Yuji Inagaki, Jun-ichi Kido, Yasufumi Nishikawa, Rie Kido, Eijiro Sakamoto, Mika Bandou, Koji Naruishi, Toshihiko Nagata and Hiromichi Yumoto : Gan-Lu-Yin (Kanroin), Traditional Chinese Herbal Extracts, Reduces Osteoclast Differentiation In Vitro and Prevents Alveolar Bone Resorption in Rat Experimental Periodontitis, Jul. 2021.
学術論文(審査論文):
1.
Rie Kido, Yuka Hiroshima, Jun-ichi Kido, Mika Bandou, Kaya Yoshida, Yuji Inagaki and Hiromichi Yumoto : Lipocalin 2 inhibits the expressions of interleukin-8 and macrophage inflammatory protein-1α in human neutrophil-like cells, Journal of Oral Biosciences, 67, 1, 100624, 2025.
(要約)
Lipocalin 2 (LCN2) is a glycoprotein with multiple functions, including antimicrobial activity, inflammatory response modulation, and cell migration. LCN2 is expressed in some cells, such as epithelial cells and neutrophils, and its levels are increased in inflammatory diseases. This study investigated the presence of LCN2 receptor (24p3R) in cells around periodontal tissues and function of LCN2 in cells with a receptor to explore the role of LCN2 in periodontal diseases. The presence of 24p3R was examined in periodontal cells, including human gingival fibroblasts, periodontal ligament fibroblasts, human oral epithelial cells (HOECs), and neutrophil-like cells (HL-60), by Western blotting. Changes in periodontal disease-associated proteins in the presence of recombinant LCN2 (rLCN2) were examined using a protein array in differentiated HL-60 (dHL-60) cells. Interleukin-8 (IL-8) and macrophage inflammatory protein-1α (MIP-1α) mRNA expressions were analyzed by qRT-PCR, and IL-8 and MIP-1α levels in dHL-60 cells treated with rLCN2 or Porphyromonas gingivalis-lipopolysaccaharide (P.g-LPS) were determined by enzyme-linked immunosorbent assay. We detected 24p3R in dHL-60 cells. IL-8 was highly expressed and MIP-1α was weakly expressed in dHL-60 cells using a protein array. rLCN2 significantly decreased IL-8 mRNA and protein levels and suppressed P.g-LPS-induced IL-8 production in dHL-60 cells. As dHL-60 cells were co-cultured with HOECs in which LCN2 was knocked down, IL-8 mRNA expression increased in dHL-60 cells. Furthermore, rLCN2 inhibited MIP-1α production in dHL-60 cells. LCN2 suppresses inflammatory responses by regulating IL-8 and MIP-1α expression in periodontal diseases.
Enggardipta Ajeng Raras, Minato Akizuki, Mika Bandou, Yuji Inagaki, Kazumitsu Sekine, Kenichi Hamada, Tomoko Sumitomo, Sato Kanta and Hiromichi Yumoto : Trimethyl chitosan: antibacterial activity on Enterococcus faecalis biofilm and cytocompatibility on human periodontal ligament fibroblasts cells, Journal of Dental Sciences, 2025.
Kenji Masutomi, Mika Bandou, Yuji Inagaki, Rie Kido, Yuta Uemura, Yukari Hatada, Ichi Jun Kido, Makoto Fukui, Daisuke Hinode and Hiromichi Yumoto : Relationship between oral hypofunction and salivary biomarkers in older adults: a cross-sectional study, BMC Oral Health, 24, 1, 766, 2024.
(要約)
Oral health problems have increased among older adults. Oral hypofunction is characterized by seven signs and symptoms: oral uncleanness, oral dryness, decline in occlusal force, decline in the movement function of the tongue and lips, decline in tongue pressure, decline in masticatory function, and decline in swallowing function, the latter being a significant risk factors for oral frailty. Recent research has suggested that salivary biomarkers can be used to assess not only oral diseases, including dental caries and periodontitis, but also systemic diseases, such as cancer and diabetes mellitus. This cross-sectional study investigated the relationship between oral hypofunction and the levels of salivary biomarkers. In total, 116 patients, aged 65 years or older, were included in this cross-sectional study. If three or more signs or symptoms in seven kinds of tests met the criteria of each test, oral hypofunction was diagnosed. The levels of biomarkers in the saliva collected from the patients were analyzed using an enzyme-linked immunosorbent assay. In total, 63.8% of patients were diagnosed with oral hypofunction. Multivariable linear regression analysis showed that calprotectin levels in the saliva were significantly related to oral moisture and masticatory function. Furthermore, 8-OHdG levels in saliva were associated with the movement function of the tongue and lips and oral hygiene level, and salivary AGE correlated only with the movement function of the tongue and lips. Multiple logistic regression analysis revealed that calprotectin levels in the saliva were significantly correlated with the prevalence of oral hypofunction, even after adjusting for age, sex, and periodontal status. However, none of the biomarker levels in the saliva had a significant relationship with the number of examinations outside the reference range. Calprotectin, 8-OHdG, and AGE levels are associated with oral hypofunction in older adults.
Yuka Hiroshima, Rie Kido, Jun-ichi Kido, Mika Bandou, Kaya Yoshida, Akikazu Murakami and Yasuo Shinohara : Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system, Odontology, 112, 4, 1103-1112, 2024.
(要約)
Secretory leukocyte protease inhibitor (SLPI) functions as a protease inhibitor that modulates excessive proteolysis in the body, exhibits broad-spectrum antimicrobial activity, regulates inflammatory responses, and plays an important role in the innate immunity. The purpose of the study was to artificially synthesize a SLPI, an antimicrobial peptide, and investigate its effect on antimicrobial activity against Porphyromonas gingivalis and interleukin-6 (IL-6) production. SLPI protein with a molecular weight of approximately 13 kDa was artificially synthesized using a cell-free protein synthesis (CFPS) system and investigated by western blotting and enzyme-linked immunosorbent assay (ELISA). Disulfide bond isomerase in the protein synthesis mixture increased the amount of SLPI synthesized. The synthesized SLPI (sSLPI) protein was purified and its antimicrobial activity was investigated based on the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells. The effect of sSLPI on IL-6 production in human periodontal ligament fibroblasts (HPLFs) was examined by ELISA. Our results showed that sSLPI significantly inhibited the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells and further inhibited IL-6 production by HPLFs. These results suggested that SLPI artificially synthesized using the CFPS system may play a role in the prevention of periodontal diseases through its antimicrobial and anti-inflammatory effects.
Yuka Hiroshima, Jun-ichi Kido, Rie Kido, Kaya Yoshida, Mika Bandou, Kazuaki Kajimoto, Hiromichi Yumoto and Yasuo Shinohara : β-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells., Odontology, 111, 830-838, 2023.
川上 歩花, 板東 美香, 髙士 友恵, 杉内 美月, Mizusa Hyodo, 三島 優奈, Masashi Kuroda, 森 博康, 黒田 暁生, 湯本 浩通, 松久 宗英, 阪上 浩, 堤 理恵 : Umami taste sensitivity is associated with food intake and oral environment in subjects with diabetes, The Journal of Medical Investigation : JMI, 70, 1.2, 241-250, 2023年.
(要約)
Dysgeusia is a serious problem in patients with diabetes because it often leads to overeating, which is associated with disease progression. This study aimed to investigate the association between taste sensitivity, eating habits, and the oral environment. In this cross-sectional study of 75 subjects with diabetes, gustatory function was assessed using the whole-mouth method, and lingual taste receptor gene expression was measured by real-time PCR. Food intake was evaluated using a food frequency questionnaire based on food groups. The oral environment was assessed using xerostomia and periodontal comprehensive examination. In total, 45.3%, 28.0%, and 18.7% of subjects showed lower umami taste sensitivity, low sweet taste sensitivity, and low salt taste sensitivity, respectively. Lower umami sensitivity correlated with lower estimated glomerular filtration rate and higher energy-source food intake. Subjects with diabetes with higher plaque control record showed significantly higher T1R3 gene expression than those with lower plaque control record. Reduced umami taste sensitivity is associated with decreased renal function and high energy food intake in diabetes. Subjects with diabetes with higher plaque control record showed significantly higher T1R3 gene expression, suggesting that the oral environment affects taste gene expression. J. Med. Invest. 70 : 241-250, February, 2023.
Yuji Inagaki, Jun-ichi Kido, Yasufumi Nishikawa, Rie Kido, Eijiro Sakamoto, Mika Bandou, Koji Naruishi, Toshihiko Nagata and Hiromichi Yumoto : Gan-Lu-Yin (Kanroin), traditional Chinese herbal extracts, reduces osteoclast differentiation in vitro and prevents alveolar bone resorption in rat experimental periodontitis, Journal of Clinical Medicine, 10, 3, 386, 2021.
(要約)
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.
Kohei Nonaka, Mika Bandou, Eijiro Sakamoto, Yuji Inagaki, Koji Naruishi, Hiromichi Yumoto and Jun-ichi Kido : 6-Shogaol inhibits advanced glycation end-products-induced IL-6 and ICAM-1 expression by regulating oxidative responses in human gingival fibroblasts, Molecules, 24, 20, e3705, 2019.
(要約)
Advanced glycation end-products (AGEs) cause diabetes mellitus (DM) complications and accumulate more highly in periodontal tissues of patients with periodontitis and DM. AGEs aggravate periodontitis with DM by increasing the expression of inflammation-related factors in periodontal tissues. 6-Shogaol, a major compound in ginger, has anti-inflammatory and anti-oxidative activities. However, the influence of shogaol on DM-associated periodontitis is not well known. In this study, the effects of 6-shogaol on AGEs-induced oxidative and anti-oxidative responses, and IL-6 and ICAM-1 expression in human gingival fibroblasts (HGFs) were investigated. When HGFs were cultured with 6-shogaol and AGEs, the activities of reactive oxygen species (ROS) and antioxidant enzymes (heme oxygenase-1 [HO-1] and NAD(P)H quinone dehydrogenase 1 [NQO1]), and IL-6 and ICAM-1 expressions were investigated. RAGE expression and phosphorylation of MAPKs and NF-κB were examined by western blotting. 6-Shogaol significantly inhibited AGEs-induced ROS activity, and increased HO-1 and NQO1 levels compared with the AGEs-treated cells. The AGEs-stimulated expression levels of receptor of AGE (RAGE), IL-6 and ICAM-1 and the phosphorylation of p38, ERK and p65 were attenuated by 6-shogaol. These results suggested that 6-shogaol inhibits AGEs-induced inflammatory responses by regulating oxidative and anti-oxidative activities and may have protective effects on periodontitis with DM.
Muneaki Hashimoto, Mika Bandou, Jun-ichi Kido, Kazumichi Yokota, Toshihiro Mita, Kazuaki Kajimoto and Masatoshi Kataoka : Nucleic acid purification from dried blood spot on FTA elute card provides template for polymerase chain reaction for highly sensitive Plasmodium detection, Parasitology International, 73, 101941, 2019.
(要約)
Polymerase chain reaction (PCR) is an essential diagnostic method for highly sensitive detection of Plasmodium-infected erythrocytes in patients with malaria. This study compared the performance of filter papers used for the preparation of dried blood spots (DBS) in detecting Plasmodium by PCR. Whole blood spiked with P. falciparum-infected erythrocytes to obtain samples with various levels of parasitemia were applied to Whatman 3MM Chr papers, FTA Cards, or FTA Elute Cards to prepare the DBS. DNA was purified from the DBS using a DNA purification kit and used as the template for nested PCR. In probit analysis, the estimated limit of detection (LoD) was 5.5 parasites/μL blood for Whatman 3MM Chr papers and FTA Cards and 1.6 parasites/μL blood for the FTA Elute Card. This result suggested that the DBS prepared on an FTA Elute Card yield the best template DNA for subsequent high-sensitivity PCR-based detection of P. falciparum-infected erythrocytes. This finding can help improve the accuracy of malarial diagnostic tests.
Kohei Nonaka, Yukari Kajiura, Mika Bandou, Eijiro Sakamoto, Yuji Inagaki, JH Lew, Koji Naruishi, Takahisa Ikuta, Kaya Yoshida, Tesuo Kobayashi, Hiromasa Yoshie, Toshihiko Nagata and Jun-ichi Kido : Advanced glycation end-products increase IL-6 and ICAM-1 expression via RAGE, MAPK and NF-kB pathways in human gingival fibroblasts, Journal of Periodontal Research, 53, 3, 334-344, 2018.
(要約)
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.
(キーワード)
Antigens, Neoplasm / Diabetes Complications / Fibroblasts / Gingiva / Glycation End Products, Advanced / Humans / Intercellular Adhesion Molecule-1 / Interleukin-6 / MAP Kinase Signaling System / Mitogen-Activated Protein Kinase Kinases / Mitogen-Activated Protein Kinases / NF-kappa B / Periodontitis / Phosphorylation / Reactive Oxygen Species / THP-1 Cells
Muneaki Hashimoto, Shouki Yatsushiro, Shohei Yamamura, Masato Tanaka, Hirokazu Sakamoto, Yusuke Ido, Kazuaki Kajimoto, Mika Bandou, Jun-ichi Kido and Masatoshi Kataoka : Hydrophilic-treated plastic plates for wide-range analysis of Giemsa-stained red blood cells and automated Plasmodium infection rate counting, Malaria Journal, 16, 1, 321, 2017.
(要約)
Malaria is a red blood cell (RBC) infection caused by Plasmodium parasites. To determine RBC infection rate, which is essential for malaria study and diagnosis, microscopic evaluation of Giemsa-stained thin blood smears on glass slides ('Giemsa microscopy') has been performed as the accepted gold standard for over 100 years. However, only a small area of the blood smear provides a monolayer of RBCs suitable for determination of infection rate, which is one of the major reasons for the low parasite detection rate by Giemsa microscopy. In addition, because Giemsa microscopy is exacting and time-consuming, automated counting of infection rates is highly desirable. A method that allows for microscopic examination of Giemsa-stained cells spread in a monolayer on almost the whole surface of hydrophilic-treated cyclic olefin copolymer (COC) plates was established. Because wide-range Giemsa microscopy can be performed on a hydrophilic-treated plate, the method may enable more reliable diagnosis of malaria in patients with low parasitaemia burden. Furthermore, the number of RBCs and parasites stained with a fluorescent nuclear staining dye could be counted automatically with a software tool, without Giemsa staining. As a result, researchers studying malaria may calculate the infection rate easily, rapidly, and accurately even in low parasitaemia. Because the running cost of these methods is very low and they do not involve complicated techniques, the use of hydrophilic COC plates may contribute to improved and more accurate diagnosis and research of malaria.
Koji Naruishi, Keiji Oishi, Yuuji Inagaki, Masumi Horibe, Mika Bandou, Masami Ninomiya, K Kawahara, J Minakuchi, S Kawashima, K Shima, Jun-ichi Kido and Toshihiko Nagata : Association between Periodontal Condition and Kidney Dysfunction in Japanese Adults: A Cross-Sectional Study, Clinical and Experimental Dental Research, 2, 2, 1-8, 2016.
Yuka Hiroshima, Mika Bandou, Yuji Inagaki, Reiko Kido, Masatoshi Kataoka, Toshihiko Nagata and Jun-ichi Kido : Effect of Hangeshashinto on calprotectin expression in human oral epithelial cells., Odontology, 104, 2, 152-162, 2016.
(要約)
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.
Jun-ichi Kido, Yukiko Bandou, Mika Bandou, Yukari Kajiura, Yuka Hiroshima, Yuji Inagaki, Hiromi Murata, Takahisa Ikuta, Reiko Kido, Koji Naruishi, Makoto Funaki and Toshihiko Nagata : YKL-40 level in gingival crevicular fluid from patients with periodontitis and type 2 diabetes, Oral Diseases, 21, 5, 667-673, 2015.
(要約)
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.
Takahisa Ikuta, Yuji Inagaki, Kazuya Tanaka, Tsuyoshi Saito, Yukiko Nakajima, Mika Bandou, Jun-ichi Kido and Toshihiko Nagata : Gene polymorphism of β-defensin-1 is associated with susceptibility to periodontitis in Japanese., Odontology, 103, 1, 66-74, 2015.
(要約)
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.
Yukari Kajiura, Mika Bandou, Yuji Inagaki, Toshihiko Nagata and Jun-ichi Kido : Glycated albumin and calprotectin levels in gingival crevicular fluid from patients with periodontitis and type 2 diabetes, Journal of Periodontology, 85, 12, 1667-1675, 2014.
(要約)
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.
Mika Bandou, Xianqiong Zou, Yuka Hiroshima, Masatoshi Kataoka, Karen F. Ross, Yasuo Shinohara, Toshihiko Nagata, Mark C. Herzberg and Jun-ichi Kido : Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line, Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1829, 9, 954-962, 2013.
(要約)
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.
(キーワード)
Base Sequence / Calgranulin B / Cell Line / DNA Primers / Epidermis / Humans / Interleukin-1alpha / Keratinocytes / Polymerase Chain Reaction / RNA Interference / Transcription, Genetic / p38 Mitogen-Activated Protein Kinases
Kaori Abe, Y Hashimoto, S Yatsushiro, S Yamamura, Mika Bandou, Yuka Hiroshima, Jun-ichi Kido, M Tanaka, Yasuo Shinohara, Toshihiko Ooie, Yoshinobu Baba and Masatoshi Kataoka : Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip., PLoS ONE, 8, 1, e53620, 2013.
(要約)
Sandwich enzyme-linked immunosorbant assay (ELISA) using a 96-well plate is frequently employed for clinical diagnosis, but is time-and sample-consuming. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. The microchip was made of cyclic olefin copolymer with 4 straight microchannels. For the construction of the sandwich ELISA for interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α), we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-IL-6 antibody or 1st anti-TNF-α antibody on the surface of the each microchannel. After the infusion of 2 µl of sample to the microchannel and a 20 min incubation, 2 µl of biotinylated 2nd antibody for either antigen was infused and a 10 min incubation. Then 2 µl of avidin-horseradish peroxidase was infused; and after a 5 min incubation, the substrate for peroxidase was infused, and the luminescence intensity was measured. Calibration curves were obtained between the concentration and luminescence intensity over the range of 0 to 32 pg/ml (IL-6: R(2) = 0.9994, TNF-α: R(2) = 0.9977), and the detection limit for each protein was 0.28 pg/ml and 0.46 pg/ml, respectively. Blood IL-6 and TNF-α concentrations of 5 subjects estimated from the microchip data were compared with results obtained by the conventional method, good correlations were observed between the methods according to linear regression analysis (IL-6: R(2) = 0.9954, TNF-α: R(2) = 0.9928). The reproducibility of the presented assay for the determination of the blood IL-6 and TNF-α concentration was comparable to that obtained with the 96-well plate. Simultaneous detection of blood IL-6 and TNF-α was possible by the deposition and fixation of each 1st antibody on the surface of a separate microchannel. This assay enabled us to determine simultaneously blood IL-6 and TNF-α with accuracy, satisfactory sensitivity, time saving ability, and low consumption of sample and reagents, and will be applicable to clinic diagnosis.
Jun-ichi Kido, Kaori Abe, Shouki Yatsushiro, Mika Bandou, Yuka Hiroshima, Toshihiko Nagata, Toshihiko Ooie, Masato Tanaka and Masatoshi Kataoka : Determination of calprotectin in gingival crevicular fluid by immunoassay on a microchip, Clinical Biochemistry, 45, 15, 1239-1244, 2012.
(要約)
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.
Yuka Hiroshima, Mika Bandou, Yuji Inagaki, Chie Wada -Mihara, Masatoshi Kataoka, Hiromi Murata, Yasuo Shinohara, Toshihiko Nagata and Jun-ichi Kido : Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils, Journal of Periodontal Research, 47, 5, 554-562, 2012.
(要約)
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.
Jun-ichi Kido, Mika Bandou, Yuka Hiroshima, Hiroyuki Iwasaka, Keisuke Yamada, Naoto Ohgami, Toshiyuki Namubu, Masatoshi Kataoka, Takenori Yamamoto, Yasuo Shinohara, Ikuko Sagawa and Toshihiko Nagata : Analysis of proteins in human gingival crevicular fluid by mass spectrometry, Journal of Periodontal Research, 47, 4, 488-499, 2012.
(要約)
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.
Jun-ichi Kido, Mami Hino, Mika Bandou and Yuka Hiroshima : Diagnosis of periodontal diseases by biomarkers, Electrical Engineering in Japan, 179, 1, 40-45, 2012.
Yuka Hiroshima, Mika Bandou, Masatoshi Kataoka, Yuji Inagaki, Mark C. Herzberg, Karen F. Ross, Kazuo Hosoi, Toshihiko Nagata and Jun-ichi Kido : Regulation of antimicrobial peptide expression in human gingival keratinocytes by interleukin-1α, Archives of Oral Biology, 56, 8, 761-767, 2011.
(要約)
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.
Yuka Hiroshima, Mika Bandou, Masatoshi Kataoka, Yasuo Shinohara, MC Herzberg, KF Ross, Yuji Inagaki, Toshihiko Nagata and Jun-ichi Kido : Shosaikoto increases calprotectin expression in human oral epithelial cells., Journal of Periodontal Research, 45, 1, 79-86, 2010.
(要約)
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.
Mika Bandou, Yuka Hiroshima, Masatoshi Kataoka, MC Herzberg, KF Ross, Yasuo Shinohara, Takenori Yamamoto, Toshihiko Nagata and Jun-ichi Kido : Modulation of calprotectin in human keratinocytes by keratinocyte growth factor and interleukin-1alpha., Immunology and Cell Biology, 88, 3, 328-333, 2010.
(要約)
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.
Yuka Hiroshima, Mika Bandou, Masatoshi Kataoka, Toshihiko Nagata and Jun-ichi Kido : Regulation of calprotectin expression in human keratinocytes in vitro, Journal of the Japanese Association of Periodontology, 49, 3, 224-232, 2007.
Mika Bandou, Yuka Hiroshima, Masatoshi Kataoka, Yasuo Shinohara, MC Herzberg, KF Ross, Toshihiko Nagata and Jun-ichi Kido : Interleukin-1alpha regulates antimicrobial peptide expression in human keratinocytes., Immunology and Cell Biology, 85, 7, 532-537, 2007.
(要約)
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.
Periodontal diseases cause an inflammation and degradation of periodontal tissues and missing of teeth. The incidence rate of periodontal diseases is high in middle-aged and elderly people.A reasonable diagnosis of periodontal diseases is very important to keep teeth, however, conventional examinations of periodontal diseases is not necessarily exact and objective. Gingival crevicular fluid (GCF) is an exudate secreted from periodontal tissues and contains many components including proteolytic enzymes, inflammatory cytokines, blood-associated proteins, cellular and bacterial fragments. Because some proteins in GCF are related to inflammation, tissue degradation and bone metabolism, those proteins have been studying as a diagnostic marker of periodontal diseases. GCF is noninvasively collected using a sterile paper strip and biomarkers are determined using enzyme-linked immunosorbent assay (ELISA) and enzyme activity assay. We identified calprotectin, an inflammationrelated protein, in GCF and calprotectin level in GCF from periodontitis sites was significantly higher than that of healthy control. Calprotectin level in GCF was positively correlated to gingival index and other biomarkers and decreased by periodontal treatments. Resistin is an adipocytokine and its level increases in some inflammatory diseases. Resistin level in GCF from periodontitis sites was high compared to the level of healthy control samples. Procollagen type I C-terminal peptide (PICP) is a biomarker for bone metabolism and its level was high in GCF collected from periodontitis sites. These results suggested that calprotectin, resistin and PICP are useful biomarkers for periodontal diseases. On the other hand, we showed that glycated albumin (GA), a marker of diabetes mellitus (DM), was contained in GCF and GA level in GCF from DM patients was significantly higher than that of non-DM individuals. Components in GCF may be biomarkers of systemic diseases as well as periodontal diseases and their determination will be useful diagnostic examination of some diseases. Recently, we have been studying the determining system of GCF calprotectin, including microchip ELISA, surface plasmon resonance assay and immuno-chromatography assay. When GCF biomarkers are determined using the determining systems, we will simply, exactly and objectively diagnose periodontal diseases at our dental offices.
Yuka Hiroshima, Mika Bandou, Takahiro Hasegawa, Yuji Inagaki and Chie Wada -Mihara : Resistin release from neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide., IADR (International Association of Dental Research) Barcelona, Spain, July 14-17, 2010,
2.
Enggardipta Ajeng Raras, Minato Akizuki, Mika Bandou, Yuji Inagaki, Kazumitsu Sekine, Kenichi Hamada, Tomoko Sumitomo and Hiromichi Yumoto : Evaluation of Trimethyl Chitosan for Root Canal Irrigation, FDCU (Faculty of Dentistry, Chulalongkorn University) International Symposium 2025, Bangkok, Thailand, May 2025.
3.
T Ikuta, Yukari Kajiura, Mika Bandou, Yuji Inagaki, Koji Naruishi, Jun-ichi Kido and Toshihiko Nagata : YKL-40 in gingival crevicular fluid from patients with periodontitis and diabetes., Euro Perio 2015, Jun. 2015.
4.
Yukari Kajiura, Mika Bandou, Jun-ichi Kido, Yuji Inagaki and Toshihiko Nagata : Determination of glycated albumin and calprotectin in gingival crevicular fluid from patients with periodontitis and diabetes., Euro Perio 2015, Jun. 2015.
5.
Yukari Kajiura, Mika Bandou, Yuji Inagaki, Hiromi Murata, Jun-ichi Kido and Toshihiko Nagata : Advanced glycation end-product increases oxidative stress in human gingival fibroblasts, 53th General Session of the Korean Academy of Periodontology Meeting, Seoul, Korea, Nov. 2013.
6.
Yukiko Nakajima, Jun-ichi Kido, Yuji Inagaki, Mika Bandou, Yuka Hiroshima, Hiromi Murata and Toshihiko Nagata : Effect of hypoxia on gene expression in human oral keratinocytes, 10th Asian Pacific Society of Periodontology Meeting, Nara, Sep. 2013.
7.
Jun-ichi Kido, Mika Bandou, Yuji Inagaki, Yuka Hiroshima, Hiromi Murata, Chie Wada -Mihara, 梶浦 由加里, 生田 貴久, 篠原 宏貴, 橋本 万里, Yukiko Nakajima, Yukiko Bandou, Makoto Funaki, Haruhiko Saito and Toshihiko Nagata : Diagnosis of diabetes-associated periodontitis using glycoalbumin and calprotectin in gingival crevicular fluid, 10th Asian Pacific Society of Periodontology Meeting, Nara, Sep. 2013.
8.
Toshihiko Nagata, Jun-ichi Kido, Mika Bandou, Yuka Hiroshima, Yuji Inagaki, Hiromi Murata, Chie Wada -Mihara, Mika Bandou and Makoto Funaki : Diagnosis of diabetes-associated periodontitis using biomarkers in gingival crevicular fluid, 91th General Session & Exhibition of the International Association for Dental Research. Seattle, USA, May 2013.
9.
Mika Bandou, Yukari Kajiura, Jun-ichi Kido, Yuji Inagaki, Masatoshi Kataoka and Toshihiko Nagata : Lipopolysaccharide induces oxidative stress and antioxidant responses in gingival fibroblasts, The 60th Annual Meeting of Japanese Association for Dental Research, Program and abstracts of papers, 79, Dec. 2012.
10.
Toshihiko Nagata, Jun-ichi Kido, Mika Bandou, Yuka Hiroshima, Yuji Inagaki, Masami Ninomiya, Chie Wada -Mihara, Hiromi Murata, Yukiko Bandou and Makoto Funaki : Diagnosis of diabetes-associated periodontitis by measuring biomarkers in gingival crevicular fluid, The 9th International Diabetes Federation Western Pacific Region Congress/ The 4th AASD Scientific Meeting, Nov. 2012.
11.
Yukiko Nakajima, Yuji Inagaki, Mika Bandou, Yuka Hiroshima, Jun-ichi Kido and Toshihiko Nagata : Advanced glycation end-product increase calcification and inflammatory factors in cultured rat dental pulp cells, European Calcified Tissue Society, 39th Annual Congress Final programme, 73, Stockholm (Sweden), May 2012.
12.
Yuji Inagaki, Yukiko Nakajima, Mika Bandou, Yuka Hiroshima, Jun-ichi Kido and Toshihiko Nagata : Relationship between pathologic calcification and osteopontin expression in dental pulp tissues of diabetic rats, European Calcified Tissue Society, 39th Annual Congress, Final Programme, 125, Stockholm (Sweden), May 2012.
国内講演発表:
1.
Enggardipta Ajeng Raras, Minato Akizuki, Mika Bandou, Yuji Inagaki and Hiromichi Yumoto : Comparison of Antibacterial Efficacy of Chitosan and Its Derivatives Against Enterococcus faecalis Biofilm, 日本歯科保存学会・2025年度春季学術大会(第162回), Jun. 2025.
2.
Raras Ajeng Enggardipta, Kanta Sato, Minato Akizuki, Mika Bandou, Yuji Inagaki and Hiromichi Yumoto : N,N,N-Trimethyl chitosan exhibits antibiofilm activity on Enterococcus faecalis biofilm and cytocompatibility on human periodontal ligament fibroblasts, 日本歯科保存学会2024年度秋季学術大会(第161回), Nov. 2024.
中島 由紀子, 木戸 淳一, 板東 美香, 稲垣 裕司, 永田 俊彦 : Effect of hypoxia and P. gingivalis-lipopolysaccharide on the expression of inflammation-related molecules in human oral keratinocytes, 第57回春季日本歯周病学会学術大会, 2014年5月.