Daisuke Nakamura, Yuji Inagaki, Yuta Uemura, Yuka Hiroshima, Yoshimune Iwata, Rie Kido, Mika Bandou, Tomoko Sumitomo and Hiromichi Yumoto : Porphyromonas gingivalis outer membrane vesicles promote alveolar bone resorption by increasing the local RANKL/OPG ratio in experimental periodontitis model rats, Scientific Reports, 16, 1, 2026.
(Summary)
Outer membrane vesicles (OMVs) released by bacteria contain bacteria-specific nucleic acids and proteins. Porphyromonas gingivalis (Pg)-OMVs exert various effects on periodontal tissue cells; however, their involvement in bone metabolism remains unclear. Therefore, this study aimed to investigate the effects of Pg-OMVs on osteoclasts and osteoblasts in vitro and elucidate their involvement in alveolar bone resorption in vivo using experimental periodontitis model rats. Tartrate-resistant acid phosphatase staining and Western blotting revealed that Pg-OMVs promoted osteoclast differentiation and increased the levels of several osteoclast differentiation markers, respectively, in RAW264.7 cells. Quantitative reverse transcription-polymerase chain reaction and Western blotting revealed that Pg-OMVs increased the receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) levels and decreased osteoprotegerin (OPG) levels in MC3T3-E1 cells. Pg-OMVs reduced alkaline phosphatase activity and calcified nodule formation in a concentration-dependent manner. Rats were divided into four groups (healthy control, Pg-OMV administration, ligature-induced periodontitis, and ligature-induced periodontitis with Pg-OMV administration groups), and the alveolar bone resorption degree was measured via micro-computed tomography. Pg-OMVs administration significantly promoted alveolar bone resorption and increased the RANKL/OPG ratio in periodontal tissues. Our results suggest that Pg-OMVs contribute to alveolar bone resorption by promoting osteoclast differentiation, suppressing osteoblast mineralization, and increasing the RANKL/OPG ratio. The online version contains supplementary material available at 10.1038/s41598-026-46625-4.
Hiroshi Ito, Yukihiro Numabe, Shuichi Hashimoto, Satoshi Sekino, Etsuko Murakashi, Hitomi Ishiguro, Daisuke Sasaki, Takashi Yaegashi, Hideki Takai, Masaru Mezawa, Yorimasa Ogata, Hisashi Watanabe, Yuichi Izumi, Jun-ichi Kido, Yuka Hiroshima and Toshihiko Nagata : Evaluation of inflammatory periodontal tissue by detecting the hemoglobin released in gingival crevicular fluid during supportive periodontal therapy., Odontology, 2025.
(Summary)
The purpose of this study was to longitudinally investigate the hemoglobin (Hb) test in gingival crevicular fluid of periodontitis patients, together with clinical parameters (plaque index: PlI, probing depth: PD, clinical attachment level: CAL, bleeding on probing: BOP, and gingival index: GI) during two-year supportive periodontal therapy (SPT). A total of 191 periodontal sites in 91 patients during the SPT period were clinically and biochemically evaluated three times (first, second, and third examinations) over two years. After the first examination of the clinical and biochemical parameters, periodontal support treatments were administered and then repeated every three months until the third examination. The values of PD, CAL, GI, and Hb decreased from the first examination to the second or third examination. Notably, the percentage reduction of Hb test values was significant. However, only the BOP value tended to increase. The decreases in these clinical and biochemical parameters suggested that the periodontitis was not exacerbated, but rather tended to recover with periodontal treatment. These reductions in PD, CAL, and Hb values from the first examination were clearly more common in the PD ≥ 5 mm group than in the PD ≤ 4 mm group for all test sites. In this study, the highly sensitive Hb test showed that periodontal treatment for two years suppressed the progression of periodontitis. This trend was significant in the deep gingival sulcus (PD ≥ 5 mm) group, suggesting that the Hb test may be a useful quantitative marker to predict recurrence of periodontitis.
Mana Mekata, Kaya Yoshida, Ayu Takai, Yuka Hiroshima, Ayu Ikuta, Kayo Yoshida and Kazumi Ozaki : Porphyromonas gingivalis outer membrane vesicles increase vascular permeability by inducing stress fiber formation and degrading vascular endothelial-cadherin in endothelial cells., The FEBS Journal, 292, 7, 1696-1709, 2025.
(Summary)
Porphyromonas gingivalis (Pg) is a keystone bacterium associated with systemic diseases, such as diabetes mellitus and Alzheimer's disease. Outer membrane vesicles (OMVs) released from Pg have been implicated in systemic diseases by delivering Pg virulence factors to host cells in distant organs and inducing cellular dysfunction. Pg OMVs also have the potential to enter distant organs via the bloodstream. However, the effects of Pg OMVs on the vascular function are poorly understood. Here, we showed that Pg OMVs increase vascular permeability by promoting stress fiber formation and lysosome/endosome-mediated vascular endothelial-cadherin (VEc) degradation in human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). F-actin, visualized via fluorescein isothiocyanate-phalloidin, became thicker and longer, leading to the formation of radical stress fibers in response to Pg OMVs in HUVECs and HPMECs. Western blotting and quantitative real-time polymerase chain reaction analyses revealed that Pg OMVs decreased VEc protein levels in a gene-independent manner. Pg OMVs enhanced vesicular VEc accumulation in the cytoplasm around lysosome-associated membrane protein 1-positive structures during pretreatment with the lysosomal inhibitor chloroquine. This suggests that Pg OMVs decrease VEc protein levels by accelerating their internalization and degradation via lysosomes and endosomes. A27632 inhibition of Rho kinases impaired the Pg OMV-induced stress fiber formation and VEc degradation, resulting in the recovery of hyperpermeability. These findings provide new insights into the pathogenesis of systemic diseases that are associated with periodontal diseases.
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.
(Summary)
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.
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.
(Summary)
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.
Kayo Yoshida, Kaya Yoshida, Mariko Seyama, Yuka Hiroshima, Mana Mekata, Natsumi Fujiwara, Yasusei Kudo and Kazumi Ozaki : Porphyromonas gingivalis outer membrane vesicles in cerebral ventricles activate microglia in mice, Oral Diseases, 29, 8, 3688-3697, 2023.
(Summary)
Objective: Porphyromonas gingivalis (Pg) is thought to be involved in the progression of Alzheimer's disease (AD). Whether Pg or its contents can reach the brain and directly affect neuropathology is, however, unknown. Here, we investigated whether outer membrane vesicles (OMVs) of Pg translocate to the brain and induce the pathogenic features of AD. Material and Methods: Pg OMVs were injected into the abdominal cavity of mice for 12 weeks. Pg OMV translocation to the brain was detected by immunohistochemistry using an anti-gingipain antibody. Tau protein and microglial activation in the mouse brain were examined by western blotting and immunohistochemistry. The effect of gingipains on inflammation was assessed by real-time polymerase chain reaction using human microglial HMC3 cells. Results: Gingipains were detected in the region around cerebral ventricles, choroid plexus, and ventricular ependymal cells in Pg OMV-administered mice. Tau and phosphorylated Tau protein increased and microglia were activated. Pg OMVs also increased the gene expression of proinflammatory cytokines in HMC3 cells in a gingipain-dependent manner. Conclusion: Pg OMVs, including gingipains, can reach the cerebral ventricle and induce neuroinflammation by activating microglia. Pg OMVs may provide a better understanding of the implications of periodontal diseases in neurodegenerative conditions such as AD.
Jun-ichi Kido, Yuka Hiroshima, Rie Kido, Kaya Yoshida, Yuji Inagaki, Koji Naruishi, Kazuaki Kajimoto, Masatoshi Kataoka, Yasuo Shinohara and Hiromichi Yumoto : Lipocalin 2, synthesized using a cell-free protein synthesis system and encapsulated into liposomes, inhibits the adhesion of Porphyromonas gingivalis to human oral epithelial cells., Journal of Periodontal Research, 58, 2, 262-273, 2023.
Yasufumi Nishikawa, Yoritoki Tomotake, Hiromichi Kawano, Koji Naruishi, Jun-ichi Kido, Yuka Hiroshima, Akikazu Murakami, Tetsuo Ichikawa and Hiromichi Yumoto : Effects of Candidalysin Derived from Candida albicans on the Expression of Pro-Inflammatory Mediators in Human Gingival Fibroblasts, International Journal of Molecular Sciences, 24, 4, 3256, 2023.
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.
Yuta Uemura, Yuka Hiroshima, Ayano Tada, Keiji Murakami, Kaya Yoshida, Yuji Inagaki, Tomomi Kuwahara, Akikazu Murakami, Hideki Fujii and Hiromichi Yumoto : Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways, Biomedicines, 10, 10, 2643, 2022.
(Summary)
() is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. -OMVs are involved in the pathogenesis of periodontitis. -OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of -OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. -OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. -OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that -OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that -OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.
Muhammad Reza Pahlevi, Keiji Murakami, Yuka Hiroshima, Akikazu Murakami and Hideki Fujii : pruR and PA0065 Genes Are Responsible for Decreasing Antibiotic Tolerance by Autoinducer Analog-1 (AIA-1) in Pseudomonas aeruginosa, Antibiotics, 11, 6, 773, 2022.
Mizuki Abe, Keiji Murakami, Yuka Hiroshima, Takashi Amoh, Mayu Sebe, Keiko Kataoka and Hideki Fujii : Autoinducer Analogs Can Provide Bactericidal Activity to Macrolides in Pseudomonas aeruginosa through Antibiotic Tolerance Reduction, Antibiotics, 11, 1, 2021.
(Summary)
Macrolide antibiotics are used in treating chronic biofilm infections despite their unsatisfactory antibacterial activity, because they display several special activities, such as modulation of the bacterial quorum sensing and immunomodulatory effects on the host. In this study, we investigated the effects of the newly synthesized quorum-sensing autoinducer analogs (AIA-1, -2) on the activity of azithromycin and clarithromycin against . In the killing assay of planktonic cells, AIA-1 and -2 enhanced the bactericidal ability of macrolides against PAO1; however, they did not affect the minimum inhibitory concentrations of macrolides. In addition, AIA-1 and -2 considerably improved the killing activity of azithromycin and clarithromycin in biofilm cells. The results indicated that AIA-1 and -2 could affect antibiotic tolerance. Moreover, the results of hydrocarbon adherence and cell membrane permeability assays suggested that AIA-1 and -2 changed bacterial cell surface hydrophobicity and accelerated the outer membrane permeability of the hydrophobic antibiotics such as azithromycin and clarithromycin. Our study demonstrated that the new combination therapy of macrolides and AIA-1 and -2 may improve the therapeutic efficacy of macrolides in the treatment of chronic biofilm infections.
Takenori Yamamoto, Mizune Ozono, Akira Watanabe, Kosuke Maeda, Atsushi Nara, Mei Hashida, Yusuke Ido, Yuka Hiroshima, Akiko Yamada, Hiroshi Terada and Yasuo Shinohara : Functional analysis of coiled-coil domains of MCU in mitochondrial calcium uptake, Biochimica et Biophysica Acta (BBA) - Bioenergetics, 148061, 2019.
(Summary)
The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca-uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca-uptake function. Thus, it is essential for the Ca uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca-uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU.
Takenori Yamamoto, Moe Tsunoda, Mizune Ozono, Akira Watanabe, Kazumasa Kotake, Yuka Hiroshima, Akiko Yamada, Hiroshi Terada and Yasuo Shinohara : Polyethyleneimine renders mitochondrial membranes permeable by interacting with negatively charged phospholipids in them, Archives of Biochemistry and Biophysics, 2018.
(Summary)
Polyethyleneimines (PEIs) are used for transfection of cells with nucleic acids. Meanwhile, the interaction of PEI with mitochondria causes cytochrome c release prior to apoptosis; the mechanisms how PEI causes this permeabilization of mitochondrial membranes and the release of cytochrome c remain unclear. To clarify these mechanisms, we examined the effects of branched-type PEI and linear-type PEI, each of which was 25 kDa in size, on mitochondria. The permeabilization potency of mitochondrial membranes by branched PEI was stronger than that by linear PEI. The permeabilization by PEIs were insensitive to permeability-transition inhibitors, indicating that PEI-induced permeabilization was not attributed to permeability transition. Meanwhile, PEIs caused permeabilization of artificial lipid vesicles; again, the permeabilization potency of branched PEI was stronger than that of linear PEI. Such a difference in this potency was close to that in the case of isolated mitochondria, signifying that the PEI-induced permeabilization of mitochondrial membranes could be attributed to PEI's interaction with the phospholipid phase. Furthermore, this PEI-induced permeabilization of the lipid vesicles was observed only in the case of lipid vesicles including negatively charged phospholipids. These results indicate that PEIs interacted with negatively charged phospholipids in the mitochondrial membranes to directly lead to their permeabilization.
Yuka Hiroshima, Takenori Yamamoto, Masahiro Watanabe, Yoshinobu Baba and Yasuo Shinohara : Effects of cold exposure on metabolites in brown adipose tissue of rats., Molecular Genetics and Metabolism Reports, 15, 36-42, 2018.
(Summary)
Brown adipose tissue (BAT) plays an important role in regulation of energy expenditure while adapting to a cold environment. BAT thermogenesis depends on uncoupling protein 1 (UCP1), which is expressed in the inner mitochondrial membranes of BAT. Gene expression profiles induced by cold exposure in BAT have been studied, but the metabolomic biological pathway that contributes to the activation of thermogenesis in BAT remains unclear. In this study, we comprehensively compared the relative levels of metabolites between the BAT of rats kept at room temperature (22 °C) and of those exposed to a cold temperature (4 °C) for 48 h using capillary electrophoresis (CE) time-of-flight mass spectrometry (TOFMS) and liquid chromatography (LC)-TOFMS. We identified 218 metabolites (137 cations and 81 anions) by CE-TOFMS and detected 81 metabolites (47 positive and 34 negative) by LC-TOFMS in BAT. We found that cold exposure highly influenced the BAT metabolome. We showed that the cold environment lead to lower levels of glycolysis and gluconeogenesis intermediates and higher levels of the tricarboxylic acid (TCA) cycle metabolites, fatty acids, and acyl-carnitine metabolites than control conditions in the BAT of rats. These results indicate that glycolysis and β-oxidation of fatty acids in BAT are positive biological pathways that contribute to the activation of thermogenesis by cold exposure, thereby facilitating the generation of heat by UCP1. These data provide useful information for understanding the basal metabolic functions of BAT thermogenesis in rats in response to cold exposure.
Yuka Hiroshima, Eijiro Sakamoto, Kaya Yoshida, Kaori Abe, Koji Naruishi, Takenori Yamamoto, Yasuo Shinohara, Jun-ichi Kido and Carolyn L Geczy : Advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide increase calprotectin expression in human gingival epithelial cells., Journal of Cellular Biochemistry, 119, 2, 1591-1603, 2018.
(Summary)
Accumulation of advanced glycation end-products (AGEs) in periodontal tissues of patients with diabetes mellitus aggravates periodontitis, but the mechanisms are unknown. Calprotectin, a heterocomplex of S100A8 and S100A9 proteins, is a constitutive cytoplasmic component of healthy gingival epithelial cells. This study aimed at investigating the effects of AGE and Porphyromonas gingivalis lipopolysaccharide (PgLPS) on calprotectin expression in the human gingival epithelial cell line OBA-9. AGE and PgLPS increased the expression of S100A8 and S100A9 mRNAs, and AGE + PgLPS co-stimulation amplified their expression in OBA-9 cells. A higher concentration of calprotectin in cell lysates was also induced by stimulation with AGE and/or PgLPS. S100A8 was mainly translocated from the nucleus to the cytoplasm by AGE stimulation, while cytoplasmic localization of S100A9 was not altered following stimulation with AGE and/or PgLPS. Calprotectin was found in the cytoplasm of BSA-treated cells, but cytoplasmic and nuclear localization was observed following stimulation with AGE and/or PgLPS. AGE-induced S100A8, and S100A9 mRNA expression was partially suppressed by RAGE-specific siRNA. In contrast, PgLPS-induced S100A8 and S100A9 mRNA expression was strongly suppressed by TLR2-specific siRNA. Furthermore, the inhibition of p38, JNK MAPK, and NF-κB attenuated AGE- and PgLPS-induced S100A8 and S100A9 mRNA expression. Taken together, these results demonstrate that AGE acts in synergy with PgLPS to stimulate RAGE and TLR2 expression and activate p38, JNK MAPK, and NF-κB signaling pathways, resulting in increased activation of calprotectin (S100A8/S100A9) in human gingival epithelial cells. Our results suggest that calprotectin may be involved in the pathogenesis of diabetic periodontitis. This article is protected by copyright. All rights reserved.
Kaya Yoshida, Hirohiko Okamura, Yuka Hiroshima, Kaori Abe, Jun-ichi Kido, Yasuo Shinohara and Kazumi Ozaki : PKR induces the expression of NLRP3 by regulating the NF-κB pathway in Porphyromonas gingivalis-infected osteoblasts, Experimental Cell Research, 354, 1, 57-64, 2017.
(Summary)
The double-stranded RNA-dependent kinase (PKR), which is activated by double stranded RNA, induces inflammation by regulating NF-κB signaling. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome also modulates inflammation in response to infection. Porphyromonas gingivalis (P.gingivalis) is an oral bacterium which is implicated in the pathogenesis of periodontal diseases. We previously reported that PKR is a key modulator of bone metabolism and inflammation in the periodontal tissue. PKR was also reported to induce inflammation in response to microbes by regulating the NLRP3 inflammasome, suggesting that PKR could affect inflammation along with NLRP3 in periodontal diseases. In this study, we investigated the effects of PKR on NLRP3 expression and NF-κB activity in P. gingivalis infected osteoblasts. We first constructed a SNAP26b-tagged P.gingivalis (SNAP-P. g.) and traced its internalization into the cell. SNAP-P. g. increased the activity of PKR and NF-κB and also induced NLRP3 expression in osteoblasts. Inhibition of NF-κB attenuated SNAP-P. g.-induced NLRP3 expression. The knockdown of PKR using shRNA decreased both the activity of NF-κB and the expression of NLRP3 induced by SNAP-P.g.. We therefore concluded that in osteoblasts, P. gingivalis activated PKR, which in turn increased NLRP3 expression by activating NF-κB. Our results suggest that PKR modulates inflammation by regulating the expression of the NLRP3 inflammasome through the NF-κB pathway in periodontal diseases.
Yuka Hiroshima, Kenneth Hsu, Nicodemus Tedla, Sze Wing Wong, Sharron Chow, Naomi Kawaguchi and Carolyn L Geczy : S100A8/A9 and S100A9 reduce acute lung injury., Immunology and Cell Biology, 95, 5, 461-472, 2017.
(Summary)
S100A8 and S100A9 are myeloid cell-derived proteins that are elevated in several types of inflammatory lung disorders. Pro- and anti-inflammatory properties are reported and these proteins are proposed to activate TLR4. S100A8 and S100A9 can function separately, likely through distinct receptors but a systematic comparison of their effects in vivo are limited. Here we assess inflammation in murine lung following S100A9 and S100A8/A9 inhalation. Unlike S100A8, S100A9 promoted mild neutrophil and lymphocyte influx, possibly mediated in part, by increased mast cell degranulation and selective upregulation of some chemokine genes, particularly CXCL-10. S100 proteins did not significantly induce proinflammatory mediators including TNF-, IL-1, IL-6 or serum amyloid A3 (SAA3). In contrast to S100A8, neither preparation induced S100A8 or IL-10 mRNA/protein in airway epithelial cells, or in tracheal epithelial cells in vitro. Like S100A8, S100A9 and S100A8/A9 reduced neutrophil influx in acute lung injury (ALI) provoked by LPS challenge but were somewhat less inhibitory, possibly because of differential effects on expression of some chemokines, IL-1, SAA3 and IL-10. Novel common pathways including increased induction of an NAD(+)-dependent protein deacetylase sirtuin-1 (SIRT1) that may reduce NF-B signalling, and increased STAT3 activation may reduce LPS activation. Results suggest a role for these proteins in normal homeostasis and protective mechanisms in the lung.Immunology and Cell Biology accepted article preview online, 11 January 2017. doi:10.1038/icb.2017.2.
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.
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.
(Summary)
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.
(Summary)
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.
Yuka Hiroshima, Linda Garthwaite, Kenneth Hsu, Hyouna Yoo, Sang-Ho Park, L Carolyn Geczy, K Rakesh Kumar and Cristan Herbert : ISU201 enhances the resolution of airway inflammation in a mouse model of an acute exacerbation of asthma., Mediators of Inflammation, 2015, 405629, 2015.
(Summary)
Glucocorticoids are commonly used for treating asthma and its exacerbations but have well-recognised adverse effects and are not always effective. Few alternative treatments exist. Using a murine model of an acute exacerbation of asthma, we assessed the ability of ISU201, a novel protein drug, to suppress the inflammatory response when administered after induction of an exacerbation. Sensitised mice were chronically challenged with a low mass concentration of aerosolised ovalbumin, and then received a single moderate-level challenge to simulate an allergen-induced exacerbation. ISU201 was administered to mice 2 and 8 hours later, while pulmonary inflammation and expression of mRNA for chemokines and proinflammatory cytokines were assessed after 4, 12, and 24 hours. Relative to vehicle-treated controls, ISU201 suppressed accumulation of pulmonary neutrophils and eosinophils, while accelerating the decline in CXCL1, TNF-, and IL-6 in lavage fluid and lung tissue. ISU201 significantly reduced peak expression of mRNA for the chemokines Cxcl9 and Cxcl10, the adhesion molecules Icam1 and Vcam1, and the proinflammatory cytokines Il1b, Il12p40, and Csf1. The ability of ISU201 to promote resolution of inflammation suggests that it may have potential as an alternative to glucocorticoids in the management of asthma, including when administered after the onset of an acute exacerbation.
Javkhlan Purevjav, Xu Guangfei, Chen Gang, Chenjuan Yao, Yuka Hiroshima, Hiroshi Yoshimura, Toshihiko Nagata and Kazuo Hosoi : 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, 2015, 290259, 2015.
Hiroshi Ito, Yukihiro Numabe, Satoshi Sekino, Etsuko Murakashi, Hitomi Iguchi, Shuichi Hashimoto, Daisuke Sasaki, Takashi Yaegashi, Kazushi Kunimatsu, Hideki Takai, Masaru Mezawa, Yorimasa Ogata, Hisashi Watanabe, Satsuki Hagiwara, Yuichi Izumi, Yuka Hiroshima, Jun-ichi Kido and Toshihiko Nagata : Evaluation of bleeding on probing and gingival crevicular fluid enzyme activity for detection of periodontally active sites during supportive periodontal therapy, Odontology, 102, 1, 50-56, 2014.
(Summary)
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.
Yuka Hiroshima, Kenneth Hsu, Nicodemus Tedla, Ming Yuen Chung, Sharron Chow, Cristan Herbert and L Carolyn Geczy : S100A8 induces IL-10 and protects against acute lung injury., The Journal of Immunology, 192, 6, 2800-2811, 2014.
(Summary)
S100A8 is considered proinflammatory by activating TLR4 and/or the receptor for advanced glycation end products. The aim was to investigate inflammatory effects of S100A8 in murine lung. S100A8 was administered to BALB/c mice by nasal inhalation and genes induced over a time-course assessed. LPS was introduced intranasally either alone or 2 h after pretreatment of mice with intranasal application of S100A8 or dexamethasone. A Cys(42)-Ala(42) mutant S100A8 mutant was used to assess whether S100A8's effects were via pathways that were dependent on reactive oxygen species. S100A8 induced IL-10 mRNA, and expression was apparent only in airway epithelial cells. Importantly, it suppressed acute lung injury provoked by LPS inhalation by suppressing mast-cell activation and induction of mediators orchestrating leukocyte recruitment, possibly by reducing NF-B activation via an IB/Akt pathway and by downmodulating pathways generating oxidative stress. The Cys(42)-Ala(42) S100A8 mutant did not induce IL-10 and was less immunosuppressive, indicating modulation by scavenging oxidants. S100A8 inhibition of LPS-mediated injury was as potent, and outcomes were remarkably similar to immunosuppression by dexamethasone. We challenge the notion that S100A8 is an agonist for TLR4 or the receptor for advanced glycation end products. S100A8 induced IL-10 in vivo and initiates a feedback loop that attenuates acute lung injury.
L Carolyn Geczy, Ming Yuen Chung and Yuka Hiroshima : Calgranulins may contribute vascular protection in atherogenesis., Circulation Journal, 78, 2, 271-280, 2013.
(Summary)
S100A8, S100A9 and S100A12 are considered proinflammatory mediators of atherosclerosis. Known as calgranulins, they are major components of neutrophils and are upregulated in macrophages and foam cells. They influence leukocyte recruitment, and may propagate inflammation by binding TLR4 and/or receptor for advanced glycation endproducts (RAGE). However, the receptors for calgranulins remain an enigma; we have no evidence for TLR4 or RAGE activation by S100A8 or S100A12. Moreover, gene regulation studies suggest antiinflammatory functions for S100A8 and emerging reports indicate pleiotropic roles. Unlike S100A9, S100A8 effectively scavenges oxidants generated by the myeloperoxidase system in vivo, forming novel thiol modifications. S100A8 is also readily S-nitrosylated, stabilizing nitric oxide and transporting it to hemoglobin. S100A8-SNO reduces leukocyte transmigration in the vasculature. S-glutathionylation of S100A9 modifies its effects on leukocyte adhesion. Both S100A8 forms inhibit mast cell activation, at least partially by scavenging reactive oxygen species required for signaling. Conversely, S100A12 activates and sequesters mast cells. However S100A12 suppresses proinflammatory cytokine induction by SAA-activated monocytes and macrophages, and inhibits matrix metalloprotease activity. We propose that the abundance and types of cells expressing calgranulins in particular microenvironments, their relative concentrations and post-translational modifications may have distinct functional outcomes, including those that are protective, at different stages of atherogenesis.
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.
(Summary)
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.
(Keyword)
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.
(Summary)
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.
Yukiko Nakajima, Yuji Inagaki, Yuka Hiroshima, Jun-ichi Kido and Toshihiko Nagata : Advanced glycation end-products enhance calcification in cultured rat dental pulp cells, Journal of Endodontics, 39, 873-878, 2013.
(Summary)
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.
Chie Wada -Mihara, 徳永 格, Hiroyuki Seto, Eijiro Sakamoto, Yuka Hiroshima, Jun-ichi Kido and Toshihiko Nagata : Topical injection of osteoprotegerin prevents alveolar bone loss in rat experimental periodontitis, Journal of the Japanese Association of Periodontology, 54, 2, 167-174, 2012.
(Keyword)
osteoprotegerin / experimental periodontitis / alveolar bone / osteoclast
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.
(Summary)
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.
(Summary)
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.
(Summary)
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.
Purevjav Javkhlan, Yuka Hiroshima, Ahmad Azlina, Takahiro Hasegawa, Chenjuan Yao, Tetsuya Akamatsu, Jun-ichi Kido, Toshihiko Nagata and Kazuo Hosoi : Lipopolysaccharide-mediated induction of calprotectin in the submandibular and parotid glands of mice, Inflammation, 34, 6, 668-680, 2011.
(Summary)
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.
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.
(Summary)
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.
Ahmad Azlina, Purevjav Javkhlan, Yuka Hiroshima, Takahiro Hasegawa, Chenjuan Yao, Tetsuya Akamatsu and Kazuo Hosoi : Roles of lysosomal proteolytic systems in AQP5 degradation in the submandibular gland of rats following chorda tympani parasympathetic denervation, American Journal of Physiology, Gastrointestinal and Liver Physiology, 299, 5, G1106-G1117, 2010.
(Summary)
Chorda tympani denervation (CTD) of rats was earlier shown to result in loss of submandibular gland (SMG) weight (at only 1 wk) and in continued reduction in aquaporin 5 (AQP5) protein expression (until 4 wk), without affecting its mRNA synthesis (Li X, Azlina A, Karabasil MR, Purwanti N, Hasegawa T, Yao C, Akamatsu T, Hosoi K. Am J Physiol Gastrointest Liver Physiol 295: G112-G123, 2008). The present study indicated that despite elevation of bax, a proapoptosis protein, by CTD, the operation also increased the level of bcl-2, an antiapoptosis protein, in the SMG. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay) showed no increase in the number of apoptotic cells in the SMG. CTD, however, induced strongly and transiently (at 1-3 days) the protein expression of LC3B-II, a marker protein of autophagosomes, suggesting that the reduction in the gland weight was due to onset of autophagy by CTD. Upon CTD, Lamp2, a lysosomal marker, gradually increased in amount, reaching a peak at the 14th day. Immunohistochemical analysis revealed an increase in the number of lysosome-like structures positive for both AQP5 and Lamp2 in the acinar cells of the SMG after CTD; similar changes were observed also for AQP5 and LC3Bs. These data suggest that AQP5 in the SMG entered autophagosomes and/or lysosomes for degradation upon CTD. In vitro AQP5-degrading activity was found in the SMG extracts, and such activity was shown to be increased by CTD. Inhibitor experiments implied cathepsins B and L to be candidate enzymes for this degradation under normal and CTD conditions, respectively.
Yuji Inagaki, Mika Bandou, 中島 由紀子, Yuka Hiroshima, Jun-ichi Kido and Toshihiko Nagata : Osmotic Stress Affects Calcium Deposition and Osteopontin Expression in Cultured Rat Dental Pulp Cells, The Japanese Journal of Conservative Dentistry, 53, 4, 367-375, 2010.
(Summary)
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.
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.
(Summary)
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.
(Summary)
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.
Jun-ichi Kido, 日野 真美, Mika Bandou and Yuka Hiroshima : バイオマーカーを用いた歯周病診断, IEEJ Transactions on Electronics, Information and Systems, 129, 2, 233-237, 2009.
Yuka Hiroshima, Yuji Inagaki, Mika Bandou, Jun-ichi Kido and Toshihiko Nagata : Long-term Observation of Marked Overfilling in Root Canal Therapy : A Report of Three Cases, The Japanese Journal of Conservative Dentistry, 51, 5, 485-492, 2008.
(Summary)
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.
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.
(Summary)
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.
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.
(Summary)
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.
Jun-ichi Kido, Yuka Hiroshima, Rie Kido, Mika Bandou, Yuji Inagaki and Hiromichi Yumoto : ---, Journal of the Japanese Association of Periodontology, 68, 2, 39-47, Jul. 2026.
Jun-ichi Kido, Yuka Hiroshima, Rie Kido, Mika Bandou, Yuji Inagaki and Hiromichi Yumoto : バイオマーカーを用いた歯周病スクリーニング・診断の基本事項, Journal of the Japanese Association of Periodontology, 68, 2, 39-47, Jun. 2026.
3.
Eijiro Sakamoto, Yuka Hiroshima, Jun-ichi Kido, Yasufumi Nishikawa, Koji Naruishi, Rie Kido and Hiromichi Yumoto : Various roles of calprotectin in periodontal disease and its possibility as a diagnostic marker for periodontal disease, Journal of the Japanese Association of Periodontology, 62, 4, 193-199, Dec. 2020.
E Sakamoto, Chie Wada -Mihara, K Tokunaga, H Seto and Yuka Hiroshima : Topical application of osteoprotegerin inhibits alveolar bone loss in rat experimental periodontitis, International Joint Symposium on Oral Science, Bali, Indonesia, Dec 17-19, 2010,
2.
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,
3.
Sirigeri Prakruti, Santiago Fernando, Yuka Hiroshima, Hsu Kenneth, Geczy Carolyn, Herbert Cristan, Thomas Paul, Kaakoush O Nadeem and Tedla Nicodemus : Leukocyte immunoglobulin-like receptor A3 protects against LPS induced acute lung injury, The 53rd Annual Scientific Meeting of The Australian and New Zealand Society for Immunology, Perth, Dec. 2025.
4.
Fenwick Jemma, Tedla Nicodemus, Raftery Mark, Zhong Ling, Yuka Hiroshima, Geczy Carolyn and Passam Freda : Investigating the role of platelet chemokines in the development of acute lung injury, Blood 2023, 123, Melbourne, Nov. 2023.
5.
Yuka Hiroshima, Eijiro Sakamoto, Kaori Abe, Kaya Yoshida, Koji Naruishi, Toshihiko Nagata, Yasuo Shinohara, Geczy Carolyn and Jun-ichi Kido : Advanced Glycation End-Products Increase Calprotectin in Human Gingival Epithelial Cells, The 95th General Session & Exhibition of the International Association for Dental Research (IADR), San Francisco, Mar. 2017.
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.
Yukiko Nakajima, Yuji Inagaki, Yuka Hiroshima, Jun-ichi Kido and Toshihiko Nagata : AGE induces calcification and inflammatory factors in dental pulp tissues, 91th General Session & Exhibition of the International Accociation for Dental Research, Seattle, USA, May 2013.
10.
Yukiko Nakajima, Yuji Inagaki, Yuka Hiroshima, Jun-ichi Kido and Toshihiko Nagata : AGE induces calcification- and inflammation-related factors in dental pulp cells, The 60th Annual Meeting of Japanese Association for Dental Research, Program and abstracts of papers, 92, Dec. 2012.
11.
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.
12.
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.
13.
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.
14.
Purevjav Javkhlan, Yuka Hiroshima, Azlina Ahmad, Takahiro Hasegawa, Chenjuan Yao, Tetsuya Akamatsu, Jun-ichi Kido, Toshihiko Nagata and Kazuo Hosoi : Induction of calprotectin mRNAs by lipopolysaccharide in the salivary gland of mice, The Journal of Medical Investigation : JMI, 56, Suppl, 287-289, Tokushima, Dec. 2009.
(Summary)
Calprotectin is a major cytosolic calcium-binding protein of leukocytes which belongs to the S100 protein family. S100A8 and S100A9, major types of calprotectin are heterodimeric complexes being composed of light- and heavy-chain subunits. The calprotectin levels in the plasma, feces, synovial fluid, gingival crevicular fluid, dental calculus and saliva change when the host animal suffers from several inflammatory diseases. Members of Toll-like receptor (TLR) family are pattern-recognition receptors for lipopolysaccharide (LPS) and other pathogens. Here we examined if the biological role of TLR receptor is reflected to the calprotectin expression in the salivary gland. Time course study by using real-time RT-PCR detected higher levels of S100A8 and S100A9 mRNA at 1.5-3 h after injection of LPS in both the submandibular gland (SMG) and parotid gland (PG) of C3H/HeN mice but not in the same tissues of C3H/HeJ, a TLR-4 mutant strain, indicating that this induction is mediated via the TLR-4. These results indicate that, an inflammatory marker, calprotectin, is expressed in the mouse salivary gland and that LPS stimulated its synthesis. Calprotectin (S100A8/A9) showed minimum expression in all cellular segments in the SMG except excretory duct cells, which showed strong signal at the cytoplasm. LPS induced their expressions in the granular convoluted tubular cells and striated duct cells. In the PG, these proteins were expressed very weakly in both duct and acinar cells with a little stronger staining for the former cells. LPS injection induced calprotectin (S100A8/A9) in both duct and acinar cells especially in the former cells.
Kaya Yoshida, Kunihiro Otsuka, Yuka Hiroshima and Kazumi Ozaki : Porphyromonas gingivalis由来外膜小胞の肝移行とクッパー細胞活性化によるMASH発症機構の検討, 第68回歯科基礎医学会学術大会, Sep. 2026.
2.
Kaya Yoshida, 高井 彩有, Yuka Hiroshima, 生田 あゆ, Mariko Seyama, Yuta Uemura, Hiromichi Yumoto and Kazumi Ozaki : P. gingivalis外膜小胞DNAはIL-6を誘導しアストロサイトの炎症を惹起する, 第12回日本細胞外小胞学会学術大会, Oct. 2025.
3.
Kaya Yoshida, Yuka Hiroshima, Ayu Takai, 生田 あゆ, Kayo Yoshida and Kazumi Ozaki : 歯周病菌外膜小胞が血管透過性を亢進する機構, 第11回日本細胞外小胞学会学術集会, Oct. 2024.
4.
Rie Kido, Yuka Hiroshima, Jun-ichi Kido, Kaya Yoshida, Takahisa Ikuta and Hiromichi Yumoto : Antimicrobial activity of artificially synthesized secretory leukocyte protease inhibitor and its encapsulation into liposomes, 第66回春季日本歯周病学会学術大会, May 2023.
5.
Yuka Hiroshima : Porphyromonas gingivalis由来メンブレンベシクルが歯周組織に及ぼす影響, ダイバーシティ推進研究交流発表会オンライン2022, Mar. 2023.
Yuta Uemura, Yuka Hiroshima, Keiji Murakami, Yuji Inagaki, Hiromichi Yumoto and Hideki Fujii : ヒト歯肉上皮細胞におけるPorphyromonas gingivalis由来メンブレンベシクルの炎症性サイトカイン産生誘導機構の解明, 第19回四国免疫フォーラム, Jun. 2021.
18.
Jun-ichi Kido, Yuka Hiroshima, Rie Kido, Kaya Yoshida, Yuji Inagaki, Koji Naruishi and Hiromichi Yumoto : リポソームに封入したリポカリン2の口腔上皮細胞への送達, 第64回春季日本歯周病学会学術大会, May 2021.
19.
Yuka Hiroshima, Yuta Uemura, Yuji Inagaki, Jun-ichi Kido and Hiromichi Yumoto : ヒト歯肉上皮細胞におけるPorphyromonas gingivalis由来メンブレンベシクルの炎症性サイトカイン産生誘導機構の解明, 第64回春季日本歯周病学会学術大会, May 2021.
Pahlevi Reza Muhammad, Keiji Murakami, Yuta Kita, Rina Murata, Yuka Hiroshima and Hideki Fujii : Screening for the genes influenced by AIA-1 in Pseudomonas aeruginosa, 第73回日本細菌学会中国・四国支部総会, 31, Oct. 2020.
Jun-ichi Kido, Yuka Hiroshima, Rie Kido, Yuji Inagaki, Koji Naruishi and Hiromichi Yumoto : 無細胞蛋白質合成系を用いた抗菌ペプチドの合成とリポソーム封入, 日本歯科保存学会2020年度春季学術大会(第152回)(神戸), Jun. 2020.
29.
Yuka Hiroshima, Jun-ichi Kido, Rie Kido, Kaya Yoshida, Yuji Inagaki, Koji Naruishi and Hiromichi Yumoto : オーラルケアへの応用に関連するリポソームのデリバリー法の検討, 第63回春季日本歯周病学会学術大会, May 2020.
30.
Rie Kido, Yuka Hiroshima, Takahisa Ikuta, Yuji Inagaki, Mika Bandou, Koji Naruishi, Jun-ichi Kido and Hiromichi Yumoto : 最終糖化産物による口腔上皮細胞の Lipocalin2 発現誘導は好中球の遊走性とサイトカイン発現を 調節する, 第63回春季日本歯周病学会学術大会, May 2020.
Yuka Hiroshima, Jun-ichi Kido, Eijiro Sakamoto, 阿部 佳織, Kaya Yoshida, Toshihiko Nagata and Yasuo Shinohara : 最終糖化産物はヒト歯肉上皮細胞におけるS100A8およびS100A9発現を上昇する, 第59回秋季日本歯周病学会学術大会, Oct. 2016.
51.
Yuka Hiroshima, Jun-ichi Kido, Kaya Yoshida, Kaori Abe, Yasuo Shinohara and Toshihiko Nagata : Hypoxic condition down-regulates S100A8 expression in human oral epithelial cells, 第59回春季日本歯周病学会学術大会, May 2016.
52.
Kaya Yoshida, Natsumi Fujiwara, Yuka Hiroshima, Kaori Abe, Jun-ichi Kido and Kazumi Ozaki : Porphyhepatic cellsromonas gingivalis inhibits insulin signaling by regulating SOCS3 and IRS-1 in, 第59回春季日本歯周病学会学術大会, May 2016.
53.
Yukari Kajiura, Jun-ichi Kido, Yukiko Nakajima, Mika Bandou, Yuji Inagaki, Koji Naruishi, Eijiro Sakamoto, Kaori Abe, Yuka Hiroshima and Toshihiko Nagata : 低酸素環境が誘導するヒト口腔上皮細胞における遺伝子発現の解析, 第58回秋季日本歯周病学会学術大会, Sep. 2015.
54.
Yukari Kajiura, Yukiko Bandou, Jun-ichi Kido, Mika Bandou, Yuji Inagaki, Yukiko Nakajima, 生田 貴久, Hiromi Murata, Yuka Hiroshima, Koji Naruishi and Toshihiko Nagata : 歯周病および糖尿病患者の歯肉溝滲出液中のYKL-40レベルの検討, 第57回秋季日本歯周病学会学術大会, Oct. 2014.
55.
Mika Bandou, Yuka Hiroshima, Yuji Inagaki, Jun-ichi Kido and Toshihiko Nagata : ヒト上皮細胞におけるInterleukin-1aによるS100A9遺伝子の転写調節, 第140回日本歯科保存学会春季学術大会, Jun. 2014.
Yuka Hiroshima, Mika Bandou, Jun-ichi Kido, Masatoshi Kataoka and Toshihiko Nagata : ヒト口腔上皮細胞におけるカルプロテクチン発現に及ぼす半夏瀉心湯の影響, 日本歯周病学会会誌 春季特別号, 53, 114, May 2011.
(Keyword)
periodontitis / 口腔上皮細胞 / 半夏瀉心湯 / カルプロテクチン
81.
Chie Wada -Mihara, Jun-ichi Kido, Satoshi Yoneda, Yuka Hiroshima, Hiroyuki Seto and Toshihiko Nagata : 上顎前歯の著しいフレアアウトを伴った薬物性歯肉増殖症患者の一症例, Journal of the Japanese Association of Periodontology, May 2011.
82.
Yukiko Nakajima, Yuji Inagaki, Mika Bandou, Yuka Hiroshima, Jun-ichi Kido and Toshihiko Nagata : グルコースが糖尿病ラット由来歯髄細胞の石灰化物形成とオステオポンチン産生に及ぼす影響, 第133回日本歯科保存学会秋季学術大会(岐阜), Oct. 2010.
Yuta Uemura, Yuka Hiroshima, Keiji Murakami, 多田 彩乃, Tomomi Kuwahara, Hideki Fujii and Hiromichi Yumoto : Porphyromonas gingivalis由来メンブレンベシクルがヒト歯肉上皮細胞に及ぼす影響, 徳島大学研究クラスター Joint Meeting on Microbiology 2020, Nov. 2020.
3.
Pahlevi Reza Muhammad, Keiji Murakami, Yuta Kita, Rina Murata, Yuka Hiroshima and Hideki Fujii : Screening for the genes influenced by AIA-1 in Pseudomonas aeruginosa, 徳島大学研究クラスター Joint Meeting on Microbiology 2020, Nov. 2020.
The role of DNA of Periodontal bacterium OMVs in Alzheimer's diseases. (Project/Area Number: 23K09505 )
Elucidation of the role of periodontal pathogenic bacteria-derived vesicles in periodontal medicine and development of preventive agents for the worsening of periodontal disease (Project/Area Number: 21K09896 )
Study of exosomes from cells in periodontal tissues with diabetes-associated periodontitis (Project/Area Number: 21K09895 )
Basic Research on Oral Care for the Elderly (Project/Area Number: 20K09941 )
Investigation of molecular mechanism of S100A8 in diabetes-associated periodontitis (Project/Area Number: 17K17352 )
Basic study for oral care system using an artificial cell (Project/Area Number: 17H04418 )
Investigation of molecular mechanism of S100A8 in diabetes-associated periodontitis (Project/Area Number: 15H06451 )