ブドウ球菌の表皮ケラチノサイトからの選択的抗菌ペプチドの誘導

Staphylococcus aureus (S. aureus) is one of the most clinically important inflammation-inducing pathogens, while Staphylococcus epidermidis (S. epidermidis) is nonpathogenic and hardly causes inflammation on skin. β-defensins, antimicrobial peptides, are secreted from keratinocytes constitutively or upon induction by various microorganisms. However, the difference between S. aureus and S. epidermidis is still unclear in terms of their influences on the production of β-defensins. In this study, we focused on the influences of S. aureus and S. epidermidis on the keratinocyte innate immune response. Pathogenic S. aureus mainly induced human β-defensin (hBD) 1 and hBD3, but not hBD2, and nonpathogenic S. epidermidis mainly induced hBD2 from human keratinocytes. Molecular weight fractions of >10 kDa prepared from S. aureus supernatants induced the production of hBD1 and hBD3. On the other hand, molecular weight fraction of >100 kDa prepared from S. epidermidis supernatants induced the production of hBD2.Furthermore, the secreted products of S. epidermidis used the toll-like receptor (TLR) 2 pathway in the induction of hBD2 production. The secreted products of S. aureus and S. epidermidis differentially induced subtypes of hBD through different receptors, which may be associated with the difference in virulence between these two bacteria.博士（医学）・甲第643号・平成28年3月15日Copyright © 2012 Elsevier Ltd. All rights reserved

ヒト細胞傷害性γδT細胞は膠芽腫細胞株を殺傷する：膠芽腫患者に対する免疫細胞治療の意義

Glioblastoma (GBM) is a highly aggressive brain tumor for which novel therapeutic approaches, such as immunotherapy, are urgently needed. Zoledronate (ZOL), an inhibitor of osteoclastic activity, is known to stimulate peripheral blood-derived γδT cells and sensitize tumors to γδT cell-mediated killing. To investigate the feasibility of γδT cell-based immunotherapy for patients with GBM, we focused on the killing of GBM cell lines by γδT cells and the molecular mechanisms involved in these cell–cell interactions. Peripheral blood mononuclear cells were expanded in ZOL and interleukin (IL)-2 for 14 days, and γδT cells were enriched in the expanded cells by the immunomagnetic depletion of αβT cells. Gliomas are resistant to NK cells but susceptible to lymphokine-activated killer cells and some cytotoxic T lymphocytes. When the γδT cell-mediated killing of three GBM cell lines (U87MG, U138MG and A172 cells) and an NK-sensitive leukemia cell line (K562 cells) were tested, 32 % U87MG, 15 % U138MG, 1 % A172, and 50 % K562 cells were killed at an effector:target ratio of 5:1. The γδT cell-mediated killing of all three GBM cell lines was significantly enhanced by ZOL and this ZOL-enhanced killing was blocked by an anti-T cell receptor (TcR) antibody. These results indicated that TcR γδ is crucial for the recognition of ZOL-treated GBM cells by γδT cells. Since the low level killing of GBM cells by the γδT cells was enhanced by ZOL, γδT cell-targeting therapy in combination with ZOL treatment could be effective for patients with GBM.博士（医学）・甲第635号・平成27年5月28日© Springer Verlag. The definitive version is available at " http://dx.doi.org/10.1007/s11060-013-1258-4

柿タンニンは新型コロナウイルスに対する抗ウイルス効果を持ち、シリアンハムスターモデルにおける新型コロナウイルス感染症の重症度および感染伝播を抑制する

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread across the world. Inactivating the virus in saliva and the oral cavity represents a reasonable approach to prevent human-to-human transmission because the virus is easily transmitted through oral routes by dispersed saliva. Persimmon-derived tannin is a condensed type of tannin that has strong antioxidant and antimicrobial activity. In this study, we investigated the antiviral effects of persimmon-derived tannin against SARS-CoV-2 in both in vitro and in vivo models. We found that persimmon-derived tannin suppressed SARS-CoV-2 titers measured by plaque assay in vitro in a dose- and time-dependent manner. We then created a Syrian hamster model by inoculating SARS-CoV-2 into hamsters' mouths. Oral administration of persimmon-derived tannin dissolved in carboxymethyl cellulose before virus inoculation dramatically reduced the severity of pneumonia with lower virus titers compared with a control group inoculated with carboxymethyl cellulose alone. In addition, pre-administration of tannin to uninfected hamsters reduced hamster-to-hamster transmission of SARS-CoV-2 from a cohoused, infected donor cage mate. These data suggest that oral administration of persimmon-derived tannin may help reduce the severity of SARS-CoV-2 infection and transmission of the virus.博士（医学）・甲第817号・令和4年3月15日© 2021. The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/

Pure Solid Pattern of Non-Small Cell Lung Cancer and Clustered Circulating Tumor Cells

There are two solid patterns of non-small cell lung cancer (NSCLC) on computed tomography (CT): pure or mixed with ground-glass opacities (GGOs). They predict the degree of invasiveness, which may suggest the presence of clustered circulating tumor cells (CTCs), a predictor of poor prognosis. In this study, we assessed the implications of the solid patterns on CT and the preoperative clustered CTCs in surgically resected NSCLC. CTCs were detected using a size selection method. The correlation between the presence of preoperative clustered CTCs and the solid pattern and the prognostic implications were evaluated using co-variables from the clinical-pathological findings. Of the 142 cases, pure solid lesions (Group PS) and mixed GGOs (Group G) were observed in 92 (64.8%) and 50 (35.2%) patients, respectively. In Groups PS and G, clustered CTCs were detected in 29 (31.5%) and 1 (2.0%) patient (p < 0.01), respectively. The PS appearance was an independent predictor of preoperative clustered CTCs in the multivariable analysis, and preoperative clustered CTCs were an independent predictor of poor recurrence-free survival; the solid pattern was not an independent variable. Thus, the PS pattern of NSCLC on CT is an indicator of preoperative clustered CTCs, which is an independent poor prognosis predictor

ヒト脂肪由来間葉型幹細胞は、間葉上皮転換によりエラスターゼ誘発マウス肺気腫を改善する

Purpose: Chronic obstructive pulmonary disease (COPD) is a worldwide problem because of its high prevalence and mortality. However, there is no fundamental treatment to ameliorate their pathological change in COPD lung. Recently, adipose-derived mesenchymal stem cells (ADSCs) have attracted attention in the field of regenerative medicine to repair damaged organs. Moreover, their utility in treating respiratory diseases has been reported in some animal models. However, the detailed mechanism by which ADSCs improve chronic respiratory diseases, including COPD, remains to be elucidated. We examined whether human ADSCs (hADSCs) ameliorated elastase-induced emphysema and whether hADSCs differentiated into alveolar epithelial cells in a murine model of COPD. Methods: Female SCID-beige mice (6 weeks old) were divided into the following four groups according to whether they received an intratracheal injection of phosphate-buffered saline or porcine pancreatic elastase, and whether they received an intravenous injection of saline or hADSCs 3 days after intratracheal injection; Control group, hADSC group, Elastase group, and Elastase-hADSC group. We evaluated the lung function, assessed histological changes, and compared gene expression between hADSCs isolated from the lung of Elastase-hADSC group and naïve hADSCs 28 days after saline or elastase administration. Results: hADSCs improved the pathogenesis of COPD, including the mean linear intercept and forced expiratory volume, in an elastase-induced emphysema model in mice. Furthermore, hADSCs were observed in the lungs of elastase-treated mice at 25 days after administration. These cells expressed genes related to mesenchymal-epithelial transition and surface markers of alveolar epithelial cells, such as TTF-1, β-catenin, and E-cadherin. Conclusion: hADSCs have the potential to improve the pathogenesis of COPD by differentiating into alveolar epithelial cells by mesenchymal-epithelial transition.博士（医学）・甲第825号・令和4年3月15日© 2021 Fujioka et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php)

Abrogated Caveolin-1 expression via histone modification enzyme Setdb2 regulates brain edema in a mouse model of influenza-associated encephalopathy

Abstract Influenza-associated encephalopathy (IAE) is a serious complication that can follow influenza virus infection. Once a cytokine storm is induced during influenza virus infection, tight junction protein disruption occurs, which consequently leads to blood-brain barrier (BBB) breakdown. However, the details of IAE pathogenesis are not well understood. Here, we established a murine IAE model by administration of lipopolysaccharide following influenza virus infection. Brains from IAE model mice had significantly higher expression of type I interferons and inflammatory cytokines. In addition, the expression of Caveolin-1, one of the key proteins that correlate with protection of the BBB, was significantly lower in brains from the IAE group compared with the control group. We also found that, among 84 different histone modification enzymes, only SET domain bifurcated 2 (Setdb2), one of the histone methyltransferases that methylates the lysine 9 of histone H3, showed significantly higher expression in the IAE group compared with the control group. Furthermore, chromatin immunoprecipitation revealed that methylation of histone H3 lysine 9 was correlated with repression of the Caveolin-1 promoter region. These studies identify Caveolin-1 as a key regulator of BBB permeability in IAE and reveal that it acts through histone modification induced by Setdb2

Persimmon-derived tannin has bacteriostatic and anti-inflammatory activity in a murine model of Mycobacterium avium complex (MAC) disease.

Nontuberculous mycobacteria (NTM), including Mycobacterium avium complex (MAC), cause opportunistic chronic pulmonary infections. Notably, MAC susceptibility is regulated by various factors, including the host immune system. Persimmon (Ebenaceae Diospyros kaki Thunb.) tannin is a condensed tannin composed of a polymer of catechin groups. It is well known that condensed tannins have high antioxidant activity and bacteriostatic properties. However, it is hypothesized that condensed tannins might need to be digested and/or fermented into smaller molecules in vivo prior to being absorbed into the body to perform beneficial functions. In this study, we evaluated the effects of soluble persimmon-derived tannins on opportunistic MAC disease. Soluble tannins were hydrolyzed and evaluated by the oxygen radical absorbance capacity (ORAC) method. The ORAC value of soluble tannin hydrolysate was approximately five times greater than that of soluble tannin powder. In addition, soluble tannin hydrolysate exhibited high bacteriostatic activity against MAC in vitro. Furthermore, in an in vivo study, MAC infected mice fed a soluble tannin-containing diet showed significantly higher anti-bacterial activity against MAC and less pulmonary granuloma formation compared with those fed a control diet. Tumor necrosis factor α and inducible nitric oxide synthase levels were significantly lower in lungs of the soluble tannin diet group compared with the control diet group. Moreover, proinflammatory cytokines induced by MAC stimulation of bone marrow-derived macrophages were significantly decreased by addition of soluble tannin hydrolysate. These data suggest that soluble tannin from persimmons might attenuate the pathogenesis of pulmonary NTM infection

CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma

Abstract Background Glioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs. Methods The GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further detected in vivo antitumor effects using xenograft brain tumor mice. Results We successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice. Conclusion The findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM

Antiviral Effect of Candies Containing Persimmon-Derived Tannin against SARS-CoV-2 Delta Strain

Inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the mouth has the potential to reduce the spread of coronavirus disease 2019 (COVID-19), due to the virus being readily transmitted by dispersed saliva. Persimmon-derived tannin has strong antioxidant and antimicrobial activity owing to its strong adhesion to proteins, and it also exhibited antiviral effects against non-variant and Alpha-variant SARS-CoV-2 in our previous study. In this study, we first demonstrated the antiviral effects of persimmon-derived tannin against the Delta variant of SARS-CoV-2 in vitro via the plaque assay method. We then examined the effects of candy containing persimmon-derived tannin. Remarkably, the saliva samples provided by healthy volunteers while they were eating tannin-containing candy showed that the virus titers of the SARS-CoV-2 Delta variant were suppressed. In addition, we found that the SARS-CoV-2 viral load in saliva from patients with COVID-19 collected immediately after they had eaten the tannin-containing candy was below the level of detection via PCR for SARS-CoV-2. These data suggest that adding persimmon-derived tannin to candy and holding such candy in the mouth is an effective method for inactivating SARS-CoV-2 in saliva, and the application of this approach shows potential for inhibiting the transmission of COVID-19

急性呼吸促迫症候群の病態におけるヒストン修飾酵素Setdb2の重要な役割

Introduction: Acute respiratory distress syndrome (ARDS) is a severe hypoxemic respiratory failure with a high in-hospital mortality. However, themolecular mechanisms underlying ARDS remain unclear. Recent findings have indicated that the onset of severe inflammatory diseases, such as sepsis, is regulated by epigenetic changes. We investigated the role of epigenetic changes in ARDS pathogenesis using mouse models and human samples. Methods: Acute respiratory distress syndrome was induced in a mouse model (C57BL/6 mice, myeloid cell or vascular endothelial cell [VEC]–specific SET domain bifurcated 2 [Setdb2]–deficient mice [Setdb2ffLyz2Cre+ or Setdb2ffTie2Cre+], and Cre− littermates) by intratracheal administration of lipopolysaccharide (LPS). Analyses were performed at 6 and 72 h after LPS administration. Sera and lung autopsy specimens from ARDS patients were examined. Results: In the murine ARDS model, we observed high expression of the histone modification enzyme SET domain bifurcated 2 (Setdb2) in the lungs. In situ hybridization examination of the lungs revealed Setdb2 expression in macrophages and VECs. The histological score and albumin level of bronchoalveolar lavage fluid were significantly increased in Setdb2ffTie2Cre+ mice following LPS administration compared with Setdb2ffTie2Cre- mice, whereas there was no significant difference between the control and Setdb2ffLyz2Cre+ mice. Apoptosis of VECs was enhanced in Setdb2ffTie2Cre+ mice. Among the 84 apoptosis-related genes, the expression of TNF receptor superfamily member 10b (Tnfrsf10b) was significantly higher in Setdb2ffTie2Cre+ mice than in control mice. Acute respiratory distress syndrome patients' serum showed higher SETDB2 levels than those of healthy volunteers. SETDB2 levels were negatively correlated with the partial pressure of oxygen in arterial blood/fraction of inspiratory oxygen concentration ratio. Conclusion: Acute respiratory distress syndrome elevates Setdb2, apoptosis of VECs, and vascular permeability. Elevation of histone methyltransferase Setdb2 suggests the possibility to histone change and epigenetic modification. Thus, Setdb2 may be a novel therapeutic target for controlling the pathogenesis of ARDS.権利情報：© 2023 The Author(s). Published byWolters Kluwer Health, Inc. on behalf of the American College of Sports Medicine. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal