Lysophospholipase D activity on oral mucosa cells in whole mixed human saliva involves in production of bioactive lysophosphatidic acid from lysophosphatidylcholine.

We reported that lysophosphatidic acid (LPA) is present at 0.8 μM in mixed human saliva (MS). In this study, we examined the distribution, origin, and enzymatic generation pathways of LPA in MS. LPA was distributed in the medium and cell pellet fraction; a true level of soluble LPA in MS was about 150 nM. The soluble LPA was assumed to be generated by ecto-type lysophospholipase D on exfoliated cells in MS from LPC that originated mainly from the major salivary gland saliva. Our results with the albumin-back extraction procedures suggest that a significant pool of LPA is kept in the outer layer of the plasma membranes of detached oral mucosal cells. Such pool of LPA may contribute to wound healing in upper digestive organs including oral cavity. We obtained evidence that the choline-producing activity in MS was mainly due to Ca-activated lysophospholipase D activity of glycerophosphodiesterase 7.


Utility of a haemoglobin test of gingival crevicular fluid: A multicentre, observational study.


Dental students' awareness after clinical training between dental treatment and systemic health: A questionnaire-based survey


Gan-Lu-Yin (Kanroin), traditional Chinese herbal extracts, reduces osteoclast differentiation in vitro and prevents alveolar bone resorption in rat experimental periodontitis

Gan-Lu-Yin (GLY), a traditional Chinese herbal medicine, shows therapeutic effects on periodontitis, but that mechanism is not well known. This study aims to clarify the precise mechanism by investigating the inhibitory effects of GLY extracts on osteoclastogenesis in vitro and on bone resorption in periodontitis in vivo. RAW264.7 cells are cultured with soluble receptor activator of nuclear factor-kappa B (sRANKL) and GLY extracts (0.01-1.0 mg/mL), and stained for tartrate-resistant acid phosphatase (TRAP) to evaluate osteoclast differentiation. Experimental periodontitis is induced by placing a nylon ligature around the second maxillary molar in rats, and rats are administered GLY extracts (60 mg/kg) daily for 20 days. Their maxillae are collected on day 4 and 20, and the levels of alveolar bone resorption and osteoclast differentiation are estimated using micro-computed tomography (CT) and histological analysis, respectively. In RAW264.7 cells, GLY extracts significantly inhibit sRANKL-induced osteoclast differentiation at a concentration of more than 0.05 mg/mL. In experimental periodontitis, administering GLY extracts significantly decreases the number of TRAP-positive osteoclasts in the alveolar bone on day 4, and significantly inhibits the ligature-induced bone resorption on day 20. These results show that GLY extracts suppress bone resorption by inhibiting osteoclast differentiation in experimental periodontitis, suggesting that GLY extracts are potentially useful for oral care in periodontitis.


A largescale observational study to investigate the current status of diabetic complications and their prevention in Japan (JDCP study 6): baseline dental and oral findings

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.


糖尿病合併症の実態とその抑制に関する大規模観察研究ベースライン時の口腔所見JDCP study 6

Journal of the The Japan Diabetes Society


A low level of lysophosphatidic acid in human gingival crevicular fluid from patients with periodontitis due to high soluble lysophospholipase activity: Its potential protective role on alveolar bone loss by periodontitis.

We previously detected a submicromolar concentration of lysophosphatidic acid (LPA) in human saliva. Here, we compare LPA concentrations in human gingival crevicular fluid (GCF) from patients with periodontitis and healthy controls, and examine how the local LPA levels are regulated enzymatically. The concentrations of LPA and its precursor lysophospholipids in GCF was measured by liquid chromatography-tandem mass spectrometry. The LPA-producing and LPA-degrading enzymatic activities were measured by quantifying the liberated choline and free fatty acid, respectively. The concentration of LPA in GCF of periodontitis patients was lower than that of healthy controls, due to higher soluble lysophospholipase activity toward LPA. LPA was found to prevent survival of Sa3, a human gingival epithelium-derived tumor cell line, activate Sa3 through Ca mobilization, and release interleukin 6 from Sa3 in vitro. Furthermore, local injection of LPA into the gingiva attenuated ligature-induced experimental alveolar bone loss induced by oral bacteria inoculation in a rat model of periodontitis in vivo. A high concentration of LPA in human GCF is necessary to maintain normal gingival epithelial integrity and function, protecting the progression of periodontitis.

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids


Relationship of oral conditions to the incidence of infective endocarditis in periodontitis patients with valvular heart disease: a cross-sectional study.

The patients with IE had fewer remaining teeth, more advanced bone resorption compared with those of patients without IE. These findings suggest a possible association between the occurrence of IE and periodontal infection.

No significant differences were observed between patients with or without VHD in oral conditions. A significant increase in the percentage of alveolar bone loss in VHD patients with IE was observed compared with that of patients without IE. The ratio of both Porphyromonas gingivalis (Pg) IgG titer > 1.68 and Pg fimA type II genotype in patients with IE was significantly higher than in patients without IE. There was a significant correlation between the occurrence of IE and clinical oral findings (number of remaining teeth: OR, 0.17; rate of alveolar bone loss > 40%: OR, 11.8).


Advanced glycation end-product 2 and Porphyromonas gingivalis lipopolysaccharide increase sclerostin expression in mouse osteocyte-like cells

Sclerostin is a secreted glycoprotein that is mainly expressed in osteocytes, exerts negative effects on bone formation, and is present at elevated levels in diabetes mellitus (DM). Periodontitis is an infectious disease caused by periodontopathic bacteria, a complication of DM, and sometimes associated with severe inflammation and alveolar bone resorption. Advanced glycation end-products (AGEs) are a major pathogen in DM complications and adversely influence periodontitis in DM patients. In the present study, the effects of AGE2 and Porphyromonas gingivalis lipopolysaccharide (P-LPS) on the expression of sclerostin in mouse osteocyte-like cells (MLO-Y4-A2 cells) and its function in osteoblast differentiation were investigated. AGE2 and P-LPS up-regulated the expressions of receptor of AGE (RAGE) and Toll-like receptor 2 (TLR2), respectively, and significantly up-regulated that of sclerostin and interleukin 6 (IL-6) in osteocytes. Sclerostin, RAGE and TLR2 levels were synergistically increased by AGE2 and P-LPS. The siRNAs of RAGE and TLR2 significantly inhibited AGE2- and P-LPS-induced sclerostin expression. AGE2 up-regulated sclerostin expression in osteocyte-like cells via the RAGE, ERK and JNK, and NF-κB signal pathways. On the other hand, P-LPS elevated sclerostin levels via the TLR2, JNK and p38, and NF-κB signal pathways. When osteocytes pre-treated with AGE2 and P-LPS and osteoblastic cells (MC3T3-E1) were co-cultured in the medium with a sclerostin-neutralizing antibody, AGE2- and P-LPS-induced decreases in alkaline phosphatase activity and Runx2 expression in osteoblastic cells were significantly inhibited by the sclerostin-neutralizing antibody. These results suggest that AGE2 and P-LPS influence bone metabolism and inflammation through the regulation of sclerostin expression, and may aggravate periodontitis with DM.


High Glucose-Mediated Cytokine Regulation in Gingival Fibroblasts and THP-1 Macrophage: a Possible Mechanism of Severe Periodontitis with Diabetes.

Diabetic conditions such as HG induce IL-1 and sIL-6R production from macrophages in inflammatory periodontal tissues and may exacerbate the periodontitis synergistically via MMP-1 production from HGFs.


Advanced glycation end-products increase IL-6 and ICAM-1 expression via RAGE, MAPK and NF-kB pathways in human gingival fibroblasts

Diabetes mellitus (DM) is a risk factor for periodontal diseases and may exacerbate the progression of the pathogenesis of periodontitis. Advanced glycation end-products (AGEs) cause DM complications relative to levels of glycemic control and larger amounts accumulate in the periodontal tissues of patients with periodontitis and DM. In the present study, we investigated the effects of AGEs on the expression of inflammation-related factors in human gingival fibroblasts (HGFs) to elucidate the impact of AGEs on DM-associated periodontitis. HGFs were cultured with or without AGEs. Cell viability was examined, and RNA and protein fractions were isolated from AGE-treated cells. The expression of interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1), and the receptor for AGE (RAGE) was investigated using reverse transcription-polymerase chain reaction, quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and reactive oxygen species activity was measured using a kit with 2',7'-dichlorofluorescin diacetate. Human monocytic cells (THP-1) labeled with a fluorescent reagent were co-cultured with HGFs treated with AGEs and IL-6 siRNA, and the adhesive activity of THP-1 cells to HGFs was assessed. The expression of IL-6 and ICAM-1 was examined when HGFs were pretreated with recombinant human IL-6, the siRNAs of RAGE and IL-6, and inhibitors of MAPK and NF-κB, and then cultured with and without AGEs. The phosphorylation of MAPK and NF-κB was assessed using western blotting. AGEs increased the mRNA and protein expressions of RAGE, IL-6, ICAM-1 and reactive oxygen species activity in HGFs, and promoted the adhesion of THP-1 cells to HGFs, but had no effect on cell viability until 72 hours. Recombinant human IL-6 increased ICAM-1 expression in HGFs, while the siRNAs of RAGE and IL-6 inhibited AGE-induced IL6 and ICAM1 mRNA expression, and IL-6 siRNA depressed AGE-induced THP-1 cell adhesion. AGEs increased the phosphorylation of p38 and ERK MAPKs, p65 NF-κB and IκBα, while inhibitors of p38, ERK MAPKs and NF-κB significantly decreased AGE-induced IL-6 and ICAM-1 expression. AGEs increase IL-6 and ICAM-1 expression via the RAGE, MAPK and NF-κB pathways in HGFs and may exacerbate the progression of the pathogenesis of periodontal diseases.


The KCNQ1 gene polymorphism as a shared genetic risk for rheumatoid arthritis and chronic periodontitis in Japanese adults: A pilot case-control study

A number of studies have suggested a bidirectional relationship of periodontitis with rheumatoid arthritis (RA) and type 2 diabetes mellitus (T2DM). However, the genetic factors that underlie these relationships have not been elucidated. We conducted a multicenter case-control study that included 185 patients with RA and chronic periodontitis (CP), 149 patients with T2DM and CP, 251 patients with CP, and 130 systemically and periodontally healthy controls from a cohort of Japanese adults to assess the shared genetic risk factors for RA and CP as well as for T2DM and CP. A total of 17 candidate single nucleotide polymorphisms (SNPs) associated with RA, T2DM, and CP were genotyped. Multiple logistic regression analyses revealed that the KCNQ1 rs2237892 was significantly associated with comorbidity of RA and CP (P = 0.005) after adjustment for age, sex, and smoking status. The carriers of the T allele among patients with RA and CP showed significantly higher disease activity scores including 28 joints using C-reactive protein values than the non-carriers (P = 0.02), although the age, female percentage, and smoking status were comparable. Other SNPs were not associated with comorbidity of RA and CP, T2DM and CP, or susceptibility to CP. The results of the present pilot study suggest for the first time that the KCNQ1 rs2237892 may constitute a shared genetic risk factor for RA and CP, but not for T2DM and CP in Japanese adults.


Relationship of aspiration pneumonia to cognitive impairment and oral condition: a cross-sectional study.

Early and simple evaluation of the oral condition and cognitive function can predict the risk of aspiration pneumonia.


b-carotene suppresses Porphyromonas gingivalis lipopolysaccharide-mediated cytokine production in THP-1 monocytes cultured with high glucose condition.

Periodontitis is associated with development of diabetes mellitus. Although lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg), a major pathogen of periodontitis, may lead the progression of diabetes complications, the precise mechanisms are unclear. We, therefore, investigated the effects of β-carotene on production of Pg LPS-induced inflammatory cytokines in human monocytes cultured high glucose (HG) condition. THP-1 cells were cultured under 5.5 mM or 25 mM glucose conditions, and cells were stimulated with Pg LPS. To investigate the productivity of TNF-α, IL-6, and MCP-1, cell supernatants were collected for ELISA. To examine the effects of NF-kB signals on cytokine production, Bay11-7082 was used. HG enhanced Pg LPS-induced production of TNF-α, IL-6, and MCP-1 via NF-kB signals in THP-1. β-carotene suppressed the enhancement of the Pg LPS-induced cytokine production in THP-1 via NF-κB inactivation. Our results suggest that β-carotene might be a potential anti-inflammatory nutrient for circulating Pg LPS-mediated cytokine production in diabetic patients with periodontitis.


Local administration of calcitonin inhibits alveolar bone loss in an experimental periodontitis in rats.

Calcitonin (CTN), a calcium regulatory hormone, promotes calcium diuresis from the kidney and suppresses bone resorption. The objective of this study was to evaluate whether the topical and intermittent application of CTN inhibits alveolar bone resorption using ligature-induced experimental periodontitis in rats. Experimental periodontitis was induced by placing a nylon ligature around maxillary molars of 8-week-old male Wistar rats for 20 days. Thirty-two rats were divided into four groups: basal sham control group, periodontitis group, periodontitis plus 0.2 U CTN (low dose), and periodontitis plus 1.0 U CTN (high dose) group. To investigate the effects of CTN on alveolar bone resorption, CTN was topically injected into the palatal gingivae every 2 days after ligature removal (day 0). Micro-computed tomography (CT) analysis was performed for linear parameter assessment of alveolar bone on day 5 and day 14. Periodontal tissues were examined histo-pathologically to assess the differences among the study groups. Micro-CT images showed that alveolar bone resorption was induced statistically around the molar of ligatured rats on day 5 and day 14. The amount of bone resorption was more severe on day 14 than that on day 5. On day 5, only high-dose CTN treatment significantly suppressed bone resorption. In addition, both doses of CTN significantly suppressed bone resorption on day 14. Histological examination clarified that there were fewer TRAP-positive cells in the CTN treatment groups than in the periodontitis group on day 5. Local administration of CTN decreased alveolar bone resorption by regulating osteoclast activation in rats with periodontitis.


Useful Immunochromatographic Assay of Calprotectin in Gingival Crevicular Fluid for Diagnosis of Diseased Sites in Patients with Periodontal Diseases.

Determination of GCF calprotectin using a novel IC chip system is useful for diagnosis of periodontal diseases.


Clinical Significance of GCF sIL-6R and Calprotectin to Evaluate the Periodontal Inflammation.

Both GCF sIL-6R and calprotectin levels are significant bio-markers to evaluate the periodontal inflammation.


Calprotectin Induces IL-6 and MCP-1 Production via Toll-Like Receptor 4 Signaling in Human Gingival Fibroblasts.

Calprotectin, a heterodimer of S100A8 and S100A9 molecules, is associated with inflammatory diseases such as inflammatory bowel disease. We have reported that calprotectin levels in gingival crevicular fluids of periodontitis patients are significantly higher than in healthy subjects. However, the functions of calprotectin in pathophysiology of periodontitis are still unknown. The aim of this study is to investigate the effects of calprotectin on the productivity of inflammatory cytokines in human gingival fibroblasts (HGFs). The HGFs cell line CRL-2014® (ATCC) were cultured, and total RNAs were collected to examine the expression of TLR2/4 and RAGE mRNA using RT-PCR. After the cells were treated with S100A8, S100A9, and calprotectin, supernatants were collected and the levels of IL-6 and MCP-1 were measured using ELISA methods. To examine the intracellular signals involved in calprotectin-induced cytokine production, several chemical inhibitors were used. Furthermore, after the siRNA-mediated TLR4 down-regulated cells were treated with S100A8, S100A9, and calprotectin, the levels of IL-6 and MCP-1 were also measured. HGFs showed greater expression of TLR4 mRNA, but not TLR2 and RAGE mRNA compared with human oral epithelial cells. Calprotectin increased significantly the production of MCP-1 and IL-6 in HGFs, and the cytokine productions were significantly suppressed in the cells treated with MAPKs, NF-B, and TLR4 inhibitors. Furthermore, calprotectin-mediated MCP-1 and IL-6 production were significantly suppressed in TLR4 down-regulated cells. Taken together, calprotectin induces IL-6 and MCP-1 production in HGFs via TLR4 signaling that involves MAPK and NF-B, resulting in the progression of periodontitis. J. Cell. Physiol. 232: 1862-1871, 2017. © 2016 Wiley Periodicals, Inc.


Correlation Between Gingival Crevicular Fluid Hemoglobin Content and Periodontal Clinical Parameters.

Hb was observed in more than 60% of GCF samples in BOP(-) gingival sulci in both periodontally stable and periodontal-management-required groups. These results suggest inspection of Hb derived from microbleeding in gingival sulci may serve as an index for preclinical diagnosis.


Association between Periodontal Condition and Kidney Dysfunction in Japanese Adults: A Cross-Sectional Study

Clinical and Experimental Dental Research


Inhibitory effects of advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide on the expression of osteoblastic markers of rat bone marrow cells in culture

Diabetes is a major risk factor for periodontitis and there is a close relationship between the degree of hyperglycemia and the severity of periodontitis. Advanced glycation end-products (AGEs) accumulate in various tissues under diabetic conditions. AGEs in the periodontal tissues probably play a role in upregulating periodontal inflammation; however, the association of AGEs with the severity of periodontitis has not been fully clarified. Lipopolysaccharide from Porphyromonas gingivalis (P-LPS) is a potent pathogenic factor in periodontitis. Although the independent effect of AGE or P-LPS on osteoblastic cells has been reported in vitro, the effect of adding both has not been clearly elucidated. In this study, to explore factors aggravating diabetic periodontitis, we investigated the effects of AGE and P-LPS on the expression of osteoblastic markers and the expression of inflammation-related markers in vitro. Rat bone marrow cells were cultured, and alkaline phosphatase activity and bone nodule formation were evaluated as osteoblastic markers. Reverse transcription-polymerase chain reaction was performed to determine the mRNA expression of molecules associated with bone and inflammation. Protein levels of osteocalcin and interleukin-1β (IL-1β) were measured using enzyme-linked immunosorbent assay. AGEs and P-LPS independently reduced alkaline phosphatase activity and bone nodule formation. The addition of both AGE and P-LPS (AGE+P-LPS) further decreased these markers. Reverse transcription-polymerase chain reaction analysis revealed that AGE+P-LPS markedly decreased the mRNA expression of osteoblast-related molecules such as type 1 collagen, osteocalcin and Cbfa1, and markedly increased that of inflammation-related molecules such as IL1β and S100A8. AGE and P-LPS decreased the protein level of osteocalcin and increased that of IL-1β, and a further increase of IL-1β was detected for AGE+P-LPS. AGEs and P-LPS inhibited the expression of osteoblastic markers and increased the levels of inflammatory markers in rat bone marrow cells, suggesting that both AGE and P-LPS may be important factors associated with the aggravation of diabetic periodontitis.


Effect of Hangeshashinto on calprotectin expression in human oral epithelial cells.

Oral epithelial cells produce antimicrobial peptides (AMPs) to prevent microbial infection. Calprotectin (S100A8/S100A9) is one of these AMPs in oral epithelial cells, the expression of which is up-regulated by interleukin-1α (IL-1α). Hangeshashinto (HST) is a traditional Japanese herbal medicine that has anti-inflammatory effects. The purpose of this study was to investigate the effect of HST on the expression of calprotectin through the regulation of IL-1α in oral epithelial cells. Human oral epithelial cells (TR146) were cultured with HST in the presence or absence of anti-IL-1α antibody or IL-1 receptor antagonist, or with six major components of HST (3,4-dihydroxybenzaldehyde, baicalin, ginsenoside Rb1, glycyrrhizin, oleanolic acid and berberine). The expression of S100A8, S100A9, other AMPs and cytokine mRNAs was examined by RT-PCR and quantitative real-time PCR. Calprotectin expression and IL-1α secretion were investigated by ELISA. HST (6 μg/ml) increased the expression of S100A8/S100A9 mRNAs and calprotectin protein, and also up-regulated β-defensin 2 (DEFB4) and S100A7 expression. The expression of IL-1α mRNA and its protein was slightly but significantly increased by HST. A neutralizing antibody against IL-1α and IL-1 receptor antagonist inhibited HST-up-regulated S100A8/S100A9 mRNA expression. Although 3,4-dihydroxybenzaldehyde, baicalin and ginsenoside Rb1 as HST components increased S100A8/S100A9 expression, oleanolic acid and berberine decreased their expression. These results suggest that HST increases the expression of calprotectin, DEFB4 and S100A7 in oral epithelial cells. In response to HST, up-regulation of calprotectin expression may be partially induced via IL-1α.


PKR regulates LPS-induced osteoclast formation and bone destruction in vitro and in vivo.

PKR plays a pivotal role in LPS-induced bone loss in PD, and, thus, has potential as a therapeutic target for PD. This article is protected by copyright. All rights reserved.


Development of the Diabetes Oral Health Assessment Tool © for Nurses


Double stranded RNA-dependent protein kinase is necessary for TNF--induced osteoclast formation in vitro and in vivo.

Double-stranded RNA-dependent protein kinase (PKR) is involved in cell cycle progression, cell proliferation, cell differentiation, tumorgenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification in osteoblasts. TNF- plays a key role in osteoclast differentiation. However, it is unknown about the roles of PKR in the TNF--induced osteoclast differentiation. The expression of PKR in osteoclast precursor RAW264.7 cells increased during TNF--induced osteoclastogenesis. The TNF--induced osteoclast differentiation in bone marrow-derived macrophages and RAW264.7 cells was markedly suppressed by the pre-treatment of PKR inhibitor, 2-aminopurine (2AP), as well as gene silencing of PKR. The expression of gene markers in the differentiated osteoclasts including TRAP, Calcitonin receptor, cathepsin K and ATP6V0d2 was also suppressed by the 2AP treatment. Bone resorption activity of TNF--induced osteoclasts was also supressed by 2AP treatment. Inhibition of PKR supressed the TNF--induced activation of NF-B and MAPK in RAW264.7 cells. 2AP inhibited both the nuclear translocation of NF-B and its transcriptional activity in RAW264.7 cells. 2AP inhibited the TNF--induced expression of NFATc1 and c-fos, master transcription factors in osteoclastogenesis. TNF--induced nuclear translocation of NFATc1 in mature osteoclasts was clearly inhibited by the 2AP treatment. The PKR inhibitor C16 decreased the TNF--induced osteoclast formation and bone resorption in mouse calvaria. The present study indicates that PKR is necessary for the TNF--induced osteoclast differentiation in vitro and in vivo. This article is protected by copyright. All rights reserved.


Inhibitory Effects of Cryptotanshinone on Inflammation-related Molecules in Human Gingival Fibroblasts

培養歯肉線維芽細胞におけるクリプトタンシノンによる炎症関連分子の産生抑制効果

Purpose: Cryptotanshinone (CPT) isolated from the root of an Asian medicinal plant, Salvia miltiorrhiza bunge, acts as a potent anti-tumor or anti-inflammatory agent in vitro. However, the effects of CPT on the progression of periodontitis are still unknown. This study investigated the effects of CPT on the production of inflammation-related molecules in human gingival fibroblasts. Methods: Human gingival fibroblasts (HGFs), CRL-2014TM (ATCC), were used. Cytotoxicity of the cells by CPT was examined. Dead cells stained by trypan blue were counted and analyzed. Next, the levels of IL-6 and VEGF in IL-1β-treated cells with or without CPT were measured using ELISA kits. The productivity of cathepsin L in IL-1β-treated cells with or without CPT was examined using Western blotting. Results: The ratio of viable cell numbers was significantly decreased by 100 μmol/l CPT. Next, the increase of IL-6 and cathepsin L induced by IL-1β was significantly suppressed by the pretreatment of 10 μmol/l CPT in HGFs. On the other hand, productivity of VEGF induced by IL-1β was not changed by the pretreatment of 10 μmol/l CPT. Conclusion: CPT suppresses IL-1β-induced IL-6 and cathepsin L production in HGFs, and might regulate the progression of periodontitis.

目的 : 漢方薬 (生薬) タンジンの主成分であるクリプトタンシノンは, 抗がん作用や抗炎症作用をもつことが知られている. しかしながら, クリプトタンシノンが歯周組織の炎症に対してどのような影響を及ぼすのかは不明である. 本研究の目的は, 歯肉線維芽細胞を標的として, その炎症関連分子の産生に及ぼすクリプトタンシノンの影響を調べることである. 方法 : 細胞は歯肉線維芽細胞CRL-2014TM (ATCC) を用いた. クリプトタンシノンの濃度依存的な細胞障害作用の有無は, トリパンブルー染色法を用いて各濃度間における生細胞率の差を比較検討して評価した. 歯肉線維芽細胞のサイトカイン産生は, 市販のELISAキットを用いて, インターロイキン (IL) -1βで細胞を24時間刺激した後のIL-6およびVEGF産生量を測定して検討した. 一方, タンパク質分解酵素カテプシンLの産生は, IL-1βで細胞を24時間刺激した後に回収した全細胞タンパク質を用いて, ウエスタンブロット法によって検討した. なお, クリプトタンシノンはIL-1β添加の1時間前に細胞培養系に添加した. 各群における差異は, ANOVA Tukey-KramerのHSD検定によって有意差を検討した. 結果 : クリプトタンシノンは, 10μmol/lまでは歯肉線維芽細胞の生細胞率に影響は与えないが, 100μmol/lでは有意に生細胞率を低下させた. また, 歯肉線維芽細胞におけるIL-1β誘導性のIL-6産生およびカテプシンL産生は, 10μmol/lクリプトタンシノンの添加によって著明に減少した. 一方, IL-1β誘導性のVEGF産生は, クリプトタンシノンの添加によっても変化しなかった. 結論 : クリプトタンシノンは, 歯肉線維芽細胞におけるIL-1β誘導性IL-6およびカテプシンLの産生を抑制することで, 歯周組織の炎症を抑制する可能性が示唆された.

The Japanese Journal of Conservative Dentistry


YKL-40 level in gingival crevicular fluid from patients with periodontitis and type 2 diabetes

YKL-40 is a chitin-binding glycoprotein, the level of which increases in inflammatory diseases, diabetes mellitus (DM), cardiovascular diseases, and tumors. Gingival crevicular fluid (GCF) contains many proteins and markers of periodontitis. The purpose of this study was to investigate YKL-40 level in GCF from patients with periodontitis and DM and the association between YKL-40 level and chronic periodontitis (CP) or DM. The subjects were 121 patients with DM, CP, DM and periodontitis (DM-P), and healthy subjects (H). GCF was collected using paper strips after the sites for GCF collection were clinically evaluated for probing depth (PD), gingival index (GI), and bleeding on probing (BOP). YKL-40 in GCF was identified by Western blotting, and its level was determined by ELISA. YKL-40 was contained in GCF samples from H, DM, CP, and DM-P sites, and its levels (amount and concentration) in CP and DM-P were significantly higher than those in H and DM. GCF YKL-40 level significantly correlated with PD and GI, and its level in BOP-positive sites was significantly higher than that in BOP-negative ones. GCF YKL-40 level was elevated in periodontitis, but not DM. YKL-40 in GCF may be an inflammatory marker for periodontitis.


Gene polymorphism of β-defensin-1 is associated with susceptibility to periodontitis in Japanese.

Periodontitis is a multifactorial disease associated with genetic and environmental factors. Single-nucleotide polymorphisms (SNPs) are associated with susceptibility to common diseases such as diabetes and periodontitis. Although the oral cavity is exposed to various organisms, the conditions are well controlled by innate and acquired immune systems. Antimicrobial peptides (AMPs) play an important role in the innate immune system; however, the association of AMP-SNPs with periodontitis has not been fully elucidated. This study investigated the relationship between AMP-SNPs and periodontitis in Japanese. One hundred and five Japanese subjects were recruited, which included patients with aggressive, severe, moderate and mild periodontitis, and age-matched healthy controls. Genomic DNA was isolated from peripheral blood and genotypes of SNPs of β-defensin-1 and lactoferrin genes (DEFB1: rs1799946, rs1800972 and rs11362; and LTF: rs1126478) were investigated using the PCR-Invader assay. Protein level of AMPs in gingival crevicular fluid (GCF) was quantified by ELISA. Case-control studies revealed that the -44 CC genotype of DEFB1 (rs1800972) was associated with periodontitis (OR 2.51), particularly with severe chronic periodontitis (OR 4.15) and with combined severe and moderate chronic periodontitis (OR 4.04). No statistical differences were found in other genotypes. The β-defensin-1 concentrations in GCF were significantly lower in subjects with the -44 CC genotype of DEFB1 than in those without this genotype. No significant differences between GCF concentrations of AMPs and other genotypes were detected. The -44 CC genotype of the β-defensin-1 gene (DEFB1 rs1800972) may be associated with susceptibility to chronic periodontitis in Japanese.


肥満糖尿病ラット歯周炎の歯肉組織におけるオステオポンチンの局在

肥満糖尿病ラット歯周炎の歯肉組織におけるオステオポンチンの局在

Journal of the Japanese Association of Periodontology


Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-a expression upon lipopolysaccharide stimulation in human monocytes.

Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. We identified TDP-43 as one of the novel TNF-α factors and found that it bound to the LPS-responsive element in the TNF-α promoter to increase TNF-α expression.


Advanced glycation end-products increase expression of S100A8 and A9 via RAGE-MAPK in rat dental pulp cells

Advanced glycation end products (AGE) are involved in the progression of diabetic complications. Although our previous reports show that AGE increased dental pulp calcification, AGE accumulation is also associated with inflammation. This study examined AGE effect on the expression of inflammation factors using rat dental pulp tissues and cell cultures. Receptor for AGE (RAGE), S100A8, S100A9, and interleukin (IL)-1β were selected as inflammation parameters. Rat dental pulp cells were cultured and treated with AGE, and the effects were determined by real-time PCR. An anti-RAGE antibody or MAPK pathway inhibitors (PD98059, SB203580, and SP60012) were used to investigate AGE signaling pathway. The mRNA levels of RAGE, S100A8, S100A9, and IL-1β were higher in diabetic pulp tissues. AGE increased mRNA expressions of S100A8, S100A9, and IL-1β in cultured dental pulp cells. In the presence of anti-RAGE antibody, AGE did not increase in S100A8 or S100A9 expressions. The AGE-induced increases in S100A8 and S100A9 were inhibited by PD98059 and SB203580, respectively. Advanced glycation end products increased mRNA expression of S100A8, S100A9, and IL-1β under diabetic pulp conditions, and AGE-induced increases in S100A8 and S100A9 expressions may be associated with the RAGE-MAPK signaling pathway.


Expression and LPS-Induced Elevation of Nod2 and Calprotectin in the Submandibular Gland of Wild-Type and TLR4-Knockout Male Mice

Journal of Research and Practice in Dentistry


Predictors of improved functional outcome in elderly inpatients after rehabilitation: a retrospective study.

Age and disorder of oral function may be significant predictors of improved functional capacity after rehabilitation for elderly inpatients.


Glycated albumin and calprotectin levels in gingival crevicular fluid from patients with periodontitis and type 2 diabetes

Patients with diabetes mellitus (DM) have a high prevalence of periodontitis. Periodontitis in these patients is characterized by severe inflammation and tissue breakdown, and its diagnosis is important for cures of periodontitis and DM. The purpose of this study is to investigate the levels of glycated albumin (GA), a DM marker, and calprotectin, an inflammatory marker, in gingival crevicular fluid (GCF) from patients with periodontitis and DM (DM-P). The 78 participants in this study were patients with DM, chronic periodontitis (CP), DM-P, and healthy individuals (H). GCF and blood were collected, and GA and calprotectin in GCF were analyzed using Western blotting and enzyme-linked immunosorbent assay. Levels were compared among H, DM, CP, and DM-P groups. Blood GA and glycated hemoglobin (HbA1c) were measured, and the correlation among GCF GA and blood HbA1c or GA levels was investigated. Receiver operating characteristic (ROC) analysis for GCF GA to predict DM was performed. GA was identified in GCF, and its amount/concentration in GCF samples from DM and DM-P were significantly higher than those of non-DM groups (H and CP). Calprotectin amounts in GCF from CP and DM-P were significantly higher than in H and DM groups. GCF GA level was positively correlated with blood HbA1c and GA level. ROC analysis of GCF GA showed an optimal cutoff value to predict DM. GA showed a high level in GCF from patients with DM. Examination of GA and calprotectin in GCF may be useful for predicting DM-P.


Evaluation of bleeding on probing and gingival crevicular fluid enzyme activity for detection of periodontally active sites during supportive periodontal therapy

This study aimed to analyze the enzyme activity in gingival crevicular fluid (GCF) and its association with clinical parameters, especially bleeding on probing (BOP), and thus reconsider the significance and accuracy of recording BOP. A total of 184 patients who had entered supportive periodontal therapy were selected and GCF was collected from 401 sites before recording the clinical parameters, probing pocket depth (PPD), BOP, clinical attachment level, gingival index and plaque index. The enzyme activity of neutrophil elastase and aspartate aminotransferase and amount of protein in GCF were also analyzed. In the clinical parameters for biochemical data, amount of GCF showed the most correlation. A cut-off value for BOP and PPD were determined by the ROC curve and Youden index. Analysis was performed with all clinical parameters and biochemical data. Of the 401 sites, 51 were less than the cut-off value and were BOP-negative. On the other hand, 29 sites had values more than the cut-off value, with 14 BOP-negative sites and 15 BOP-positive sites. A conclusion is as follows: twenty-nine sites with values more than the cut-off value were diagnosed as sites requiring periodontal management, however, 14 of these were BOP-negative. These results suggest that combining other biochemical tests with examination of BOP and PPD may improve the validity of periodontal disease diagnosis. In future studies, it will be essential to find a marker that can precisely detect periodontal disease activity, and to develop a diagnostic tool for chair-side use.


Resveratrol improves oxidative stress and prevents the progression of periodontitis via the activation of the Sirt1/AMPK and the Nrf2/antioxidant defense pathways in a rat periodontitis model.

Oxidative stress is a key factor regulating the systemic pathophysiological effects associated with periodontitis. Resveratrol is a phytochemical with antioxidant and anti-inflammatory properties that can reduce oxidative stress and inflammation. We hypothesized that resveratrol may prevent the progression of periodontitis and reduce systemic oxidative stress through the activation of the Sirtuin1 (Sirt1)/AMP-activated protein kinase (AMPK) and the nuclear factor-E2 related factor 2 (Nrf2)/antioxidant defense pathways. Three groups of male Wistar rats (periodontitis treated with melinjo resveratrol, periodontitis without resveratrol, and control rats with no periodontitis or resveratrol treatment) were examined. A ligature was placed around the maxillary molars for 3 weeks to induce periodontitis, and the rats were then given drinking water with or without melinjo resveratrol. In rats with periodontitis, ligature placement induced alveolar bone resorption, quantified using three-dimensional images taken by micro-CT, and increased proinflammatory cytokine levels in gingival tissue. Melinjo resveratrol intake relieved alveolar bone resorption and activated the Sirt1/AMPK and the Nrf2/antioxidant defense pathways in inflamed gingival tissues. Further, melinjo resveratrol improved the systemic levels of 8-hydroxydeoxyguanosine, dityrosine, nitric oxide metabolism, nitrotyrosine, and proinflammatory cytokines. We concluded that oral administration of melinjo resveratrol may prevent the progression of ligature-induced periodontitis and improve systemic oxidative and nitrosative stress.


Buccal bone resorption around posterior implants after surgery: a 1-year prospective study.

This prospective study aimed to examine postoperative dimensional changes in the buccal bone and mucosa around single-stage implants placed in the posterior region. The dimensions of peri-implant tissue around screw-type implants placed in the posterior region were examined at surgery (baseline) and 6 months and 1 year after surgery. The lateral contour of the buccal bone and mucosa was horizontally measured at five vertical heights at 1-mm intervals (+1 to -3 mm from the implant platform) using custom-designed instruments. Bone resorption on the proximal sides was assessed on radiographs. Mucosal recession was measured on plaster casts of the dentition. Sixty-six implants placed in 30 patients were examined. All implants were clinically osseointegrated and stable throughout the study period. The buccal bone exhibited horizontal resorption throughout the study period, even at the most apical height measured. Assessed at each height, thicker bone (> 2 mm thick) tended to exhibit horizontal resorption during the first 6 months after surgery. However, the bone resorbed horizontally by approximately 0.4 mm during the final 6 months, irrespective of its contour. Vertical resorption of the buccal marginal bone was approximately 1 mm during the period from 6 months to 1 year. The bone-retaining group at the 1-year time point was found to have thicker bone walls at baseline compared with the bone-loss group. The thickness of the buccal mucosa showed little change. There was no obvious correlation between buccal bone resorption and mucosal recession. The buccal bone exhibited both horizontal and vertical resorption over the year after surgery. The initial contour of the bone was significantly associated with bone retention or loss at 1 year. However, mucosal recession was not directly affected by buccal bone resorption.

The International Journal of Oral & Maxillofacial Implants


Successful case of periodontal tissue repair by fibroblast growth factor-2. -Long-term follow-up and comparison with enamel matrix derivative-


Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line

S100A9 is a calcium-binding protein and subunit of antimicrobial calprotectin complex (S100A8/A9). Produced by neutrophils, monocytes/macrophages and keratinocytes, S100A9 expression increases in response to inflammation. For example, IL-1α produced by epithelial cells acts autonomously on the same cells to induce the expression of S100A8/A9 and cellular differentiation. Whereas it is well known that IL-1α and members of the IL-10 family of cytokines upregulate S100A8 and S100A9 in several cell lineages, the pathway and mechanism of IL-1α-dependent transcriptional control of S100A9 in epithelial cells are not established. Modeled using human epidermal keratinocytes (HaCaT cells), IL-1α stimulated the phosphorylation of p38 MAPK and induced S100A9 expression, which was blocked by IL-1 receptor antagonist, RNAi suppression of p38, or a p38 MAPK inhibitor. Transcription of S100A9 in HaCaT cells depended on nucleotides -94 to -53 in the upstream promoter region, based upon the use of deletion constructs and luciferase reporter activity. Within the responsive promoter region, IL-1α increased the binding activity of CCAAT/enhancer binding protein β (C/EBPβ). Mutated C/EBPβ binding sequences or C/EBPβ-specific siRNA inhibited the S100A9 transcriptional response. Hence, IL-1α is strongly suggested to increase S100A9 expression in a human epidermal keratinocyte cell line by signaling through the IL-1 receptor and p38 MAPK, increasing C/EBPβ-dependent transcriptional activity.

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms


Advanced glycation end-products enhance calcification in cultured rat dental pulp cells

Amorphous calcification frequently appears in dental pulp tissues of diabetic patients; however, its pathologic process has not been fully elucidated. We previously found that pulp stones and thickened predentin occurred more frequently in diabetic rats. Recent findings demonstrated that accumulation of advanced glycation end-products (AGE) might be involved in vascular calcification complicated with diabetes. The aim of this study was to determine the effect of AGE on calcified nodule formation by rat dental pulp cells in culture. Rat dental pulp cells and gingival fibroblasts were independently cultured with 50 and 100 μg/mL AGE. Alkaline phosphatase activity and calcified nodule formation were measured. Expressions of receptor for AGE, osteopontin (OPN), and osteocalcin (OCN) mRNA were determined by quantitative real-time polymerase chain reaction. Protein levels of OPN and OCN secreted in culture medium were quantified by enzyme-linked immunosorbent assay. AGE (50 and 100 μg/mL) markedly increased both alkaline phosphatase activity and calcified nodule formation in dental pulp cells (P < .01), whereas it did not affect those in gingival fibroblasts. Real-time polymerase chain reaction analysis revealed that AGE increased mRNA expressions of receptor for AGE, OPN, and OCN in dental pulp cells (P < .05). Enzyme-linked immunosorbent assay analysis revealed that the protein levels of OPN and OCN produced by dental pulp cells were higher in AGE-treated than in untreated cells (P < .05). AGE enhanced the calcification potentials of rat dental pulp cells, suggesting that it may stimulate pathologic calcification of diabetic dental pulp tissues.


Association of gingival recession and other factors with the presence of dentin hypersensitivity.

Dentin hypersensitivity (DH) may be present in association with gingival recession. The aim of this study was to determine quantitatively the association of gingival recession and other factors with the presence of DH. One hundred and four Japanese subjects with or without gingival recession were randomly selected. Intact canines and/or first premolars in both maxillary and mandibular quadrants were analyzed. Gingival recession was measured as a vertical length at the buccal site of the teeth. DH was recorded as an ordered categorical variable registering four increasing levels of pain after cold stimulation; from no discomfort to severe pain during and after stimulation (DH1, 2, 3, and 4). Association of DH with periodontal parameters and daily lifestyle was also investigated. Tooth-based analysis of 446 teeth from 104 subjects revealed that DH level was significantly higher in recessive teeth (1, 2, 3, and 4-8 mm) than in non-recessive teeth (0 mm). DH-positive rate in non-recessive teeth was only 18 % (DH1; 14 %, DH2; 3 %, and DH3; 1 %). Highest DH level was observed in teeth with severe recession (4-8 mm), showing DH0; 21 %, DH1; 33 %, DH2; 31 %, and DH3; 15 %. Recession-dependent increase in DH was observed, showing 18, 49, 52, 60, and 79 % DH-positive in teeth with 0, 1, 2, 3, and 4-8 mm recession, respectively. Plaque-free teeth showed a higher DH level than plaque-stained teeth, suggesting that good plaque control may be associated with the presence of DH. There were no significant differences in DH of teeth on the basis of smoking, probing depth, and bleeding on probing. Multiple logistic regression analysis revealed that gingival recession [odds ratio (OR) = 10.2, 95 % confidence interval (CI) = 5.5-18.9] and plaque deposition (OR = 0.3, 95 % CI = 0.2-0.5) were significant contributors to DH. Multilevel modeling analysis revealed that not only gingival recession and plaque deposition but also V-shaped cervical notch and tooth brushing frequency were associated with DH. These results demonstrate that the progression of gingival recession, plaque-free teeth, V-shaped cervical notch, and frequent brushing may be significant predictors of DH in canines and first premolars.


Assessment of the plasma/serum IgG test to screen for periodontitis.

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients.


Topical injection of osteoprotegerin prevents alveolar bone loss in rat experimental periodontitis

ラット実験的歯周炎におけるオステオプロテジェリンの局所投与による歯槽骨吸収抑制効果

Journal of the Japanese Association of Periodontology


Determination of calprotectin in gingival crevicular fluid by immunoassay on a microchip

Gingival crevicular fluid (GCF) contains calprotectin, which appears to be a useful biomarker for periodontal diseases because of its high level in GCF from periodontally diseased pockets. To determine calprotectin in GCF that has a very small volume, sandwich enzyme-linked immunosorbent assay (ELISA) on a microchip was performed and its utility was estimated. Anti-calprotectin primary antibody was discharged on a microchip using a piezoelectric inkjet printing system. Calprotectin standard and calprotectin in GCF samples from eleven subjects were determined by the ELISA method with the prepared microchip and their values were compared with those obtained by conventional ELISA. Using the ELISA on a microchip, a reasonable standard curve of calprotectin protein (1.56-100 ng/mL) was obtained. Calprotectin in GCF samples was quantified and showed reasonable values in accordance with the condition of periodontal diseases. The values determined by the microchip method and conventional ELISA showed a significant linear relationship (R(2)=0.981). Calprotectin in GCF was determined using the ELISA on a microchip with high efficiency and this ELISA method for calprotectin determination may become a useful method for diagnosing periodontal diseases.


Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils

Resistin is an adipocytokine that induces insulin resistance and is predominantly expressed in adipocytes and peripheral blood mononuclear cells. Resistin expression increases in inflammatory diseases as well as diabetes mellitus, and is upregulated by bacterial pathogens and proinflammatory cytokines. The aim of this study was to identify resistin in human gingival crevicular fluid, to compare the resistin levels in gingival crevicular fluid between subjects with and without periodontitis and diabetes mellitus and to investigate the regulation of resistin release from human neutrophils by Porphyromonas gingivalis lipopolysaccharide (P-LPS). Gingival crevicular fluid samples were collected from patients with chronic periodontitis (n = 24), patients with diabetes mellitus-related periodontitis (n = 18) and healthy subjects (n = 21). Resistin in gingival crevicular fluid was determined using western blot analysis and an ELISA kit. The glycated hemoglobin (HbA(1c)) value was obtained from patients with diabetes mellitus-related periodontitis by a medical interview. Human neutrophils were cultured with P-LPS (0-1000 ng/mL), or incubated with inhibitors of actin or microtubule polymerization in the absence or presence of P-LPS. The medium and cellular fractions were used for determination of resistin by ELISA. The resistin level in gingival crevicular fluid from patients with periodontitis or diabetes mellitus-related periodontitis was significantly higher than that of healthy subjects. The resistin level in gingival crevicular fluid was correlated with gingival index score, but not blood HbA(1c) value. The P-LPS increased resistin release from human neutrophils, and its induction was decreased by actin polymerization inhibitors. We show, for the first time, the presence of resistin in gingival crevicular fluid. A high resistin level in gingival crevicular fluid samples from periodontitis patients may to some extent be related to P-LPS-induced resistin release from neutrophils.


Analysis of proteins in human gingival crevicular fluid by mass spectrometry

Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.


Lipopolysaccharide-mediated induction of calprotectin in the submandibular and parotid glands of mice

S100A8 and S100A9 constitute a heterodimeric protein, calprotectin. The mRNAs of S100A8 and S100A9, being expressed at minimal levels in the submandibular and parotid glands (SMG and PG, respectively) of C3H/HeN mice, were induced strongly and transiently by lipopolysaccharide (LPS). Among the mRNAs of members of the S100 protein family examined, those of S100A8 and S100A9 were specifically induced by LPS in the salivary glands. The induction was assumed to be mediated via toll-like receptor 4 (TLR4), since their elevation was limited in C3H/HeJ mice, a TLR4-mutant strain. These proteins became expressed in the granular convoluted tubular cells and striated duct cells in the SMG, and in both acinar and duct cells in the PG (all in the cytoplasm). The salivary calprotectin level was not increased by LPS treatment, implying that elevated calprotectin was not secreted into the saliva and that they may function in microcellular environment of the salivary gland.


Topical and intermittent application of parathyroid hormone recovers alveolar bone loss in rat experimental periodontitis.

Periodontitis is characterized by periodontal tissue inflammation and alveolar bone loss. The intermittent administration of parathyroid hormone (PTH), a major regulator of bone remodeling, has been demonstrated to stimulate osteoblastic activity. Although the systemic administration of PTH has been reported to protect against periodontitis-associated bone loss, the effect of the topical administration of PTH is unclear. In this study, the effect of intermittent administration of PTH on osteoblastic differentiation was examined in cultured calvaria cells and then the effect of topical and intermittent administration of PTH was determined by measuring the recovery of alveolar bone loss after inducing experimental periodontitis in rats. Alkaline phosphatase activity and bone nodule formation were measured in fetal rat calvaria cells. Experimental periodontitis was induced by placing nylon ligature around rat maxillary molars for 20 d. After ligature removal (day 0), PTH was topically injected into buccal gingiva three times a week for 10 wk. Micro-computed tomography analysis and histological examination were performed on days 35 and 70. Intermittent exposure of PTH in calvaria cells increased alkaline phosphatase activity and bone nodule formation by 1.4- and 2.4-fold, respectively. Ligature procedures induced marked alveolar bone loss around the molars on day 0 and greater bone recovery was observed in the PTH-treated rats on day 70. An increase in osteoid formation on the surface of alveolar bone was detected in the PTH-treated rats. Intermittent treatment with PTH stimulated osteoblastic differentiation in fetal rat calvaria cell cultures, and topical and intermittent administration of PTH recovered alveolar bone loss in rat experimental periodontitis.


Regulation of antimicrobial peptide expression in human gingival keratinocytes by interleukin-1α

In the oral cavity, mucosal keratinocytes resist bacterial infection, in part, by producing broad-spectrum antimicrobial peptides (AMPs) including defensin, adrenomedullin and calprotectin. Epidermal keratinocyte expression of many AMPs increases in response to interleukin-1α (IL-1α). IL-1α is produced by epidermal keratinocytes and regulates cell differentiation. To better understand innate immunity in the oral cavity, we sought to determine how IL-1α might regulate expression of AMPs by human gingival keratinocytes (HGKs) using DNA microarray and Western blot analyses. HGKs from three subjects expressed eleven AMPs, including S100A7, S100A8, S100A9, S100A12, secretory leucocyte protease inhibitor, lipocalin 2 (LCN2), cystatin C and β-defensin 2. Of the expressed AMPs, S100A7, S100A12 and LCN2 were up-regulated by IL-1α (inducible AMPs); the other AMPs were considered to be constitutive. Human gingival keratinocytes, therefore, express constitutive and IL-1α-inducible AMPs to provide a rapid and robust innate response to microbial infection.


Osmotic Stress Affects Calcium Deposition and Osteopontin Expression in Cultured Rat Dental Pulp Cells

オスモティックストレスが培養歯髄細胞の硬組織形成能とオステオポンチン産生に及ぼす影響

Osmotic stress is a stimulus caused by osmotic pressure, which is known to affect physiological functions in various tissues and cells. However, there is little information on the effect of osmotic stress on dental pulp tissues and cells. In this study, we investigated the relationship between osmotic stress and calcium deposition in cultured rat dental pulp cells. We also analyzed the effect of osmotic stress on the expression and phosphorylation of osteopontin (OPN), a multi-functional bone matrix protein, in the dental pulp cells. Both hypo- and hyper-osmotic stress affected cell-shape and -growth, and decreased calcium deposition in cultured rat dental pulp cells. Hypoosmotic stress increased OPN secretion and its protein ratio in the culture medium. Also, the secreted OPN was highly phosphorylated. On the other hand, hyper-osmotic stress decreased OPN secretion and its protein ratio and most of the OPN in the hypertoinc medium was dephosphorylated. Both hypo- and hyper-osmotic stress decreased calcium deposition in the cultured rat dental pulp cells. However, the mechanism of the inhibitory action seemed to be different in the two groups, because the amount and phosphorylation pattern of OPN were not similar between the hypo- and hyper-osmotic conditions. We concluded that osmotic stress affects calcium deposition in cultured rat dental pulp cells and that OPN expression and its phosphorylation may be a control factor of osmotic stress-associated mineralization.

浸透圧変化が惹起する刺激源は一般にオスモティックストレス(osmotic stress)と呼ばれており,種々の組織や細胞に影響をもたらすことが知られている.しかし,オスモティックストレスが歯髄の組織や細胞にどのような影響を与えるのかを論じた成書や研究報告はぼとんどない.本研究ではラット由来の歯髄細胞培養系を用いて,オスモティックストレスと歯髄細胞の硬組織形成の関連性を検証した.また,オスモティックストレスによって硬組織形成関連タンパクであるオステオポンチン(OPN)の発現やリン酸化に変化が生じるかも検証した.その結果,歯髄細胞培養系においては,低張および高張のいずれのオスモティックストレスによっても細胞の形態や増殖に影響が認められ,硬組織形成も低下した.低張ストレスを付与するとOPNの発現量や総タンパク量に対するOPNの比率は上昇し,また発現したOPNはリン酸化されていた.一方,高張ストレスを付与するとOPNの発現量や総タンパク量に対するOPNの比率は低下し,脱リン酸化されたOPNが主に発現した.このように低張と高張のオスモティックストレスではOPNの発現量やリン酸化による修飾の程度が異なっていたことから,低張および高張のいずれのオスモティックストレスによっても石灰化は抑制されたが,その抑制のメカニズムは一様でないことが明らかとなった.以上より,オスモティックストレスは歯髄細胞の硬組織形成に影響を与え,OPNの発現およびそのリン酸化が制御因子として機能している可能性が示唆された.

The Japanese Journal of Conservative Dentistry


Shosaikoto increases calprotectin expression in human oral epithelial cells.

BACKGROUND AND OBJECTIVE: Oral epithelial cells help to prevent against bacterial infection in the oral cavity by producing antimicrobial peptides (AMPs). A broad-spectrum AMP, calprotectin (a complex of S100A8 and S100A9 proteins), is expressed by oral epithelial cells and is up-regulated by interleukin-1alpha (IL-1alpha). Shosaikoto (SST) is a traditional Japanese herbal medicine that has immunomodulatory effects and is reported to enhance the levels of IL-1alpha in epithelial cells. The purpose of this study was to investigate the effect of SST on the expression of calprotectin and other AMPs through the regulation of IL-1alpha in oral epithelial cells. MATERIAL AND METHODS: Human oral epithelial cells (TR146) were cultured with SST (at concentrations ranging from 10 to 250 microg/mL) in the presence or absence of anti-IL-1alpha or IL-1 receptor antagonist. The expression of S100A8- and S100A9-specific mRNAs was examined by northern blotting. Calprotectin expression and IL-1alpha secretion were investigated by immunofluorescent staining or ELISA. The expression of other AMPs and IL-1alpha was analyzed by RT-PCR and by quantitative real-time PCR. RESULTS: Shosaikoto (25 microg/mL) significantly increased the expression of S100A8- and S100A9-specific mRNAs and calprotectin protein. Shosaikoto increased S100A7 expression, but had no effect on the expression of other AMPs. The expression of IL-1alpha-specific mRNA and its protein were slightly increased by SST. A neutralizing antibody against IL-1alpha or IL-1 receptor antagonist inhibited SST up-regulated S100A8/S100A9 mRNA expression. CONCLUSION: These results suggest that SST increases the expression of calprotectin and S100A7 in oral epithelial cells. In response to SST, up-regulation of calprotectin may be partially induced via IL-1alpha.


High glucose levels increase osteopontin production and pathologic calcification in rat dental pulp tissues.

INTRODUCTION: Pulp stones are frequently formed as a pathologic calcification product in dental pulp tissues, but the pathogenesis is poorly understood. We previously found that osteopontin (OPN) was produced by dental pulp cells, and its expression was associated with formation of the pulp stone matrix. It was reported that amorphous calcification appeared in the dental pulp of diabetic patients. The aim of this study was to determine the relationship between OPN expression and pathologic calcification in rat diabetic pulp. METHODS: The effect of glucose on OPN production and alkaline phosphatase activity in cultured rat dental pulp cells (RPC-C2A) was investigated, and then dental pulp calcification and OPN expression in diabetic rats were determined and compared with those in healthy rats by histologic and immunohistochemical analyses. RESULTS: In RPC-C2A cells, biochemical analysis showed that a high concentration of glucose (50 mmol/L) increased OPN protein production and alkaline phosphatase activity 1.3-fold and 1.5-fold, respectively. Histologic observations showed more calcified particles in dental pulp tissues in diabetic than in nondiabetic rats. Moreover, a thickened layer of predentin was formed in the radicular pulp of diabetic rats. OPN was more strongly stained around the calcified particles and in the odontoblast zone under the thickened predentin in diabetic rats. CONCLUSIONS: OPN might be a key molecule involved in the increase of pathologic pulp calcifications, which are frequently observed in diabetic patients.


Modulation of calprotectin in human keratinocytes by keratinocyte growth factor and interleukin-1alpha.

Calprotectin is an antimicrobial complex composed of the S100A8 and S100A9 protein family subunits. Contributing to innate immunity, calprotectin expression is increased by interleukin-1alpha (IL-1alpha), which modulates keratinocyte differentiation. Keratinocyte growth factor (KGF) is produced by mesenchymal cells and has a mitogenic activity for epithelial cells. In this study, we investigated the effect of KGF on calprotectin expression in keratinocytes and modulation by IL-1alpha. Human keratinocytes were cultured with KGF in the presence or absence of a KGF receptor (KGFR) inhibitor or mitogen-activated protein kinase (MAPK) inhibitors. Calprotectin (S100A8/S100A9) expression was determined by northern blotting and enzyme-linked immunosorbent assay, respectively, whereas MAPK phosphorylation was analyzed by western blot analysis. KGF significantly decreased the expression of S100A8/S100A9-specific mRNAs and calprotectin protein. In the presence of KGF, KGFR inhibitor or extracellular-regulated kinase inhibitor restored KGF-downregulated expression of S100A8/S100A9. KGF increased IL-1alpha expression in keratinocytes, whereas IL-1alpha increased KGF expression in fibroblasts. Cocultured fibroblast and keratinocytes showed lower S100A8/S100A9 mRNA expression than keratinocytes alone in the presence or absence of IL-1alpha or KGF. These results suggest that fibroblast-derived KGF reduces or restricts calprotectin expression in keratinocytes, which supports our hypothesis that calprotectin expression in keratinocytes is modulated by factors associated with epithelial-mesenchymal interactions.


Ca2+-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions.

Yeast mitochondria have generally been believed not to undergo the permeability transition (PT) by the accumulation of Ca(2+) within the mitochondrial matrix, unlike mammalian mitochondria. However, the reason why the yeast PT is not induced by Ca(2+) has remained obscure. In this study, we examined in detail the effects of Ca(2+) on yeast mitochondria under various conditions. As a result, we discovered that the PT could be induced even in yeast mitochondria by externally added Ca(2+) under optimized experimental conditions. The 2 essential parameters for proper observation of the PT-inducing effects of Ca(2+) were the concentrations of the respiratory substrate and that of inorganic phosphate (Pi) in the incubation medium. The yeast mitochondrial PT induced by Ca(2+) was found to be insensitive to cyclosporin A and suppressed in the presence of a high concentration of Pi. Furthermore, when the PT was induced in yeast mitochondria by Ca(2+), the release of cytochrome c from mitochondria was also observed.

Biochimica et Biophysica Acta (BBA) - Bioenergetics


徳島大学パイロット事業支援プログラム「歯科医療系学生の口腔保健・福祉体験実習による健康長寿支援」

Journal of University Education Research


Differential permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes as revealed by proteomics analysis of proteins released from mitochondria.

It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca2+. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224-5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca2+-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca2+-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes are discussed.


Long-term Observation of Marked Overfilling in Root Canal Therapy : A Report of Three Cases

著明な過剰根管充填の経過を長期観察した3症例

In endodontic therapy, overfilling of gutta-percha point in root canals sometimes occurs due to wrong measurement of working length and overextension of gutta-percha point. Although a slight overfilling to periapical tissues (within 2 mm past the apical foramen) is tolerable, marked overfilling should be checked by postoperative recheck radiographs and/or clinical symptoms. In this report, three clinical cases of marked overfilling are described and long-term radiographic observation (6-14 years) was continued. Overfilled gutta-percha points on the radiographic observation were 3.0 (case 1), 2.5 (case 2), and 3.5 mm (case 3) in the left maxillary molar of a 47-year-old woman, in the left mandibular molar of a 45-year-old woman, and in the left mandibular premolar of a 39-year-old woman, respectively. In case 1, an overfilled point was cut after 1.5 years and disappeared after 3.5 years, and then apical radiolucency was improved after 6 years. In case 2, overfilled points started to minimize after 3 years and disappeared after 14 years. In case 3, an overfilled point was minimized after 13 years accompanied with improvement of apical lesion. None of the cases showed any significant clinical symptoms after the overfilling. These observations are interesting to longitudinally evaluate the disappearance of overfilled gutta-percha and the improvement of apical lesion.

歯内治療において,ときどき過剰根管充填(過剰根充)に遭遇する.一般に,過剰根充は作業長の測定ミスや根充操作時のガッタパーチャポイントの根尖孔外への突き出しが主な原因であるといわれている.根尖孔から2mm以内のわずかな過剰根充であれば根尖歯周組織に為害性はないが,それ以上の著しい過剰根充の場合は,術後の症状やX線写真によるチェックを行い注意深く対応する必要がある.本報告では,X線写真上で2.5∼3.5mmのポイントの突出が認められた3症例(症例1:上顎第二大臼歯,3.0mm,症例2:下顎第一大臼歯,2.5mm,症例3:下顎第一小臼歯,3.5mm)について,最長6∼14年の経過観察を行った結果を提示した.症例1では,突出ポイントは1年5カ月後に切断され3年8カ月後に消失し,6年8カ月後に根尖透過像も改善した.症例2では,突出ポイントは3年後に縮小傾向を示し,14年7カ月後には消失しており,根尖透過像の消失,歯槽硬線の明瞭化が観察された.症例3では,突出ポイントは13年6カ月後にわずかな部分を残して,ほとんどが吸収しており,同時に根尖透過像も消失していた.臨床症状は,症例1で打診痛が長期残存したほかには特記事項はほとんど認められなかった.当該歯は現在も健常に機能している.今回の症例は,術前診断,術中術後の情報を詳細に提示しながら過剰根充されたガッタパーチャポイントの経過と根尖病巣の治癒経過を長期間追跡したものであり,過剰根充の転帰を知るうえで興味深い臨床報告である.


Regulation of calprotectin expression in human keratinocytes in vitro

Calprotectin, a heterodimeric complex of S100A8 and S100A9, is an antimicrobial peptide (AMP) expressed in healthy or inflamed epithelium and epidermis. The expression of calprotectin and other AMPs in keratinocytes is regulated by proinflammatory cytokines, bacterial products and keratinocyte differentiation modulators. Interleukin-1α (IL-1α), an autonomous cytokine in keratinocytes, induces keratinocyte differentiation and up-regulates calprotectin expression. Since keratinocyte differentiation is also specified<KG>by interactions with the basement membrane and connective tissue proteins, we sought to learn whether calprotectin expression is affected by extracellular matrix (ECM) proteins in the presence or absence of IL-1α. Human immortalized epidermal keratinocytes (HaCaT cells) were grown in vitro on different ECM substrates including type I collagen (Col I), type IV collagen (Col IV), fibronectin (FN) and laminin (LM) with or without IL-1α and were then cultured in basement membrane extract (Matrigel)-coated dishes or on a feeder layer of fibroblasts. Calprotectin expression in the cultured cells was investigated using immunohistochemical and northern blot analyses, and an enzyme-linked immunosorbent assay. The four ECM proteins (Col I, Col IV, FN and LM) had little effect on calprotectin expression. IL-1α significantly increased the expression of S100A8/ S100A9 mRNAs and calprotecin protein in the cells cultured on each of the ECM(Col I, Col IV, FN and LM)-coated dishes,but these ECM protein substrates did not show a further effect on calprotectin expression in the presence of IL-1α. On the other hand, Matrigel and the fibroblast-feeder layer slightly decreased the expression of S100A8/S100A9 mRNAs in HaCaT cells. These results suggest that calprotectin expression in keratinocytes is regulated by cytokines during epithelial-mesenchymal interactions.<KG>Nihon Shishubyo Gakkai Kaishi (J Jpn Periodontol) 49 : 224-232,2007.

カルプロテクチンはS100A8とS100A9の2つの蛋白複合体から構成される抗菌ペプチドであり，上皮において発現している．カルプロテクチン発現は，炎症性サイトカイン，細菌産物や上皮細胞分化調節因子により調節されている．上皮細胞で恒常的に産生されるインターロイキン-1α( IL-1α) は上皮細胞分化を誘導し，カルプロテクチン発現を上昇させる．上皮細胞の分化は基底膜や結合組織の相互作用により制御されている．本研究では，細胞外基質(ECM)蛋白やIL-1αによるカルプロテクチン発現について検討した．ヒト上皮細胞株HaCaTをECM蛋白(I型とIV型コラーゲン，フィブロネクチン，ラミニン)コートディッシュに播種し，IL-1αの存在，非存在下で培養した．また，基底膜抽出物(マトリゲル)コートディッシュや線維芽細胞フィーダー層でも培養した．カルプロテクチンの発現は，免疫組織染色，ノーザンブロット法およびELISA法により分析した．その結果，4種のECM蛋白ディッシュで培養を行った場合，カルプロテクチン発現に変化は認められなかった．一方，IL-1αは培養ディッシュに関わらずカルプロテクチン発現を有意に上昇させたが，ECM蛋白はこの効果に影響を及ぼさなかった．また，マトリゲルや線維芽細胞フィーダー層ではカルプロテクチン発現の軽度の減少が認められた．これらの結果より，上皮細胞でのカルプロテクチン発現は上皮-間葉系においてサイトカインにより調節されていることが示唆された．

Journal of the Japanese Association of Periodontology


Calyculin A induces apoptosis and stimulates phosphorylation of p65NF-κB in human osteoblastic osteosarcoma MG63 cells

Previously, we reported that okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in human osteoblastic cells. However, it is not clear whether calyculin A, another inhibitor of protein phosphatases, would induce apoptosis in human osteoblastic cells and if so, which mechanisms are involved and whether the phosphorylation status of NF-kappaB could be affected by the treatment with calyculin A. In this report, we demonstrate that calyculin A induced apoptosis in MG63 cells, as judged by WST-8 assay, nuclear fragmentation, and DNA ladder formation. Expression of PTEN, FasL, and FasR mRNA was stimulated by calyculin A treatment in MG63 cells. Calyculin A also enhanced the phosphorylation level of NF-kappaB, as judged from the results of Western blot analysis and an in vitro dephosphorylation assay. Western blot analysis with anti-phospho-p65NF-kappaB antibody disclosed that the NF-kappaB was phosphorylated on serine 536 in cytosol and translocated into nucleus with calyculin A-treatment. The phosphorylation status of p65NF-kappaB was further confirmed by using the phosphorylation site-mutated p65NF-kappaB gene transfected into HEK293 cells. Unlike TNF-alpha, calyculin A treatment did not degraded IkappaBalpha within 10 min, while it degraded IkappaBalpha at 2-h treatment. Our findings indicate that calyculin A elicit phosphorylation of NF-kappaB on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappaB to the nucleus, thereby promoting transcriptional activity of NF-kappaB-related genes.


Interleukin-1alpha regulates antimicrobial peptide expression in human keratinocytes.

Human epidermis and epithelium serve as physiologic barriers to protect against noxious and infectious agents. Contributing to the defense against infection, epithelial cells express antimicrobial peptides (AMPs). The expression of AMPs in keratinocytes is generally regulated directly by bacteria and indirectly by proinflammatory cytokines. Bacteria may also regulate AMP expression by inducing keratinocyte expression of the autonomous proinflammatory cytokine, interleukin-1alpha (IL-1alpha). To test the hypothesis that AMP expression may be regulated by cell autonomous cytokines, we investigated the effect of IL-1alpha on the expression of AMPs in human keratinocytes (HaCaT cells) by microarray, northern blot, reverse transcriptase (RT)-PCR and western blot analyses. IL-1alpha increased expression of mRNA in a dose- and time-dependent manner specific for lipocalin 2, S100A8, S100A9 and secretory leukocyte protease inhibitor (SLPI) more than twofold relative to nonstimulated cells (control), and slightly upregulated S100A7 and beta-defensin-2. Furthermore, the expression of lipocalin 2, S100A7, S100A8, S100A9 and SLPI proteins were upregulated by IL-1alpha. On the other hand, HaCaT cells expressed mRNA specific for other AMPs, including cystatin 3, adrenomedullin, RNase-7 and mucin 5, which were unaffected by IL-1alpha treatment. These results suggest that the autonomous keratinocyte cytokine, IL-1alpha, selectively upregulates the expression of AMPs which may modulate innate epithelial cell immunity in skin and mucosa.


Establishment and characterization of rat dental epithelial derived ameloblast-lineage clones.

Teeth are the hardest tissues covered with enamel produced by ameloblasts. The ameloblast differentiation is controlled by sequential epithelial-mesenchymal interactions during tooth morphogenesis. However, the molecular mechanism of ameloblast differentiation remains unclear. To address this question, we developed an in vitro assay system to evaluate the molecular mechanism of amelogenesis. First, we established dental epithelium-derived clones from 6-day-old rat incisors and established that cells of the clone SRE-G5 were the largest producers of amelogenin mRNA. Next, we analyzed the effects of several chemicals on the amelogenin expression in SRE-G5 cells. Only mitogen-activated protein kinase (MAPK) activators enhanced amelogenin mRNA expression. This finding corresponded to the immunohistochemical data showing the presence of phosphorylated forms of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) during ameloblast differentiation. To examine the roles of MAPK signals, we compared the effects of anisomycin and sodium salicylate on the expression of tooth-related differentiation markers. Both anisomycin and sodium salicylate induced amelogenin, Abcg2, and Bmp4 mRNA and down-regulated p75NGFR mRNA. On the other hand, ALP, ectodin, Bmp2 and Fgf8 mRNA were up-regulated only by anisomycin. These results indicate that MAPK signaling functions, at least in part, as the inducer of ameloblast differentiation.

Journal of Bioscience and Bioengineering


Regulation of Calprotectin Expression by IL-1α and TGF-β in Human Gingival Keratinocytes

Calprotectin, a heterodimer of S100A8 and S100A9 with antimicrobial properties, is expressed in gingival keratinocytes and plays an important role in innate immunity. Because calprotectin expression is localized in the spinous cell layer of the gingival epithelium, we hypothesized that the expression of calprotectin in keratinocytes is related to the differentiation stage. The aim of the present study was to investigate the relationship between calprotectin expression and keratinocyte differentiation using some factors that regulated its differentiation. Normal human gingival keratinocytes were isolated from gingival tissues obtained at the extraction of wisdom teeth, and were cultured in serum-free keratinocyte medium supplemented with interleukin-1alpha or calcium, which promote keratinocyte differentiation, and transforming frowth factor-beta (TGF-beta) or retinoic acid, which suppress its differentiation. The expression of S100A8/A9 mRNA and the production of calprotectin in normal human gingival keratinocytes were examined by northern blotting and enzyme-linked immunosorbent assay, respectively. The expression of cytokeratin 14, involucrin and filaggrin (marker proteins of keratinocyte differentiation) was investigated by immunohistochemical staining, and the DNA-binding activity of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor, was examined by electrophoretic mobility shift assay. The expression of S100A8/A9 mRNA and the production of calprotectin were increased by interleukin-1alpha and calcium, but decreased by TGF-beta. RA inhibited the expression of S100A8/A9 and keratinocyte differentiation, which were induced by interleukin-1alpha. C/EBPalpha DNA-binding activity in normal human gingival keratinocytes was enhanced by interleukin-1alpha and calcium, but suppressed by TGF-beta. The present study suggests that calprotectin expression is related to keratinocyte differentiation and that C/EBPalpha is a regulator of calprotectin expression in keratinocytes.


Arachidonic acid inhibits osteoblast differentiation through cytosolic phospholipase A2-dependent pathway.

Arachidonic acid, a precursor of prostaglandins (PGs), is released by phospholipase A2 (PLA2) and plays an important role in biological reactions. We examined the roles of arachidonic acid on the pathway of PG synthesis and osteoblast differentiation by using clone MC3T3-E1 cells. The effect of arachidonic acid was evaluated by the measurement of alkaline phosphatase activity, cells shape, production of arachidonic acid and the expression of cyclooxygenase (COX). Arachidonic acid dose dependently decreased alkaline phosphatase activity and increased PGE2 production in MC3T3-E1 cells. The cell shape changed from polygonal to fibroblastic following treatment with arachidonic acid. These effects were recovered by the treatment of NS-398 and indomethacin. Arachidonic acid increased the expression of COX-2 mRNA and the PGE2 production. The exogenous arachidonic acid induced the release of cellular arachidonic acid in MC3T3-E1 cells. Moreover, methylarachidonyl fluorophosphonate suppressed the arachidonic acid release and the expression of COX-2 mRNA. The present results indicate that exogenous arachidonic acid stimulated the activity of PLA2, leading to the new release of membranous arachidonic acid. The amplified arachidonic acid enhanced PGE2 production by COX-2, which inhibits the differentiation of MC3T3-E1 cells. Our results provide a new insight into the molecular mechanisms by which exogenous arachidonic acid plays a role as a paracrine/autocrine amplifier of PGE2 biosynthesis by coupling with PLA2 and COX-2.


VDAC1, having a shorter N-terminus than VDAC2 but showing the same migration in an SDS-polyacrylamide gel, is the predominant form expressed in mitochondria of various tissues.

The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.


Norepinephrine Stimulates Calprotectin Expression in Human Monocytic Cells

Calprotectin is composed of two proteins, S100A8 and S100A9, which are S100 family members, and is detected in gingival crevicular fluid and gingival tissue with inflammation. The release and production of calprotectin are regulated by lipopolysaccharides of periodontopathic bacteria and cytokines. Emotional or psychological stress, a risk factor of periodontal disease, is transmitted by stress modulators including norepinephrine and cortisol. The aim of the present study was to investigate the effect of stress on calprotectin expression using norepinephrine and cortisol. U-937 cells, a human monocytic cell line, were incubated with norepinephrine in the presence or absence of beta- or alpha-adrenergic receptor antagonists, or with cortisol. The expression of S100A8/S100A9 mRNAs was examined by northern blotting and the amount of calprotectin was measured by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha (CCAAT enhancing binding protein), a transcription factor, was examined by electrophoretic mobility shift assay. Norepinephrine stimulated the expression of S100A8/S100A9 mRNAs via beta-adrenergic receptors in U-937 cells and significantly increased calprotectin production to about 3.6-fold that of the control. However, cortisol had no effect on calprotectin expression at the mRNA and protein levels. Norepinephrine elevated C/EBPalpha DNA binding activity, but cortisol did not increase the activity. Norepinephrine, a stress modulator, stimulated calprotectin expression in human monocytic cells. Calprotectin expression may be regulated by stress in addition to inflammatory factors.


High-turnover osteoporosis is induced by cyclosporin A in rats.

Cyclosporin A (CsA) is used widely as an immunosuppressive agent, but it induces osteoporosis as a prominent side effect. To elucidate the mechanisms involved in CsA-induced osteoporosis, the effects of CsA on bone metabolism were investigated in a rat experimental model. Fifteen-day-old rats were fed a powdered diet containing or lacking CsA for 8-30 days. Analysis was performed by micro-computed tomography (muCT) and light microscopy to examine histomorphometric changes in rat tibiae on days 8, 16, and 30. Plasma parathyroid hormone (PTH) and osteocalcin (OCN) levels were determined by enzyme-linked immunosorbent assay (ELISA) on days 8, 16, and 30. The expression of OCN, osteopontin (OPN), and cathepsin K mRNAs in tibial bone marrow was examined by Northern blot analysis on days 8 and 16. Although no significant differences were observed in tibial length during the experimental periods, or in histomorphometric parameters on day 8, an apparent decrease in bone volume was observed in the CsA-treated group after day 16. Histologic analysis showed that the number of osteoblasts and osteoclasts on the surface of trabecular bone in the CsA-treated group had increased significantly on day 16. Plasma PTH and OCN levels in CsA-treated rats were significantly higher than those in control animals on day 8. Northern blot analysis revealed that the CsA-treated group showed an increase in the expression of OCN, OPN, and cathepsin K mRNAs on day 8 compared with the controls. These findings suggest that bone resorption in CsA-treated rats is induced by high-turnover osteoporosis and that bone remodeling activity may be activated by PTH.


Alpha 2 integrin +807 polymorphism in drug-induced gingival overgrowth

Alpha2 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 alpha2 integrin gene is associated with high/low alpha2 integrin expression. The aim of this study was to test the hypothesis that expression of alpha2 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 alpha2 +807 C allele is one of the genetic risk factors for drug-induced gingival overgrowth.


ラット実験的歯周炎におけるアレンドロネートの歯槽骨吸収抑制作用

ラット実験的歯周炎におけるアレンドロネートの歯槽骨吸収抑制作用


Calprotectin expression in human monocytes: induction by porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1beta

Calprotectin is a major cytosolic protein of monocytes and granulocytes. It is increased in inflammatory tissues and is detected at high levels in the gingival crevicular fluid (GCF) of periodontitis patients. We previously reported that lipopolysaccharide of Porphyromonas gingivalis (P-LPS) and cytokines induced the release of calprotectin from monocytes isolated from human peripheral blood. The mechanisms of calprotectin expression and presence of its regulation factors in periodontal disease are unknown. On the other hand, P-LPS and cytokines are significant etiologic factors in the initiation and progression of periodontal diseases. In this study, we investigated the expression and production of calprotectin from human monocytes by examining the effects of lipopolysaccharide of P-LPS, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Monocytes were isolated from the peripheral blood of healthy donors and cultured in the presence or absence of P-LPS, TNF-alpha, or IL-1beta. The expressions of calprotectin mRNAs (MRP8 and MRP14) were detected by Northern blotting. The contents of calprotectin in the cells and medium fractions were determined by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha, a transcription factor of MRP14, was assessed by electrophoretic mobility shift DNA-binding assay (EMSA). P-LPS, TNF-alpha, and IL-1beta induced MRP8/14 mRNAs and calprotectin production in monocytes. These factors also induced DNA CEBPalpha binding activity in monocytes. P-LPS increased MRP14 mRNA expression in monocytes to the maximum level, about two times the control level after 24 hours treatment, but did not enhance the basal level of MRP8. When the effects of TNF-alpha and IL-1beta on those mRNAs were investigated, both MRP8 and MRP14 significantly increased to about 2- and 2.5-fold the control level, respectively. Increases of MRP8/14 mRNA expression were followed by their protein production at about 2-fold the basal amount. DNA binding activity of C/EBPalpha was increased in P-LPS, TNF-alpha, and IL-1beta-treated monocytes. These results demonstrate that P-LPS, TNF-alpha, and IL-1beta induce calprotectin production from human monocytes and that this production is associated with the activation of DNA C/EBPalpha binding complex.


A Study on Community Periodontal Index of Treatment Needs (CPITN) in Type 2 Diabetic Patients

2型糖尿病患者の歯周治療必要度指数(CPITN)

Diabetic patients tend to suffer from severe periodontitls, which is aggravated in poorly controlled longterm diabetes. We calculated the community periodontal index of treatment needs (CPITN) and the number of missing teeth in 49 type 2 diabetic patients (DM group) and 30 nondiabetic patients (controls) . The DM group was further divided into 4 groups by HbA<1C>^^ score (DM1:<5.7, DM2:5.8-6.4,DM3:6.5-7.9,DM4:>8.O), and CPITN and the number of teeth lost were measured in each group. The CPITN for all diabetic patients ranged from I to 4, but no O, indicating required oral hygiene instruction.

糖尿病患者は歯周病にかかりやすく,血糖コントロールが不良な場合や罹患期間が長くなると歯周病の程度も悪化すると言われている．本研究では,49名の2型糖尿病患者(DM群)および30名の非糖尿病患者(対照群)の歯周治療必要度(CPITN)と喪失歯数を調べるとともに,糖尿病患者をHbA1c値によって4群に分け,血糖コントロールの程度と歯周治療必要度との関連についても調べた．その結果,DM群ではすべての人が口腔清掃の改善を必要としていた(コード値1-4)．4mm以上の歯周ポケットを有する人(コード値3,4)は対照群で56%であるのに対しDM群では94%と歯周病罹患者が多かった．6mm以上の歯周ポケットを有する人,すなわち口腔清掃の改善やスケーリング,ルートプレーニングだけでなく複雑な歯周治療が必要な人(コード値4)は,対照群での23%に対しDM群では59%と高い数値を示した．HbA1c値によってDM群をさらに4群に分けた場合,4群いずれにおいてもコード値3と4の合計比率が非常に高く,HbA1c値8.0以上の群ではコード値4の比率が60%であった．しかしながら,これら4群間での統計学的有意差は認められなかった．1人あたりの平均喪失歯数は,DM群で7.8歯,対照群で1.7歯であり,糖尿病患者では歯の喪失が著明であることが確認された．DM群を4群に分けた場合の喪失歯は,HbA1c値5.8未満の群で6.7歯,HbA1c値8.0以上の群で10.8歯であったが,統計学的有意差は認められなかった．以上の結果から,2型糖尿病患者のCPITNすなわち歯周治療必要度指数は非糖尿病患者より高く,喪失歯数も糖尿病患者で多いことが明らかとなり,HbA1c値と歯周炎の程度との関連はさらなる研究が必要であることが示唆された．

Journal of the Japanese Association of Periodontology


Regulation of tenascin expression in cultured rat dental pulp cells

Tenascin (TN) is a glycoprotein of extracellular matrix abundantly present in embryonic mesenchymal tissues. Transforming growth factor-beta1 (TGF-beta1), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and retinoic acid (RA) are important regulators of dentinogenesis. Dental pulp cells have the capacity to differentiate into odontoblast-like cells. In this study, we investigated the effects of growth factors on TN expression and adhesive function using rat clonal dental pulp cells, RPC-C2A. Analyses of reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting revealed that RPC-C2A cells expressed TN molecules and that TGF-beta1, HGF, and RA increased expression of TN at the mRNA and protein level, while bFGF and EGF showed a weak effect. An adhesion assay revealed that treatment with TGF-beta1, HGF, and RA induced a marked reduction of cell attachment to fibronectin (FN)-coated surfaces, whereas there was no change with bFGF and EGF. Functional blocking of growth factor-stimulated TN protein by pretreating cells with anti-TN antibodies restored cell attachment to control levels. These findings suggest that TGF-beta1, HGF, and RA may regulate pulpal cell adhesion to FN-coated surfaces and that this effect is mediated by TN.


Substance P Enhances the Inhibition of Osteoblastic Cell Differentiation Induced by Lipopolysaccharide From Porphyromonas gingivalis

Substance P (SP) is a multifunctional neuropeptide that transmits pain signals, regulates the immune system, and may modulate emotional stress. SP stimulates bone resorption activity of osteoclasts, and SP level in gingival crevicular fluid is correlated with the degree of periodontal inflammation. However, the exact roles of SP in bone metabolism and periodontal diseases are poorly understood. To elucidate the effect of stress on bone metabolism, we investigated the effect of SP on osteoblastic cell differentiation in the presence of lipopolysaccharide from Porphyromonas gingivalis (P-LPS). The primary osteoblastic cells were isolated from fetal rat calvaria (RC) and cultured with SP, P-LPS, and an SP antagonist (SPa). The effects of SP on bone nodule (BN), alkaline phosphatase (ALPase) activity, mRNA expressions of SP receptor, bone matrix proteins, and Cbfa 1 were investigated. SP stimulated the expression of SP receptor mRNA in RC cells and enhanced its expression in the presence of P-LPS (50 ng/ml). SP inhibited BN formation and ALPase activity in a dose-dependent manner (10(-7) to 10(-5) M) and further suppressed mRNA expression of bone sialoprotein, osteopontin, and osteocalcin but not of type I collagen mRNA. The inhibitory effects were enhanced in the presence of P-LPS and blocked by Spantide III. Furthermore, the expression of Cbfa 1 mRNA was also markedly suppressed in the presence of SP and P-LPS. These findings suggest that SP inhibits osteoblastic cell differentiation and may be related to bone metabolism in periodontal diseases under conditions of stress.


Induction of calprotectin release by Porphyromonasgingivalis lipopolysaccharide in human neutrophils

Calprotectin, a major cytosolic protein of leukocytes, is detected in neutrophils, monocytes/macrophages, and epithelial cells. This protein is known to be a marker for several inflammatory diseases and is detected in inflammatory gingival tissue with periodontal disease. Recently, we found that the calprotectin level in gingival crevicular fluid from periodontitis patients was significantly higher than that of healthy subjects. However, the regulation of calprotectin in periodontal disease is unclear. In the present study, we investigated the effect of lipopolysaccharides of periodontopathic bacteria on calprotectin release from human neutrophils. Neutrophils from healthy donors were treated with lipopolysaccharides from Porphyromonas gingivalis (P-LPS), Actinobacillus actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, and Escherichia coli. Calprotectin of neutrophil was identified by immunoblotting and calprotectin amount was determined by ELISA. Two subunits (10 and 14 kDa) of calprotectin were observed in the cell and medium fractions from neutrophils. P-LPS increased calprotectin release from seven to 16 times the control level after 30 min and its effect appeared in a dose-dependent manner (10-1000 ng/ml). Lipopolysaccharides from A. actinomycetemcomitans, P. intermedia, F. nucleatum, and E. coli also induced calprotectin release from neutrophils. These results suggest that lipopolysaccharides from periodontopathic bacteria induce calprotectin release from human neutrophils.


エナメル基質タンパクの歯の移植への応用

エナメル基質タンパクの歯の移植への応用