression may lead to decreased degradation of gingival connective tissue, a net increase in matrix proteins, and gingival overgrowth, the effects of cyclosporin A need to be more elucidated, considering the long-term use for patients following organ transplantation. Objective: The aim of the present study was to evaluate the effects of clinically relevant doses of cyclosporin A on cultured human gingival fibroblasts. We evaluated the effects of long-term cyclosporin A exposure on cell proliferation, mRNA expression of various proteases and both cathepsin-B and -L activity in human gingival fibroblasts. Materials and Methods: Human gingival fibroblasts were isolated from three donors' healthy gingiva and cultured from five to eight passages with or without 200 ng/ml of cyclosporin A. Proliferative activity of cyclosporin A-treated cells was examined using MTT assay. Total RNA and cellular proteins were collected for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis and for measurement of the cathepsin-B and -L activity. Results: Long-term cyclosporin A exposure had no effects on cell proliferation. Accumulation of cathepsin-B, -H and -L mRNA was markedly suppressed by long-term cyclosporin A exposure, whereas accumulation of another lysosomal enzyme N-acetyl-β-D-glucosaminidase mRNA, which is involved in remodeling of gingival epithelium, was not apparently impaired in cyclosporin A-treated cells. Accumulation of matrix metalloprotease-1 (MMP-1) and tissue inhibitor of matrix metalloprotease-1 (TIMP-1) mRNA, which are involved in remodeling of extracellular matrix, also was not impaired. In addition, we demonstrated that long-term cyclosporin A exposure significantly suppressed not only the activity of the active form of cathepsin-(B + L) compared to the activity in non-treated cells (p = 0.0458), but also the activity of the active form of cathepsin-B (p < 0.0001) in human gingival fibroblasts. Conclusion: The decreased ability of protein degradation by not only cathepsin-L but also cathepsin-B is, at least, one of the several factors developing the cyclosporin A-induced gingival overgrowth.

DOI： 10.1111/j.1600-0765.2004.00746.x

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Objective: To investigate the prevalence of blood pressure levels and hypertension-related diseases and to evaluate the risks associated with dental patients in Japan. Research design: Retrospective cross-sectional. Method: Blood pressure levels and medical histories of hypertension-related diseases obtained from 3,811 adult outpatients consulting a University Dental Hospital were investigated. Blood pressure levels were compared with those reported in the Japanese national survey obtained from a standard Japanese population. The relationships between gender or age and blood pressure level or the rate of the subjects with a hypertension-related disease were evaluated. Results: Mean values of blood pressure in the present study were similar to those in the Japanese national survey. Among hypertension-related diseases, hypertension showed the highest prevalence (10.9%) in subjects. Blood pressure levels and the rates of subjects with hypertension-related diseases were significantly related to increasing age. Elderly subjects had a tendency to combine hypertension or high blood pressure with hypertension-related diseases. Further, 20.4% of subjects who had not been diagnosed with hypertension had high blood pressure. Among them, 1.5% had blood pressure more than 180/110 mmHg. Conclusions: The results suggested that blood pressure levels of dental patients would reflect the prevalence of blood pressure levels and hypertension in the Japanese general population, and that high blood pressure and increasing age are the greatest risk factors in the medical status of dental patients. Furthermore, because many subjects examined were unaware of their high blood pressure levels, caution is required prior to performing dental procedures.

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Leprosy is characterized by a wide spectrum of clinical features depending on the individual differences in Th1-type immunity. The objective of this study was to evaluate whether monocyte activation by stimulus via class II HLA molecules would be correlated with the differences in cellular immune responses among diverse clinical forms of leprosy. IL-1β and IL-12 productivity in monocyte preparations obtained from PBMCs was estimated in patients with lepromatous- and tuberculoid-type leprosy. We found that monocytes from lepromatous patients produced significantly higher (about 4-fold higher) amounts of IL-12 as compared to in patients with tuberculoid type of leprosy when class II HLA molecules were cross-linked with anti-HLA class II antibodies, whereas almost equal amounts of IL-1β were produced from each monocyte preparation by stimulus via class II HLA molecules regardless of the clinical form of leprosy. These results suggest that monocyte activation differs between lepromatous and tuberculoid patients in terms of IL-12 secretion, which might be related to individual differences in the cellular immune responses according to the clinical type of leprosy.

DOI： 10.1111/j.1600-0463.2004.apm11204-0507.x

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Diabetic patients are susceptible to severe inflammatory periodontitis manifesting as swollen gingiva with bleeding, but the underlying mechanism is not well understood. Our purpose was to determine the effect of a high glucose (HG) condition on the interleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R)-induced activation of signaling and vascular endothelial growth factor (VEGF) expression in human gingival fibroblasts (HGFs). In this study, HGFs were cultured for at least two passages under a normal glucose (NG; 5.5 mM) condition or high glucose (25 mM) condition. Importantly, the HG condition significantly induced expression of gp130 mRNA in HGFs compared with levels in control cells. Consistent with the expression of its mRNA, the HG condition also increased the expression of gp130 protein, and phosphorylation of the tyrosine residue by gp130 was enhanced significantly by IL-6/sIL-6R stimulation. Furthermore, the HG condition enhanced the IL-6/sIL-6R-induced phosphorylation of p44/42 MAPK and led to subsequent activation of CCAAT/enhancer binding protein in nuclei. In contrast, there was no significant difference in phosphorylation of JNK between the HG and NG condition. Interestingly, HGFs increased IL-6/sIL-6R-induced VEGF165 mRNA expression and VEGF165 secretion under the HG condition compared with levels under the NG condition. In contrast, the induction of VEGF165 secretion was partially inhibited by PD98059 (selective p44/42 MAPK inhibitor) under the HG condition. In addition, the VEGF165 secretion was completely inhibited by the combination of PD98059 and SP600125 (JNK inhibitor). Our findings suggest that the HG condition indirectly increases VEGF expression via activation of gp130-mediated p44/42 MAPK-CCAAT/enhancer binding protein signaling in HGFs. Thus, elevated VEGF secretion in HGFs under the HG condition may play a role in the development of the severe periodontitis observed in diabetic patients.

DOI： 10.1074/jbc.M311688200

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The aim of the present study was to investigate the relationship between periodontal bacteria infection (Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedius) and C-reactive protein (CRP) and albuminuria in non-obese Japanese type 2 diabetic patients. One hundred and thirty-four non-obese Japanese type 2 diabetic patients without evidence of current acute illness including clinically significant acute infectious disease were enrolled into the study. The degree of periodontal bacterial infection was evaluated using IgG titer against Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, or Prevotella intermedius. The bacterial sonic extracts were used as antigens. High-sensitivity CRP (hCRP), glucose, glycosylated hemoglobin (HbA _1c), and lipids were also measured after an overnight fast. Urinary albumin excretion rate as a ratio of urinary albumin and urinary creatinine was assessed in a morning spot urine sample using a commercial enzymatic immunoassay. The prevalence of Porphyromonas gingivalis infection was 52.2% and that of Actinobacillus actinomycetemcomitans and Prevotella intermedius was 7.5 and 14.2%, respectively. IgG titer against Porphyromonas gingivalis significantly correlated with CRP (r = 0.225, p < 0.001) and albuminuria (r = 0.185, p < 0.05), while IgG titer against Actinobacillus actinomycetemcomitans or Prevotella intermedius was not associated with either parameter. These results suggest that among periodontal bacteria, Porphyromonas gingivalis infection is associated with atherosclerosis in non-obese Japanese type 2 diabetic patients.

DOI： 10.1055/s-2004-814221

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Previous studies suggested that the onset of phenytoin-induced gingival overgrowth depended on serum phenytoin concentration. Cytochrome P450 2C (CYP2C) plays an important role in phenytoin metabolism. Recently, single nucleotide polymorphisms in the coding region of CYP 2C influencing phenytoin metabolism were identified. The purpose of the present study was to see if CYP 2C polymorphisms might relate to the onset and severity of phenytoin-induced gingival overgrowth. Twenty-eight epileptic patients taking phenytoin aged 15 to 75 (mean age: 42.2 years old, 20 males and 8 females) and 56 unrelated healthy subjects aged 30 to 48 (mean age: 36.8 years old, 48 males and 8 females) were examined for CYP 2C polymorphisms. All epileptic subjects were examined for the degree of gingival overgrowth, daily phenytoin dose and serum phenytoin concentration. The results indicated about 7% of the subjects including epileptic and healthy subjects examined were positive for CYP 2C9*3. However, the degree of gingival overgrowth did not directly correlate with CYP 2C polymorphisms. Nevertheless, the subjects with severer gingival overgrowth exhibited significantly higher serum phenytoin concentration, indicating that phenytoin metabolism is an important determinant for the severity of the disease. Additionally, CYP 2C9*3 carriers exhibited significantly higher serum drug concentration to drug dose. Therefore, we concluded although the gene analysis is not directly related to diagnose the disease itself, it can be utilized in estimating serum phenytoin concentration from drug dose, which in turn serves to predict the future development and clinical course of the disease.

DOI： 10.1016/j.lfs.2003.07.018

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Aspiration pneumonia is a common cause of death in older people, and the pathophysiology is a chronic respiratory failure with a mild airway inflammation. In this study, we established a mild inflammatory pneumonia model using Porphyromonas gingivalis (Pg) pathogen-infected mice. It elucidated the effects of Pg-infected pneumonia on proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), and IL-1β production in both lung tissue and serum. We also elucidated production of soluble (s) TNF receptor (R) s, because TNF-α is considered to be a dominant inflammatory mediator. Lung TNF-α levels significantly increased at 2 h after infection, and rapidly returned to basal level at 24 h. Consistent with increase of TNF-α, remarkable increase of sTNFR2 but not sTNFR1 was detected in lung tissue from 2 to 72 h. Interestingly, sTNFR2/sTNFR1 ratio was significantly enhanced at 2 h in serum. In addition, lung IL-1β and IL-6 levels also significantly increased from 2 to 24 h. Importantly, we found that IL-6 levels in serum reflected its local level. These results may suggest that systemically produced sTNFR2 and IL-6 could be a key role to modulate proinflammatory activities of TNF-α in Pg-induced lung inflammation simulated aspiration pneumonia.

DOI： 10.1016/j.yexmp.2003.09.002

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Periodontal healing requires the participation of regulatory molecules, cells, and scaffold or matrix. Here, we hypothesized that a certain set of genes is expressed in alveolar bone wound healing. Reciprocal subtraction gave 400 clones from the injured alveolar bone of Wistar rats. Identification of 34 genes and analysis of their expression in injured tissue revealed several clusters of unique gene regulation patterns, including the up-regulation at 1 wk of cytochrome c oxidase regulating electron transfer and energy metabolism, presumably occurring at the site of inflammation; up-regulation at 2.5 wks of pro-α-2 type I collagen involving the formation of a connective tissue structure; and up-regulation at 1 and 2 wks and down-regulation at 2.5 and 4 wks of ubiquitin carboxyl-terminal hydrolase 13 involving cell cycle, DNA repair, and stress response. The differential expression of genes may be associated with the processes of inflammation, wound contraction, and formation of a connective tissue structure.

DOI： 10.1177/154405910408300707

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Angiogenesis is a common complication of organ-transplant rejection. One of the primary responsible molecules for enhanced angiogenesis is vascular endothelial growth factor (VEGF). Activated protein (AP)-1 is considered to play a key role in the transcription of VEGF. c-jun N-terminal kinase (JNK), one of the MAP kinase family members, plays a critical role in AP-1 activation. Thus, we tested the effect of a novel JNK inhibitor, SP600125, on VEGF production in fibroblasts. SP600125 significantly suppressed interleukin (IL)-6-induced production of VEGF in cultured fibroblasts. Cyclosporine A (CsA), a known in vitro antiangiogenic reagent, partially mimicked this suppression. In fact, CsA suppressed IL-6-induced phosphorylation of JNK. The results indicate that although both SP600125 and CsA are anti-angiogenic by inhibiting VEGF production by way of a JNK-dependent pathway, the inhibitory effect was much stronger with the novel inhibitor of JNK than with CsA.

DOI： 10.1097/01.TP.0000085661.52980.95

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Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp^R Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10⁷ cells (10-10⁷ copies for tetQ gene), while the quantitative range for total bacteria was 10²-10⁷ cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.

DOI： 10.1016/S0928-8244(03)00224-4

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Background: Elevated levels of C-reactive protein (CRP) and decreased plasma adiponectin are associated with increased risk of atherosclerosis. Furthermore, recent observations suggested that adiponectin and tumor necrosis factor-α (TNF-α) suppressed each other's production. Since periodontal disease has been suggested to act as a risk factor for atherosclerosis, we examined the effects of antimicrobial periodontal treatment on CRP, adiponectin, and TNF-α levels. Methods: Fifteen chronic periodontitis patients with various systemic conditions at high risk for atherosclerosis were enrolled in the study. Patients were non-surgically treated with topical application of antibiotics and mechanical debridement of calculus once a week for 1 month. Before and after therapy, CRP, adiponectin, and TNF-α levels were measured. Results: Both CRP and TNF-α levels were significantly decreased after treatment (P <0.01 and P <0.03, respectively), while adiponectin levels did not change significantly. Conclusions: Periodontal treatment is effective in reducing CRP and TNF-α, while adiponectin does not appear to be influenced by periodontal treatment. Elevated levels of CRP and TNF-α may be associated with increased risk for future development of atherosclerosis in periodontitis patients.

DOI： 10.1902/jop.2003.74.8.1231

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The role of human leukocyte antigen (HLA) class II molecules on non-antigen presenting cells has been a matter of controversy. We recently reported that ligation of HLA-DR molecule with anti-HLA-DR antibodies (L243) and/or antigenic peptide/T cell receptor complex resulted in a secretion of several chemokines such as RANTES. In the present study, we aimed to detect putative signal transduction pathway leading to RANTES production from fibroblasts when the DR molecules were ligated with L243. Protein tyrosine kinase inhibitor (GF109203X) suppressed RANTES expression in a dose dependent manner for up to 50% from gingival fibroblasts (GF), while protein kinase C inhibitor (genistein) had no inhibitory effect. Ligation of DR molecules with L243 resulted in tyrosine phosphorylation of 54 kDa cellular protein. Thus, we suspected that either Jun N-terminal kinase-2 (JNK-2) or Src family proteins were involved in HLA-DR-mediated signaling. JNK inhibitor (SP600125), but not Src inhibitor (PP2), suppressed both L243 stimulated RANTES mRNA expression and protein secretion. The maximum inhibition for RANTES production by SP600125 was more than 80%. Additionally, JNK inhibitor nearly completely blocked tumor necrosis factor-α (TNF-α)-induced RANTES production in GF. Furthermore, ligation of GF HLA-DR with L243 induced selective phosphorylation of JNK-2. We concluded that JNK-2 was one of the HLA-DR-mediated signal transduction pathways.

DOI： 10.1016/S1043-4666(03)00123-6

Language：English   Publishing type：Research paper (scientific journal)  

Objectives: Tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) participate in the establishment of inflammatory lesions in periodontitis. High production of these cytokines may relate to the severity of periodontitis. There have already been several studies examining the association between periodontitis and single nucleotide polymorphisms (SNPs) that affect cytokine productivity. Recently, new SNPs of TNF-α, -1031, -863 and -857, variants of which are observed in a relatively large proportion in Japanese, have been identified. The variant alleles of these SNPs have been suggested to be related to high TNF-α production. For a better understanding of the genetic factors associated with the severity of periodontitis, further analysis including these newly identified SNPs is essential. In addition, previous reports on TNF-α or IL-1β SNPs associated with periodontitis were mainly for Caucasian populations. Therefore, the aim of this study is to examine the association between severe periodontitis in Japanese and the following SNPs: five in the TNF-α gene promoter (-1031, -863, -857, -308, -238) and three in the IL-1β gene (-511, -31, +3953). Material and Methods: A total of 128 Japanese individuals were enrolled in this study. They were 64 patients with severe adult periodontitis and 64 healthy subjects. TNF-α and IL-1β SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism for all subjects. TNF-α and IL-1β production from LPS-stimulated monocytes/macrophages was also measured for 15 healthy male subjects. Results: TNF-α production in TNF-α -1031/ -863 (linkage disequilibrated) or -857 SNP variant allele carriers tended to be elevated, and the frequency of subjects who carried at least one variant allele in TNF-α -1031, -863 or -857 SNPs among severe periodontitis patients was significantly higher than in healthy subjects. Conclusion: Since the frequency of subjects who carried at least one variant allele in TNF-α -1031, -863 or -857 SNPs was higher in periodontitis patients than in healthy subjects, TNF-α -1031, -863 and -857 SNPs appear to be associated with severe adult periodontitis in Japanese populations.

DOI： 10.1034/j.1600-051X.2003.00287.x

Language：English   Publishing type：Research paper (scientific journal)  

The aim of the present study was to investigate whether non-obese Japanese type 2 diabetic patients with porphyromonas gingivalis infection have atherosclerotic vascular diseases. A total of 134 non-obese Japanese type 2 diabetic patients (96 men and 38 women, aged 36 to 84 years, body mass index [BMI] 20.1 to 26.9 kg/m²) were studied. In conjunction with BMI, glycosylated hemoglobin (HbA_1c), fasting glucose, and serum lipids (triglycerides, total cholesterol, high-density lipoprotein [HDL] cholesterol, low-density lipoprotein [LDL] cholesterol) were measured. LDL cholesterol was calculated using the Friedewald formula. Using high-resolution B-mode ultrasound scan, we measured intimal medial thickness (IMT) in plaque-free segments of bilateral common carotid arteries, and the mean of IMT in 2 vessels was used for the analysis. Furthermore, we calculated the degree of stenosis in plaque segments of bilateral common carotid arteries. The degree of carotid atherosclerosis was expressed as a percentage ratio between the area of plaque and that of the lumen using the formula (Lumen Area Residual - Lumen Area)/Lumem Area x 100. Both the areas were automatically measured by the system on a frozen transverse scanning plane at the site of maximal narrowing. When 2 or more plaques were present in the vessel, only that causing the greatest degree of stenosis was considered for analysis. Values represent mean±SEM unless otherwise stated. Immunoglobulin G (IgG) titer against porphyromonas gingivalis was 245 ± 65 (mean ± 2 SD) in nondiabetic healthy subjects. In contrast, there was a wide variation in IgG titer against porphyromonas gingivalis in type 2 diabetic patients studied (range, 16 to 26,800). Thus, we classified our type 2 diabetic patients into 2 subpopulations according to the value of mean ± 2 SD (= 310) of nondiabetic healthy subjects: one with high IgG titer against porphyromonas gingivalis (>310) (1,422 ± 408) and the other with normal IgG titer against porphyromonas gingivalis (<310) (152 ± 10, P = .002). The populations did not differ with respect to age, sex, BMI, fasting glucose, HbA_1c, serum triglycerides, total, HDL, and LDL cholesterol levels. Although the mean IMT in plaque-free segments was not different between the 2 groups (0.73 ± 0.03 v 0.68 ± 0.02 mm, P = .098), the degree of stenosis in plaque segments was significantly higher in the high IgG titer group (12.0% ± 2.2%) than in normal one (5.5% ± 1.4%, P = .009). From these results, it can be concluded that porphyromonas gingivalis infection, although still a subclinical infection, is associated with atherosclerotic vascular disease in non-obese Japanese type 2 diabetic patients.

DOI： 10.1053/meta.2003.50001

Language：English   Publishing type：Research paper (scientific journal)  

Objectives: Denaturing gradient gel electrophoresis (DGGE) was applied to the microbiologic examination of subgingival plaque. Materials and methods: The PCR primers were designed from conserved nucleotide sequences on 16S ribosomal RNA gene (16SrDNA) with GC rich clamp at the 5′-end. Polymerase chain reaction (PCR) was performed using the primers and genomic DNAs of typical periodontal bacteria. The generated 16SrDNA fragments were separated by denaturing gel. Results: Although the sizes of the amplified DNA fragments were almost the same among the species, 16SrDNAs of the periodontal bacteria were distinguished according to their specific sequences. The microflora of clinical plaque samples were profiled by the PCR-DGGE method, and the dominant 16SrDNA bands were cloned and sequenced. Simultaneously, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were detected by an ordinary PCR method. In the deep periodontal pockets, the bacterial community structures were complicated and P. gingivalis was the most dominant species, whereas the DGGE profiles were simple and Streptococcus or Neisseria species were dominant in the shallow pockets. The species-specific PCR method revealed the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in the clinical samples. However, corresponding bands were not always observed in the DGGE profiles, indicating a lower sensitivity of the DGGE method. Conclusion: Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, it could visualize the bacterial qualitative compositions and reveal the major species of the plaque. The DGGE analysis and following sequencing may have the potential to be a promising bacterial examination procedure in periodontal diseases.

DOI： 10.1034/j.1600-0765.2003.02607.x

Language：English   Publishing type：Research paper (scientific journal)  

Objectives: The aim of this study is to isolate mechanical stress-induced genes (MSGens) from human periodontal ligament (PDL) cells and to analyze profiles of the mRNA expression of these genes. Background: Differential expression of genes in PDL cells under physiological stress such as occlusal force is thought to be orchestrated not only for the remodeling of PDL itself but also for the repair and regeneration of periodontal tissues. However, little is known about the genes expressed in PDL cells under mechanical stress. Methods: The cDNA from mechanical stress-applied human PDL cells was subtracted against the cDNA from static control cells. The subtracted cDNA was amplified by polymerase chain reaction (PCR) and cloned for further analysis. Results: Among 68 independent clones isolated, 15 contained DNA fragments greater than 250 bp. Reverse Northern analysis revealed a marked induction of MSGen-15 and MSGen-28 mRNA expression in the mechanical stress-applied cells. However, little difference in the magnitude of expression for the other MSGens was detected between the stress-applied cells and the control cells. After nucleotide sequencing and the analysis of homology with known genes, five clones were identified; ribosomal protein S27 (MSGen-9), MRG 15 (MSGen-15), androgen-binding protein (MSGen-18), cathepsin H (MSGen-28), and cytochrome c (MSGen-47). Interestingly, it has been reported that MRG 15 is a novel transcription factor involved in the regulation of cell growth and senescence. The remaining 10 clones, classified into six sequence types, had no significant homology with any known genes. Conclusions: These results suggest that many known and unknown genes are expressed in response to mechanical stress in PDL cells, and that a transcription factor, MRG 15, may be responsible for molecular events in PDL cells under mechanical stress.

DOI： 10.1034/j.1600-0765.2003.00602.x

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It is generally accepted that obesity is associated with many other multiple-risk factor syndromes such as hypertension, hyperlipidemia, type 2 diabetes mellitus, and periodontal disease. The number of obese people is increasing rapidly in both western and eastern countries. Adipocytes in the adipose tissues of obese people produce large quantities of biologically active molecules such as leptin, an important molecule regulating energy expenditure and body weight. Therefore, adipocyte-derived active molecules, named adipocytokines, are candidate molecules accounting for the close association between obesity and other multiple-risk factor syndromes. The proinflammatory cytokine tumor necrosis factor-α (TNF-α) is produced by adipocytes, and its blood concentration is elevated in obese patients and declines with weight loss. Studies have demonstrated that TNF-α suppresses insulin action via its specific receptor; hence, it exacerbates insulin resistance. In addition to adipocytes, monocytes/macrophages produce large quantities of TNF-α. Thus, TNF-α, produced from monocytic cells due to inflammatory diseases, may have an additive influence on insulin sensitivity to adipocyte-derived TNF-α. Here, we hypothesized that 1) TNF-α produced by the adipose tissues of obese patients acts as a risk factor for periodontal inflammation, and 2) TNF-α produced due to periodontal inflammation may be an additional important factor influencing insulin sensitivity in both obese and type 2 diabetic patients. We believe that this interaction is a possible mechanism accounting for a 2-way relationship between type 2 diabetes and periodontal disease.

DOI： 10.1902/jop.2003.74.1.97

Language：English   Publishing type：Research paper (scientific journal)  

Genes expressed by human periodontal ligament fibroblasts (HPFs) are likely to be associated with specific functions of the ligament. The aim of this study is to profile genes expressed highly by HPFs. A library (6 × 10 ³ pfu) was constructed, followed by subtraction of HPF cDNAs with human gingival fibroblast (HGF) cDNAs. Reverse-dot hybridization revealed that 33 clones expressed higher levels of specific mRNAs in HPFs than in HGFs. These were mRNAs for known genes, including several associated with maturation and differentiation of cells. None had been reported in PFs. One clone, PDL-29, identified as a COX assembly factor, showed much stronger mRNA expression in HPFs than in HGFs in culture. In rat periodontium, however, PDL-29 mRNA expression was similar in PFs and GFs. These results suggest that HPFs express many previously unreported genes associated with maturation and differentiation, but expression can differ in vitro and in vivo.

DOI： 10.1177/154405910308200814

Language：English   Publishing type：Research paper (scientific journal)  

Drug-induced gingival overgrowth, the chronic side effect of calcium antagonists, is frequently seen due to the increase in patients with hypertension, although the etiology of the disease is largely unknown. I-cell disease, which accompanies gingival overgrowth, is characterized by a deficiency in UDP-N- acetyl-glucosamine and is classified as one of the lysosomal storage diseases. Here, we hypothesized that a common mechanism may underlie the etiology of gingival overgrowth seen in patients treated with calcium antagonist and in patients with I-cell disease. A calcium antagonist, nifedipine, specifically suppressed cathepsin-L activity and mRNA expression, but not that of cathepsin-B in cultured gingival fibroblasts. The activity of cathepsin-L was suppressed up to 50% at 24 hours after treatment of the cells with the reagent. The selective suppression of cathepsin-L activity appeared not to be dependent on Ca
²⁺
, since treatment of the cells with thapsigargin suppressed both cathepsin-B and -L activity. Mice deficient in the cathepsin-L gene manifested enlarged gingivae. Histological observation of the gingivae demonstrated typical features of acanthosis, a phenotype very similar to that of experimentally induced gingival overgrowth. Since cathepsin-L deficiency was reported to be associated with thickening of the skin, impaired cathepsin-L activity may play a key role in the establishment of skin and gingival abnormalities seen in I-cell disease. In addition, reduced cathepsin-L activity may play an important role in inducing drug-induced gingival overgrowth.

DOI： 10.1016/S0002-9440(10)64483-5

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Fibroblasts are known to express histocompatibility leukocyte antigen DR (HLA-DR) molecules on their cell surface upon stimulation with interferon γ (IFN-γ), while the exact roles of HLA-DR on fibroblasts remain undetermined. To understand the role of HLA-DR molecules on fibroblasts, we examined whether: (1) fibroblasts act as antigen presenting cells (APC) which activate helper T (Th) cells; and/or (2) fibroblasts are activated via HLA-II molecules by making a T-cell receptor (TCR)-peptide-major histocompatibility complex (MHC) complex. We used Th₀ clone HT8.3, which recognizes an antigenic peptide (Ag53 p141-161) in the context of DRB1*1501, as well as IFN-γ-treated and irradiated periodontal ligament fibroblasts (PDL) expressing DRB1*1501 molecules. When peptide-pulsed fibroblasts were co-incubated with HT8.3 treated by the protein synthesis inhibitor emetine, peptide-induced de novo expression of lymphokines and cell-surface molecules on T cells can be neglected. The antigen presenting capacity of these fibroblasts was evaluated by examining the proliferative responses of Th cells. Possible activation of fibroblasts by stimulation via HLA-DR molecules was evaluated by quantitating secreted cytokines in the supernatants after 18-h culture with or without anti-HLA-DR monoclonal antibody (mAb) or emetine-treated HT8.3. Indeed, Th cells did not show proliferative responses when peptide-pulsed PDL were used as APC, whereas PDL produced larger amounts of interleukin (IL) 6, IL-8, monocyte chemoattractant protein 1 (MCP-1) and regulated upon activation, normal T expressed and secreted (RANTES) compared with controls, when cultured with anti-HLA-DR mAb or emetine-treated HT8.3. These findings suggest that HLA-DR expressed on fibroblasts do not present antigens to induce T-cell proliferation, but may act as receptor molecules that transmit signals into fibroblasts, based on DR-peptide-TCR interaction, resulting in the secretion of several cytokine species.

DOI： 10.1006/cyto.2001.0976

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2337/diacare.25.10.1888

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Major membrane protein II (MMP II) of Mycobacterium leprae (M. leprae) is a 22kDa protein inducing humoral immune response in leprosy patients. MMP II-specific bulk T cell lines were established from leprosy patients to determine major T cell epitopes in MMP II and to evaluate lymphokine production induced by MMP II. These bulk T cell lines reacted to one or more peptides in the locus of amino acid residues from 23 to 109 of MMP II. The proliferative responses of all T cell lines were mainly inhibited by the addition of anti-DRB1 mAb. Many bulk T cell lines induced IFN-γ, IL-5, but not IL-4. However, it was not possible to distinguish the LL or TT types of leprosy based on the pattern of T cell epitopes and the lymphokine productivity in the responses against MMP II. Thus, it appears that T cell response to MMP II is restricted by the HLA-DRB1 molecule, but not by DQ and DP molecules, which results in the induction of IFN-γ production.

DOI： 10.1016/S0264-410X(01)00354-1

Language：English   Publishing type：Research paper (scientific journal)  

Recent attention has been focused on our understanding of the negative influences of oral chronic inflammation on systemic health. Successful periodontal treatment appears to have beneficial effects on the metabolic control of type 2 diabetes. Although type 2 diabetes is a multiple-risk-factor syndrome, lowered insulin sensitivity, called insulin resistance, is essential in developing the disease. Pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), produced from adipose tissues in obese subjects, is known to play a predominant role in inducing insulin resistance. Therefore, it can be hypothesized that anti-infectious periodontal treatment may improve metabolic control of diabetes via improved insulin sensitivity by reducing peripheral TNF-α concentration. In this review, we summarize the nature of insulin resistance and discuss the mechanisms by which insulin sensitivity is influenced by chronic inflammation, such as in periodontal disease.

Language：English   Publishing type：Research paper (scientific journal)  

Background: Tumor necrosis factor-α (TNF-α) may play an important role in insulin resistance. In this study, we hypothesized that TNF-α produced due to periodontal inflammation synergistically affects insulin resistance as well as TNF-α produced from adipose tissues in insulin-resistant type 2 diabetes patients. Therefore, to understand the effects of antimicrobial periodontal therapy on serum TNF-α concentration and subsequent metabolic control of diabetes, we examined the periodontal and diabetic status on 13 type-2 diabetes patients. Methods: These patients were treated with local minocycline administration in every periodontal pocket around all existing teeth once a week for a month. Before and after treatment, the number of total bacteria in the periodontal pockets and circulating TNF-α concentration were measured and the HbA1c value was assessed. Results: Antimicrobial therapy significantly reduced the number of microorganisms in periodontal pockets (P <0.01). After treatment, the circulating TNF-α level was significantly reduced (P <0.015). The HbA1c value was also reduced significantly (P <0.007). In addition, the 6 patients who were not receiving insulin therapy demonstrated decreased fasting insulin levels (P <0.03), and HOMA-R (P <0.03) indices. The average reductions in circulating TNF-α concentration and HbA1c value were 0.49 pg/ml and 0.8%, respectively. Conclusion: The results indicate that anti-infectious treatment is effective in improving metabolic control in diabetics, possibly through reduced serum TNF-α and improved insulin resistance.

DOI： 10.1902/jop.2001.72.6.774

Language：English   Publishing type：Research paper (scientific journal)  

Background: Type 1 diabetes is caused by a destruction of pancreatic β cells due to autoimmunity. Autoantibody against glutamic acid decarboxylase (GAD) 65 expressed in pancreatic β cells is widely used as a predictive marker for pancreatic destruction. In this study, we hypothesized that if certain cells in periodontal tissues could express GAD, then it may influence GAD antibody titer. Methods: We used: 1) reverse transcription-polymerase chain reaction (PCR) analysis to detect GAD 65 mRNA in various cells; 2) nucleotide sequencing analysis to confirm that amplified PCR product is the gene encoding GAD; and 3) Western blotting to determine the expression of GAD 65 protein in human gingival fibroblasts. Immunohistochemical staining of GAD 65 protein in normal and inflamed gingiva was performed to examine the potential influence of periodontal inflammation on GAD 65 expression. GAD antibody titer in sera of periodontal patients as well as healthy subjects was measured to determine if periodontal patients could develop autoantibody against GAD 65. Results: Cultured human gingival, periodontal, and dermal fibroblasts and mesangial cells expressed GAD mRNA. Nucleotide sequencing analyses confirmed the amplified PCR product as GAD 65. Western immunoblotting analyses and immunohistochemical staining revealed that the GAD 65 protein was expressed in vitro and in vivo. The expression of GAD 65 in inflamed tissue was higher than that in normal tissues. Two of 62 periodontal patients without diabetes showed an increased antibody titer against GAD 65, while none of the systemically healthy subjects showed an increased antibody titer against this antigen. Conclusions: We concluded that periodontal inflammation may result in higher levels of GAD and influence GAD antibody titer, and, hence, affect diabetic diagnosis based upon GAD antibody production.

DOI： 10.1902/jop.2001.72.5.598

Language：English   Publishing type：Research paper (scientific journal)  

Background: The pathogenesis of early-onset periodontitis (EOP) can be explained by various host risk factors. Previous studies have focused on a single (among many possible) immunological risk factor and the association among the factors has not been assessed. We comprehensively investigated the associations among multiple host immunological risk factors in EOP patients to further elucidate their role in the pathogenesis of EOP. Methods: Sixty-eight EOP patients (50 generalized EOP, 18 localized EOP), 51 EOP-suspected patients (S-EOP), 43 adult periodontitis (AP) patients, and 36 periodontally healthy subjects (HS) participated in this cross-sectional study. We examined peripheral neutrophil functions, phenotypic and functional characterization of peripheral lymphocytes (lymphocyte subsets, T-cell proliferative activity), cytokine productivity (interleukin [IL]-1, IL-2, tumor necrosis factor [TNF]-α, interferon [IFN]-γ, IL-4 and IL-6), serum immunoglobulin G (IgG) antibody titers against 12 periodontal bacteria, and HLA class Il genotypes. Results: G-EOP, S-EOP, and AP patient groups showed significantly lower percentages of pan T cells and CD8-positive cells (P<0.02) compared with the HS group. L-EOP patients showed depressed IL-4 and TNF-α productivity compared with the HS group (P<0.02). The EOP group showed significantly elevated antibody levels against Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum compared with the HS group (P<0.05). The frequency with DQB1*0503 was significantly higher in the EOP patient group than the HS group (P = 0.045) due to the higher frequency in L-EOP patients than the HS group (P = 0.035). There were wide interindividual variations in each of the tests among patient and HS groups; however, EOP patients showed wider intradiagnostic group variations in certain host defensive cell functions than the other groups. There were some EOP patients who showed extremely low or high values in some tests; the EOP patients could be further divided into subgroups according to their host defensive and immunological profiles. However, there was heterogeneity in some of the other host immunological tests even in the subgroups. Conclusions: The association of host immunological risk factors in EOP patients is widely varied and more complex than previously thought. These results indicate the difficulty of explaining the pathogenesis of EOP based on a single host risk factor and also emphasize the importance of critical assessment of not only EOP patient groups, but also individual patients.

DOI： 10.1902/jop.2001.72.4.425

Language：English   Publishing type：Research paper (scientific journal)  

We have previously reported that Porphyromonas gingivalis FDC 381 possesses a 53-kDa protein antigen (Ag53) on its outer membrane that evokes a strong humoral immune response in many patients with periodontal disease and that the humoral immune responses to Ag53 differ greatly among patients. To understand how the individual humoral immune system against Ag53 was determined, the regions of Ag53 recognized by specific antibody (B-cell epitopes) and dominant subclasses of serum immunoglobulin G (IgG) against major B-cell epitopes were examined by enzyme-linked immunosorbent assay. This study used sera from six patients with periodontitis, which all reacted strongly with sonic extracts of P. gingivalis 381 and with purified Ag53, and sera from six periodontally healthy children, which did not react with either sonic extracts of P. gingivalis 381 or Ag53. The epitopes were identified using synthetic 5-residue overlapping decapeptides covering the entire Ag53. Thirteen of 89 synthetic decapeptides showed a strong reaction with sera from the periodontal patients, but no reaction with those from the healthy children. Four peptides of 13 exerted different immune responses among patients. Furthermore, restriction analyses of the highly antigenic regions revealed that three sequences, RAAIRAS, YYLQ and MSPARR, were identified as major B-cell epitopes. Additionally, these epitopes were recognized mainly by the IgG2 isotype. These data suggest that the difference of B-cell epitopes might influence individual differences in antibody titer against Ag53 and also that the epitopes recognized commonly by multiple antibodies are quite valuable for peptide vaccine development against P. gingivalis infection.

DOI： 10.1034/j.1399-302X.2001.016002073.x

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The present narrative review examines the scientific evidence of the biological mechanisms that may link periodontitis and diabetes, as a source of comorbidity. Publications regarding periodontitis and diabetes, in human, animals, and in vitro were screened for their relevance. Periodontal microbiome studies indicate a possible association between altered glucose metabolism in prediabetes and diabetes and changes in the periodontal microbiome. Coinciding with this, hyperglycemia enhances expression of pathogen receptors, which enhance host response to the dysbiotic microbiome. Hyperglycemia also promotes pro-inflammatory response independently or via the advanced glycation end product/receptor for advanced glycation end product pathway. These processes excite cellular tissue destruction functions, which further enhance pro-inflammatory cytokines expression and alteration in the RANKL/osteoprotegerin ratio, promoting formation and activation of osteoclasts. The evidence supports the role of several pathogenic mechanisms in the path of true causal comorbidity between poorly controlled diabetes and periodontitis. However, further research is needed to better understand these mechanisms and to explore other mechanisms.

DOI： 10.1111/prd.12298

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Type：Peer review 

Number of peer-reviewed articles in foreign language journals：10

Number of peer-reviewed articles in Japanese journals：1

Type：Competition, symposium, etc. 

Number of participants：2,900

Type：Academic society, research group, etc. 

Type：Academic society, research group, etc. 

Type：Competition, symposium, etc. 

Number of participants：200

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：10

Number of peer-reviewed articles in Japanese journals：1

Type：Competition, symposium, etc. 

Number of participants：200

Type：Competition, symposium, etc. 

Number of participants：200

Type：Academic society, research group, etc. 

歯髄で炎症が惹起されると急激な炎症が短時間に誘導され、歯髄は数日内に融解壊死を起こすがその機序は不明であった。歯髄細胞から分泌される細胞外微粒子内の活性化シグナル伝達分子がマクロファージからの炎症性サイトカイン産生を誘導することを見出し、この現象が急激な歯髄炎症の原因であることを世界で初めて見出した。

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〔研究目的及び到達目標〕
口腔の健康は咀嚼機能の回復を介した高齢者のQOLの向上に寄与することに加え、認知機能、肥満や糖尿病などの生活習慣病予防、宿主防御機構の恒常性維持など多岐にわたって健康長寿社会の実現に貢献する。さらに、口腔諸組織には幹細胞が豊富に存在し、かつ採取が比較的容易であることから、これらを用いた難治性自己免疫疾患や肝臓病などに対する治療応用の可能性についても注目されつつある。

九州大学歯学研究院は、「口腔の健康から全身の健康を推進する口腔健康科学」、「組織の再生・再建研究」を重点プロジェクトとして位置づけ、本分野における研究を強力に推進し、独創性に富む世界的研究拠点の形成を目指している。また、その達成に向け研究志向大学としての使命を自覚し、①研究マインドを有する学部学生や大学院生の醸成教育の展開、および②次世代リーダーの育成に力を注いでいる。健康長寿の実現を口腔の健康から強力に推進するために、海外の一流の研究者（研究室）と相互訪問・交流（IN-CROSS-OUT）を通じて強固なネットワークを創出して、国際ハブ拠点を目指す。

〔研究計画・方法〕
歯学研究院の掲げる重点プロジェクトによる独創的拠点形成を達成するため、頭脳循環により次世代のリーダー候補者によるプロジェクト研究の水準向上と知的財産の創出を推進する。

口腔健康科学プロジェクトの計画を示す。申請者らは、味覚受容体およびその摂食調節機構の研究において被引用論文数世界第1位（SCOPUS）であり、単一味神経・細胞応答記録など世界オンリーワンの技術を有する（生理学）。新規に発見したPRIP(IP3結合蛋白)とその関連蛋白PP1、PP2Aによる骨代謝/生殖/エネルギー代謝の包括的制御という世界に類をみない独創的な研究を展開中である（生化学）。これらの課題について、味覚・嗅覚における世界でもオンリーワンの研究機関（Monell Center）と、若手研究者を中心に継続的な共同研究を展開することで、栄養の経口摂取を介した肥満・生活習慣病予防、および超高齢者に多くみられる飢餓状態の改善が可能となる。一方、日本人に多い軽微な過体重や肥満を呈する患者の中で、重症の歯周病が合併した場合に惹起される、欧米型肥満類似の慢性炎症病態を解析する極めてユニークな歯周医学研究を展開している（歯周病学）。さらに、こうして活性化された自然免疫機構により、神経障害性疼痛の惹起によるアルツハイマー病の誘導（薬理学）、ミクリッツ病などの唾液線自己免疫疾患を介した高齢者のドライマウス発現（口腔外科学）について、卓越した研究を展開中である。この課題についてはUniversity of Pennsilvania、Harvard University免疫学部門との交流により、臨床免疫学の充実化を図り、最終的にエネルギー代謝研究と融合させることで、neurology、immunogy、endocrinologyを包含する口腔健康科学研究の拠点化を目指す。

一方、組織再生・再建プロジェクトにおいて、申請者らは、口腔組織に由来する幹細胞（以下、口腔幹細胞と略す）を用いた顎顔面口腔領域の再生医療研究に留まらず、自己免疫疾患や難治性肝疾患、先天性疾患等を含む全身の難病への臨床応用を目的とした、独創的かつ先端的なトランスレーショナルリサーチを展開し、卓越した実績を挙げつつある（解剖学）。加えて、口腔幹細胞を用いて難病の発症機序を分子/遺伝子レベルで解明・診断し、当該分子を標的とした治療法の開発を試みるユニークな研究を展開しつつある（解剖学、小児歯科学）。また従来の歯周再生治療や歯科インプラント治療の概念を凌駕する、口腔幹細胞を用いたインプラント体への付着性・シーリングの向上を目指す画期的な粘膜治療法の開発を推進している（歯科保存学、補綴学）。これら萌芽期にある口腔幹細胞を基盤とした再生医療の展開と発展のために次世代の若手研究者の育成は急務の課題であり、この取り組みに関して卓越した研究を展開しているUniversity of Southern California、University of Pennsilvania、University of Michiganと共同研究することで、その理論と実践、とりわけトランスレーショナルリサーチの実際を徹底的にマスターさせる。

特に重要な点は単に相互訪問（IN-OUT）するものではなく、若手研究者が世界のトップサイエンティストとの議論と実践を通じて成果を確実に獲得するための交流（CROSS）へとパラダイムシフトさせることである。また、海外の連携研究者を、毎年開催している国際シンポジウム「Kyudai Oral Bioscience」と「味覚嗅覚の分子神経機構」前後に招聘し、共同研究について精査・議論するとともに、派遣若手研究者の成果発表の場として、CROSSの達成度を評価し、次年度の事業計画に生かすとともに将来の国際共同研究のマイルストーンを構築する。これらの取り組みにより若手研究者を刺激し、さらなる意欲の向上を目指す。以上の頭脳循環・交流の取り組みにより九州大学歯学研究院を独創性と先端性に富むプロジェクトを展開する世界トップレベルの歯学研究拠点とする取り組みを推進する。

〔研究目的及び到達目標〕
口腔の健康は咀嚼機能の回復を介した高齢者のQOLの向上に寄与することに加え、認知機能、肥満や糖尿病などの生活習慣病予防、宿主防御機構の恒常性維持など多岐にわたって健康長寿社会の実現に貢献する。さらに、口腔諸組織には幹細胞が豊富に存在し、かつ採取が比較的容易であることから、これらを用いた難治性自己免疫疾患や肝臓病などに対する治療応用の可能性についても注目されつつある。

九州大学歯学研究院は、「口腔の健康から全身の健康を推進する口腔健康科学」、「組織の再生・再建研究」を重点プロジェクトとして位置づけ、本分野における研究を強力に推進し、独創性に富む世界的研究拠点の形成を目指している。また、その達成に向け研究志向大学としての使命を自覚し、①研究マインドを有する学部学生や大学院生の醸成教育の展開、および②次世代リーダーの育成に力を注いでいる。健康長寿の実現を口腔の健康から強力に推進するために、海外の一流の研究者（研究室）と相互訪問・交流（IN-CROSS-OUT）を通じて強固なネットワークを創出して、国際ハブ拠点を目指す。

〔研究計画・方法〕
歯学研究院の掲げる重点プロジェクトによる独創的拠点形成を達成するため、頭脳循環により次世代のリーダー候補者によるプロジェクト研究の水準向上と知的財産の創出を推進する。

口腔健康科学プロジェクトの計画を示す。申請者らは、味覚受容体およびその摂食調節機構の研究において被引用論文数世界第1位（SCOPUS）であり、単一味神経・細胞応答記録など世界オンリーワンの技術を有する（生理学）。新規に発見したPRIP(IP3結合蛋白)とその関連蛋白PP1、PP2Aによる骨代謝/生殖/エネルギー代謝の包括的制御という世界に類をみない独創的な研究を展開中である（生化学）。これらの課題について、味覚・嗅覚における世界でもオンリーワンの研究機関（Monell Center）と、若手研究者を中心に継続的な共同研究を展開することで、栄養の経口摂取を介した肥満・生活習慣病予防、および超高齢者に多くみられる飢餓状態の改善が可能となる。一方、日本人に多い軽微な過体重や肥満を呈する患者の中で、重症の歯周病が合併した場合に惹起される、欧米型肥満類似の慢性炎症病態を解析する極めてユニークな歯周医学研究を展開している（歯周病学）。さらに、こうして活性化された自然免疫機構により、神経障害性疼痛の惹起によるアルツハイマー病の誘導（薬理学）、ミクリッツ病などの唾液線自己免疫疾患を介した高齢者のドライマウス発現（口腔外科学）について、卓越した研究を展開中である。この課題についてはUniversity of Pennsilvania、Harvard University免疫学部門との交流により、臨床免疫学の充実化を図り、最終的にエネルギー代謝研究と融合させることで、neurology、immunogy、endocrinologyを包含する口腔健康科学研究の拠点化を目指す。

一方、組織再生・再建プロジェクトにおいて、申請者らは、口腔組織に由来する幹細胞（以下、口腔幹細胞と略す）を用いた顎顔面口腔領域の再生医療研究に留まらず、自己免疫疾患や難治性肝疾患、先天性疾患等を含む全身の難病への臨床応用を目的とした、独創的かつ先端的なトランスレーショナルリサーチを展開し、卓越した実績を挙げつつある（解剖学）。加えて、口腔幹細胞を用いて難病の発症機序を分子/遺伝子レベルで解明・診断し、当該分子を標的とした治療法の開発を試みるユニークな研究を展開しつつある（解剖学、小児歯科学）。また従来の歯周再生治療や歯科インプラント治療の概念を凌駕する、口腔幹細胞を用いたインプラント体への付着性・シーリングの向上を目指す画期的な粘膜治療法の開発を推進している（歯科保存学、補綴学）。これら萌芽期にある口腔幹細胞を基盤とした再生医療の展開と発展のために次世代の若手研究者の育成は急務の課題であり、この取り組みに関して卓越した研究を展開しているUniversity of Southern California、University of Pennsilvania、University of Michiganと共同研究することで、その理論と実践、とりわけトランスレーショナルリサーチの実際を徹底的にマスターさせる。

特に重要な点は単に相互訪問（IN-OUT）するものではなく、若手研究者が世界のトップサイエンティストとの議論と実践を通じて成果を確実に獲得するための交流（CROSS）へとパラダイムシフトさせることである。また、海外の連携研究者を、毎年開催している国際シンポジウム「Kyudai Oral Bioscience」と「味覚嗅覚の分子神経機構」前後に招聘し、共同研究について精査・議論するとともに、派遣若手研究者の成果発表の場として、CROSSの達成度を評価し、次年度の事業計画に生かすとともに将来の国際共同研究のマイルストーンを構築する。これらの取り組みにより若手研究者を刺激し、さらなる意欲の向上を目指す。以上の頭脳循環・交流の取り組みにより九州大学歯学研究院を独創性と先端性に富むプロジェクトを展開する世界トップレベルの歯学研究拠点とする取り組みを推進する。

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