Springer Science and Business Media LLC Acta Medica Okayama 2045-2322 16 1 2026 Pseudohypoxia induced by iron chelators preserves working memory performance in aged mice 11550 EN Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yoshiaki Iwasaki Health Service Section, Environment Health & Safety Intelligence Department, Okayama University Tomonari Kasai Department of Applied Energy, Graduate School of Engineering, Nagoya University Toru Yamashita Department of Neurology, Graduate School of Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shiho Komaki Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yusuke Hamada Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Pseudohypoxia refers to a physiological condition wherein hypoxia-inducible factor (HIF) is pharmacologically upregulated under normoxia, thereby modulating immune responses. We hypothesized that pseudohypoxia, induced by iron chelators, may similarly potentiate systemic immune responses in aged mice, concurrently triggering neuro-regenerative signaling pathways and enhancing cognitive performance. In this study, aged mice (43–48 weeks old) were orally administered two iron chelators, Super Polyphenol 10 (SP10) or Roxadustat, to induce a pseudohypoxia. An 8-week oral regimen of SP10 and Roxadustat significantly preserved working memory, as assessed by the Y-maze test (YMT). White blood cell counts and hippocampal volume, as assessed by magnetic resonance imaging (MRI), were elevated in the treatment groups relative to controls. Pseudohypoxia induced by SP10 tended to enhance neuro-regenerative signaling, specifically involving the Tau and JNK pathways, and potentially modulated Doublecortin (DCX) expression, although statistical significance was limited by sample size. Importantly, inflammatory markers, such as ionized calcium-binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP), were not elevated by treatment. Collectively, these findings suggest that pseudohypoxia induced by iron chelators preserves working memory performance accompanied by leukocytosis, without concomitant neuroinflammation. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1422-0067 27 7 2026 CXCR2-Dependent Infiltration of Tumor-Associated Neutrophils Is Linked to Enhanced CD8+ T Cell Effector Function and Reduced Lung Metastasis in 4T1 Breast Cancer 3143 EN Tiantian Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Miao Tian Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Gakushi Nishida Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chunning Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Triple-negative breast cancer (TNBC) is characterized by prominent neutrophil infiltration; however, its significance remains controversial. Here, we investigated the role of neutrophil chemoattractant receptors in TNBC progression and metastasis. In contrast to wild-type (WT), Fpr1�|/�|, and Fpr2�|/�| mice, neutrophils were almost completely absent in 4T1 tumors from Cxcr2�|/�| mice, indicating a dominant role for CXCR2 in the recruitment of tumor-associated neutrophils, leading us to use Cxcr2�|/�| mice for further studies. Primary tumor growth was comparable between WT and Cxcr2�|/�| mice, whereas lung metastasis was significantly increased in Cxcr2�|/�| mice, with reduced expression of inflammatory cytokines, chemokines and cytotoxic molecules, including granzyme B and perforin, in primary tumors and metastatic lungs of Cxcr2�|/�| mice. In vitro, WT, but not Cxcr2�|/�|, neutrophils enhanced CD8+ T cell activation, partly via ICAM-1, and directly induced tumor cell death, supporting their anti-tumor function. To assess clinical relevance, transcriptomic data were analyzed. High neutrophil infiltration combined with elevated CXCR2 expression, and to a lesser extent CXCR1 expression, was associated with improved prognosis in patients with basal-like BC that largely overlaps with TNBC. Collectively, these findings suggest that CXCR2-mediated neutrophil recruitment exerts protective, anti-tumor effects and may represent a new prognostic marker for TNBC patients. No potential conflict of interest relevant to this article was reported.

China Anti-cancer Association Acta Medica Okayama 2095-3941 2026 SPRED2 suppresses the stemness of hepatocellular carcinoma through the p53/miR-506-3p/KLF4 pathway EN Tong Gao Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sachio Ito Department of Pathophysiology and Drug Discovery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Aye Moh-Moh-Aung Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tianyi Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Objective: We previously reported that endogenous Sprouty-related, EVH1 domain-containing protein 2 (SPRED2), an inhibitor of the Ras/Raf/ERK-MAPK pathway, controls hepatocellular carcinoma (HCC) cell stemness by downregulating the expression of pluripotency factors, such as Nanog, c-Myc, and KLF4, in an ERK-dependent fashion. However, the exact mechanisms by which SPRED2 regulates HCC cell stemness have not been established.<br> Methods: Three human HCC cell lines [HepG2 (parental and SPRED2-deficient), HLE, and Hep3B] were used. Cells were transfected to downregulate or overexpress proteins. Western blot and RT-qPCR were used to evaluate the level of protein and mRNA expression. Co-immunoprecipitation and ChIP-qPCR were used to examine protein-protein interactions and the activation of gene transcription. Clinical HCC tissues were also used to validate in vitro data.<br> Results: KLF4 was identified as the major pluripotency factor responsible for SPRED2-mediated downregulation of HCC cell stemness and KLF4 expression was regulated by miR-506-3p. SPRED2 formed a protein complex with the tumor suppressor (p53) and upregulated miR-506 gene transcription by binding to the promoter region, resulting in subsequent downregulation of KLF4 mRNA expression. There was a negative correlation between KLF4 expression and miR-506-3p and a positive correlation between miR-506-3p expression and SPRED2 in human HCC samples, highlighting the relevance of the study findings.<br> Conclusions: The current study revealed a novel SPRED2/p53/miR-506-3p/KLF4 axis through which SPRED2 contributes to the suppression of HCC cell stemness and provides a potential new target to prevent HCC progression. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0167-4889 1873 2 2026 SPRED2 controls the severity of cisplatin-induced acute kidney injury by inhibiting ERK activation and TNFƒ¿ production in mice 120091 EN Xu Yang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Jiali He Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tong Gao Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Steven L. Kunkel Department of Pathology, University of Michigan Medical School Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Cisplatin is an effective chemotherapeutic agent used to treat solid tumors, but its clinical use is limited by acute kidney injury (AKI), in which ERK signaling plays a crucial role. Here, we investigated whether Sprouty-related EVH1 domain-containing protein 2 (SPRED2), an endogenous inhibitor of the Ras/Raf/ERK pathway, protects against cisplatin-induced AKI. Spred2�|/�| mice showed more severe renal injury and stronger ERK activation than wild-type (WT) mice, whereas pretreatment with the MEK inhibitor U0126 markedly attenuated the injury. In HK-2 cells (proximal tubular cells), SPRED2 knockdown enhanced cisplatin-induced apoptosis and caspase-3 activation, accompanied by decreased Bcl-2 expression. Spred2�|/�| kidneys displayed increased macrophage infiltration and elevated Tnfƒ¿, Il1b, and Ccl2 expression. Neutralization of TNFƒ¿ with anti-TNFƒ¿ antibody ameliorated renal injury and reduced the levels of Il1b and Ccl2 mRNA in Spred2�|/�| mice. In vitro, TNFƒ¿ slightly decreased the viability of control and SPRED2 knockdown HK-2 cells without cisplatin treatment, but the decreased viability was augmented in SPRED2 knockdown cells by cisplatin. Immunohistochemistry revealed that macrophages were the predominant TNFƒ¿-positive cell population. Bone marrow–derived macrophages from Spred2�|/�| mice produced higher levels of TNFƒ¿ in response to cisplatin compared with control cells, and this increase was markedly suppressed by U0126.<br> These findings indicate that endogenous SPRED2 protects kidneys from cisplatin-induced AKI by limiting ERK activation, tubular apoptosis, and TNFƒ¿-mediated inflammation. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1422-0067 26 20 2025 Neurofibromin Encoded by the Neurofibromatosis Type 1 (NF1) Gene Promotes the Membrane Translocation of SPRED2, Thereby Inhibiting the ERK Pathway in Breast Cancer Cells 10072 EN Nang Thee Su Pwint Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chunning Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tong Gao Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuze Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Neurofibromin (NF) inhibits the RAS/RAF/ERK pathway through its interaction with SPRED1 (Sprouty-related EVH1 domain-containing protein 1). Here, we investigated the functional relationship between NF and SPRED2 in breast cancer (BC). Human BC cell lines were transfected to downregulate or overexpress NF and SPRED2 and subsequently subjected to functional assays. Protein and mRNA levels were analyzed by Western blotting and RT-qPCR, respectively. Protein–protein interactions were examined by immunoprecipitation. Database analyses and immunohistochemistry (IHC) of BC tissues were performed to validate the in vitro findings. Downregulating NF or SPRED2 expression in BC cells enhanced cell proliferation, migration and invasion accompanied by RAF/ERK activation, whereas overexpression produced opposite effects. NF formed a protein complex with SPRED2 and facilitated its translocation to the plasma membrane. By IHC, SPRED2 membrane localization was absent in NF-negative luminal A and triple-negative BC (TNBC) but present in a subset of luminal A BC. By database analyses, both NF1 and SPRED2 mRNA levels were reduced in BC tissues, and luminal A BC patients with high expression of both NF1 and SPRED2 mRNA exhibited improved relapse-free survival. These results suggest a critical role for the NF–SPRED2 axis in BC progression and highlight it as a potential therapeutic target. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2045-2322 15 1 2025 Pharmacokinetics and the effectiveness of pyrogen-free bioabsorbable wet adhesives 20056 EN Risa Oshima Department of Periodontology, Faculty of Dental Medicine, Hokkaido University Kumiko Yoshihara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ko Nakanishi Department of Biomaterials and Bioengineering, Faculty of Dental Medicine, Hokkaido University Tsukasa Akasaka Department of Biomaterials and Bioengineering, Faculty of Dental Medicine, Hokkaido University Shinji Shimoji Department of Periodontology, Faculty of Dental Medicine, Hokkaido University Teppei Nakamura Department of Applied Veterinary Science, Faculty of Veterinary Medicine, Hokkaido University Takumi Okihara Division of Applied Chemistry, Graduate School of Natural Science and Technology, Okayama University Mariko Nakamura Department of Clinical Psychology, School of Clinical Psychology, Kyushu University of Medical and Science Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ikkei Tamada Department of Plastic and Reconstructive Surgery, Tokyo Metropolitan Children�fs Medical Center Bart Van Meerbeek BIOMAT, Department of Oral Health Sciences, & UZ Leuven, Dentistry, KU Leuven Tsutomu Sugaya Department of Periodontology, Faculty of Dental Medicine, Hokkaido University Yasuhiro Yoshida Department of Biomaterials and Bioengineering, Faculty of Dental Medicine, Hokkaido University Bioabsorbable materials are essential for advanced therapies, including surgical sealing, cell therapy, and drug delivery. Natural bioabsorbable materials, including collagen and hyaluronic acid, have better biocompatibility than synthetic bioabsorbable polymers; however, they are mainly derived from animals, presenting infection risks. Non-animal origin polymers have a lower molecular weight than those of animal origins. Their viscosity increases with increase in molecular weight, making endotoxin removal difficult. Here, using the phosphoryl chloride disposal method, we present a strategy for synthesizing pyrogen-free bioabsorbable adhesives with controlled molecular weight. Phosphopullulan, a polysaccharide derivative, had less than detectable endotoxin levels and controllable average molecular weight of approximately 300,000 to over 1,400,000. Furthermore, it is important to ensure the safety as well as efficacy of bio-implantable materials. We have evaluated the biosafety of polysaccharide derivatives we are developing, and have examined their cell phagocytosis and pharmacokinetics in vitro and in vivo, and have confirmed that they are safe. We have also evaluated their adhesion to wet tissue adhesions and confirmed that they leak less than existing materials. No potential conflict of interest relevant to this article was reported.

Informa UK Limited Acta Medica Okayama 1071-5762 2025 Pseudohypoxia induced by iron chelator activates tumor immune response in lung cancer EN Yusuke Hamada Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuehua Chen Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Manato Terada Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuze Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hotaka Kawai Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hypoxia-inducible factor (HIF) signaling plays a critical role in immune cell function. Pseudohypoxia is characterized as iron-mediated stabilization of HIF-1ƒ¿ under normoxic conditions, which can be induced by iron chelators. This study explored whether iron chelators exert antitumor effects by enhancing tumor immune responses and elucidating the underlying mechanisms. The iron chelators Super–polyphenol 10 (SP10) and Deferoxamine (DFO) were used to create iron-deficient and pseudohypoxia conditions. Pseudohypoxia induced by iron chelators stimulates IL-2 secretion from T cells and from both human and murine nonsmall cell lung cancer (NSCLC) cell lines (A549, PC-3, and LLC). Administration of SP10 reduced tumor growth when LLC tumors were implanted in C57BL/6 mice; however, this was not observed in immunodeficient RAG1-deficient C57BL/6 mice. SP10 itself did not directly inhibit LLC cells proliferation in vitro, suggesting an activation of the tumor immune response. SP10 synergistically enhanced the efficacy of PD-1 antibody therapy in lung cancer by increasing the number of tumor-infiltrating lymphocytes (TILs). In conclusion, iron chelation-induced pseudohypoxia activates tumor immune responses by directly upregulating HIF-1ƒ¿, augmenting T cell function, and inducing IL-2 secretion from T cells, and cancer cells, thereby amplifying the immune efficacy of the PD-1 antibody in lung cancer treatment. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1068-9265 2025 Tumor Microvessels with Specific Morphology as a Prognostic Factor in Esophageal Squamous Cell Carcinoma EN Hnin Thida Tun Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Seitaro Nishimura Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomoyoshi Kunitomo Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazuhiro Noma Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Background Angiogenesis is essential for tumor progression. Microvessel density (MVD) is a widely used histological method to assess angiogenesis using immunostained sections, but its prognostic significance in esophageal cancer remains controversial. Recently, the evaluation of microvascular architecture has gained importance as a method to assess tumor aggressiveness. The present study aimed to identify the histological characteristics of tumor microvessels that are associated with the aggressiveness of esophageal squamous cell carcinoma.<br> Patients and Methods A total of 108 esophageal squamous cell carcinoma tissues were immunohistochemically stained with blood vessel markers and angiogenesis-related markers, including CD31, alpha smooth muscle actin, vascular endothelial growth factor A (VEGF-A), CD206, and D2-40. MVD, microvessel pericyte coverage index (MPI), and tumor vascular morphology were evaluated by microscopy.<br> Results MVD was significantly associated with patient outcomes, whereas neither MPI nor VEGF-A expression throughout the tumor showed a significant correlation. In addition, the presence of blood vessels encircling clusters of tumor cells, termed C-shaped microvessels, and excessively branching microvessels, termed X-shaped microvessels, was significantly associated with poor prognosis. These vessel types were also correlated with clinicopathological parameters, including deeper invasion of the primary tumor, presence of lymph node metastasis, advanced pathological stage, and distant metastasis. Focal VEGF-A immunoexpression in tumor cells was higher in areas containing C-shaped or X-shaped microvessels compared with areas lacking these vessel morphologies.<br> Conclusions The data suggest that tumor microvessels with specific morphologies (C-shaped and X-shaped microvessels) may serve as a promising prognostic factor in esophageal squamous cell carcinoma. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 0340-7004 74 7 2025 HIF-PH inhibitors induce pseudohypoxia in T cells and suppress the growth of microsatellite stable colorectal cancer by enhancing antitumor immune responses 192 EN Yuehua Chen Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yusuke Hamada Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuze Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Miao Tian Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazuhiro Noma Department of Gastroenterological Surgery, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hiroshi Tazawa Department of Gastroenterological Surgery, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Background Recent studies have revealed that CD8+ T cells can be activated via genetic upregulation of HIF-1 alpha, thereby augmenting antitumor effector functions. HIF-1 alpha upregulation can be attained by inhibiting HIF-prolyl hydroxylase (HIF-PH) under normoxic conditions, termed pseudohypoxia. This study investigated whether pseudohypoxia induced by HIF-PH inhibitors suppresses Microsatellite stable (MSS) colorectal cancer (CRC) by affecting tumor immune response.<br> Methods The HIF-PH inhibitors Roxadustat and Vadadustat were utilized in this study. In vitro, we assessed the effects of HIF-PH inhibitors on human and murine colon cancer cell lines (SW480, HT29, Colon26) and murine T cells. In vivo experiments were performed with mice bearing Colon26 tumors to evaluate the effect of these inhibitors on tumor immune responses. Tumor and spleen samples were analyzed using immunohistochemistry, RT-qPCR, and flow cytometry to elucidate potential mechanisms.<br> Results HIF-PH inhibitors demonstrated antitumor effects in vivo but not in vitro. These inhibitors enhanced the tumor immune response by increasing the infiltration of CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs). HIF-PH inhibitors induced IL-2 production in splenic and intratumoral CD4+ T cells, promoting T cell proliferation, differentiation, and immune responses. Roxadustat synergistically enhanced the efficacy of anti-PD-1 antibody for MSS cancer by increasing the recruitment of TILs and augmenting effector-like CD8+ T cells.<br> Conclusion Pseudohypoxia induced by HIF-PH inhibitors activates antitumor immune responses, at least in part, through the induction of IL-2 secretion from CD4+ T cells in the spleen and tumor microenvironment, thereby enhancing immune efficacy against MSS CRC. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1053-2498 44 2 2024 Loss of Nr4a1 ameliorates endothelial cell injury and vascular leakage in lung transplantation from circulatory-death donor 249 260 EN Shinichi Kawana Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mikio Okazaki Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomohisa Sakaue Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine Kohei Hashimoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaro Nakata Department of Surgery, Division of Cardiovascular and Thoracic Surgery, Duke University School of Medicine Haruki Choshi Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shin Tanaka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaroh Miyoshi Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinji Ohtani Department of Cell Growth and Tumor Regulation, Proteo-Science Center (PROS), Ehime University Toshiaki Ohara Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichiro Sugimoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: Ischemia-reperfusion injury (IRI) stands as a major trigger for primary graft dysfunction (PGD) in lung transplantation (LTx). Especially in LTx from donation after cardiac death (DCD), effective control of IRI following warm ischemia (WIRI) is crucial to prevent PGD. This study aimed to identify the key factors affecting WIRI in LTx from DCD.<br> Methods: Previously reported RNA-sequencing dataset of lung WIRI was reanalyzed to identify nuclear receptor subfamily 4 group A member 1 (NR4A1) as the immediate early gene for WIRI. Dynamics of NR4A1 expression were verified using a mouse hilar clamp model. To investigate the role of NR4A1 in WIRI, a mouse model of LTx from DCD was established using Nr4a1 knockout (Nr4a1�|/�|) mice.<br> Results: NR4A1 was located around vascular cells, and its protein levels in the lungs increased rapidly and transiently during WIRI. LT‚˜ from Nr4a1�|/�| donors significantly improved pulmonary graft function compared to wild-type donors. Histological analysis showed decreased microvascular endothelial cell death, neutrophil infiltration, and albumin leakage. Evans blue permeability assay demonstrated maintained pulmonary microvascular barrier integrity in grafts from Nr4a1�|/�| donors, correlating with diminished pulmonary edema. However, NR4A1 did not significantly affect the inflammatory response during WIRI, and IRI was not suppressed when a wild-type donor lung was transplanted into the Nr4a1�|/�| recipient.<br> Conclusions: Donor NR4A1 plays a specialized role in the positive regulation of endothelial cell injury and microvascular hyperpermeability. These findings demonstrate the potential of targeting NR4A1 interventions to alleviate PGD and improve outcomes in LTx from DCD. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1661-6596 25 11 2024 SPRED2 Is a Novel Regulator of Autophagy in Hepatocellular Carcinoma Cells and Normal Hepatocytes 6269 EN Tianyi Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tong Gao Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sprouty-related enabled/vasodilator-stimulated phosphoprotein homology 1 domain containing 2 (SPRED2) is an inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and has been shown to promote autophagy in several cancers. Here, we aimed to determine whether SPRED2 plays a role in autophagy in hepatocellular carcinoma (HCC) cells. The Cancer Genome Atlas (TCGA) Liver Cancer Database showed a negative association between the level of SPRED2 and p62, a ubiquitin-binding scaffold protein that accumulates when autophagy is inhibited. Immunohistochemically, accumulation of p62 was detected in human HCC tissues with low SPRED2 expression. Overexpression of SPRED2 in HCC cells increased the number of autophagosomes and autophagic vacuoles containing damaged mitochondria, decreased p62 levels, and increased levels of light-chain-3 (LC3)-II, an autophagy marker. In contrast, SPRED2 deficiency increased p62 levels and decreased LC3-II levels. SPRED2 expression levels were negatively correlated with translocase of outer mitochondrial membrane 20 (TOM20) expression levels, suggesting its role in mitophagy. Mechanistically, SPRED2 overexpression reduced ERK activation followed by the mechanistic or mammalian target of rapamycin complex 1 (mTORC1)-mediated signaling pathway, and SPRED2 deficiency showed the opposite pattern. Finally, hepatic autophagy was impaired in the liver of SPRED2-deficient mice with hepatic lipid droplet accumulation in response to starvation. These results indicate that SPRED2 is a critical regulator of autophagy not only in HCC cells, but also in hepatocytes, and thus the manipulation of this process may provide new insights into liver pathology. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1320-5463 74 7 2024 The specific shapes of capillaries are associated with worse prognosis in patients with invasive breast cancer 394 407 EN Hnin�]Wint�]Wint Swe Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yu Komatsubara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tadahiko Shien Department of Breast and Endocrine Surgery, Okayama University Hospital Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Angiogenesis is considered essential for tumor progression; however, whether histological counting of blood vessel numbers, expressed as microvessel density (MVD), can be a prognostic factor in breast cancer remains controversial. It has been suggested that the specific morphology of blood vessels such as glomeruloid microvascular proliferation (GMP) is associated with clinical parameters. Here, we aimed to clarify the significance of MVD with revised immunohistochemistry and to identify new blood vessel shapes that predict prognosis in breast cancer. Four hundred and eleven primary breast cancer specimens were collected, and the sections were immunohistochemically stained with CD31 (single staining) and CD31 and Collagen IV (double staining). The prognosis of patients was examined based on the MVD value, and the presence of GMP and other blood vessels with other specific shapes. As a result, high MVD value and the presence of GMP were not associated with worse prognosis. By contrast, patients with deep-curved capillaries surrounding tumor cell nests (C-shaped) or excessively branched capillaries near tumor cell nests showed a significantly poor prognosis. The presence of these capillaries was also correlated with clinicopathological parameters such as Ki-67 index. Thus, the morphology of capillaries rather than MVD can be a better indicator of tumor aggressiveness. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 78 1 2024 Role of Macrophages in Liver Fibrosis 1 8 EN Cuiming Sun Department of Pathology and Experimental Medicine, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Review 10.18926/AMO/66664 Liver fibrosis, which ultimately leads to liver cirrhosis and hepatocellular carcinoma, is a major health burden worldwide. The progression of liver fibrosis is the result of the wound-healing response of liver to repeated injury. Hepatic macrophages are cells with high heterogeneity and plasticity and include tissue-resident macrophages termed Kupffer cells, and recruited macrophages derived from circulating monocytes, spleen and peritoneal cavity. Studies have shown that hepatic macrophages play roles in the initiation and progression of liver fibrosis by releasing inflammatory cytokines/chemokines and pro-fibrogenic factors. Furthermore, the development of liver fibrosis has been shown to be reversible. Hepatic macrophages have been shown to alternately regulate both the regression and turnover of liver fibrosis by changing their phenotypes during the dynamic progression of liver fibrosis. In this review, we summarize the role of hepatic macrophages in the progression and regression of liver fibrosis. No potential conflict of interest relevant to this article was reported.

BMC Acta Medica Okayama 1472-6920 24 1 2024 Influence of the coronavirus disease 2019 pandemic on the post-graduate career paths of medical students: a cross-sectional study 55 EN Ayumu Nishimura Okayama University Medical School Faculty of Medicine Tomoko Miyoshi Department of General Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Fumio Otsuka Department of General Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Background The World Health Organization first declared the coronavirus disease 2019 (COVID-19) pandemic in March 2020 and announced the end of the emergency in May 2023. Additionally, the COVID-19 pandemic significantly impacted individuals globally, including medical students. Although the COVID-19 pandemic increased online education, it restricted clinical training, extracurricular activities, and interprovincial travel. Therefore, this study aimed to examine whether the COVID-19 pandemic influenced the choice of training hospitals and career paths among 3rd- to 6th-year medical students in Japan.<br> Methods We developed a questionnaire comprising 21 multiple-choice and 1 open-ended questions, which was administered anonymously via online platforms. The survey targeted Japanese medical students to obtain insights into their preferences for training hospitals and career paths during the COVID-19 pandemic. Participants included 4th- to 6th-year medical students from 51 medical schools in Japan. The survey was conducted through student networks from 8 February 2022 to 20 March 2022.<br> Results Overall, 507 medical students participated in the survey, with representation from various academic years as follows: 102 (20.1%), 134 (26.4%), 121 (23.9%), and 150 (29.6%) students from the 3rd, 4th, 5th, and 6th year, respectively. Of these, 338 (66.6%) students reported that the COVID-19 pandemic had influenced their choice of training hospitals. The degree of the influence varied based on the university region and the student year. However, most of the students (473, 93.3%) did not change their course for clinical, basic research, or administrative pathways due to the COVID-19 pandemic. Among the clinically oriented students, 391 (77.2%) did not change their preferred speciality.<br> Conclusions The COVID-19 pandemic influenced medical students' choice of training hospitals. Although many students believed that the pandemic would not change their career choices, our results indicate a potential subconscious trend to avoid internal medicine, which is the speciality most directly involved in treating patients with COVID-19. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1091-255X 26 6 2022 Diagnostic Utility of the PD-L1 Immunostaining in Biopsy Specimens of Patients with Biliary Tract Neoplasms 1213 1223 EN Kazuyuki Matsumoto Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Toshiaki Ohara Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Akinobu Takaki Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Masahiro Takahara Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hironari Kato Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Ryuichi Yoshida Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yuzo Umeda Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Takahito Yagi Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hiroyuki Okada Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Background�@Anti-programmed death 1/programmed death ligand 1 (PD1/PD-L1) antibodies have been successfully used as treatment agents for several solid tumors; however, it is difficult to predict their effectiveness. We evaluated whether biopsy specimens could predict the positive status of PD-L1 in surgically resected tissue.<br> Methods�@Among 91 patients who underwent tissue sampling with endoscopic or liver biopsy before surgery for biliary tract neoplasms in an academic center, 45 (49%) patients were selected for retrospective analysis because the quality and quantity of their biopsy specimens were adequate for histologic evaluation. We performed immunohistochemical staining to investigate the PD-L1 expression in both resected and biopsy specimens. The percentage of the positively stained cells was calculated for subsequent use in the correlation investigation.<br> Results�@The biopsy methods were endoscopic retrograde cholangiopancreatography (ERCP) in 28 cases, percutaneous liver biopsy in 10 cases, and endoscopic ultrasound fine-needle aspiration in 7 cases. Among the 45 patients, when patients with > 10% positive tumor cells in surgically resected tissues were regarded as truly positive PD-L1, the positive and negative concordance rates between surgically resected tissues and biopsy samples were 56% (5/9) and 100% (36/36), respectively. With regard to the use of preoperative biopsy as a diagnostic tool, all (5/5) PD-L1-positive patients had a positive resected specimen. The accuracy of each biopsy method was as follows: ERCP, 89% (25/28); fine-needle aspiration, 86% (6/7); and liver biopsy, 100% (10/10).<br> Conclusions�@Biopsy samples could be a surrogate material for the assessment of the PD-L1 expression with substantial positive and high negative concordance rates. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 24 5 2023 SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells 4996 EN Tong Gao Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Xu Yang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tianyi Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nahoko Tomonobu Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44(+)CD90(+) and CD44(-)CD90(-) populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44(+)CD90(+) cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 15 2 2023 Highly Metastatic Subpopulation of TNBC Cells Has Limited Iron Metabolism and Is a Target of Iron Chelators 468 EN Yuze Wang Department of Pathology and Experimental Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuehua Chen Department of Pathology and Experimental Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yusuke Hamada Department of Pathology and Experimental Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chunning Li Department of Pathology and Experimental Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Simple Summary Excess iron is known to be a risk factor of carcinogenesis. Although iron chelators show anti-cancer effects, they have not been used successfully to treat cancer patients. Triple-negative breast cancer (TNBC) is a disease with poor prognosis without effective treatments. Thus, we aimed to evaluate the possibility of iron chelators as a therapy for TNBC. Deferasirox (DFX), an iron chelator, suppressed the growth of 4T1 murine TNBC cell line cells in vitro and in vivo lung metastatic model. We found that highly metastatic TNBC cells have limited iron metabolism and can be more effectively targeted by iron chelators. Excess iron is known to be a risk factor of carcinogenesis. Although iron chelators show anti-cancer effects, they have not been used successfully to treat cancer patients. Triple-negative breast cancer (TNBC) is a disease with poor prognosis without effective treatments. Thus, we aimed to evaluate a possibility of iron chelators as a therapy for TNBC. Deferasirox (DFX), an iron chelator, suppressed the growth of 4T1 murine TNBC cell line cells in vitro and in vivo. Lung metastasis was further significantly reduced, leading to the hypothesis that iron metabolism between metastatic and non-metastatic cells may be different. An analysis of existing database demonstrated that the expression of iron-uptake genes was significantly suppressed in TNBC cells that metastasized to lymph nodes or lungs compared to those in primary tumors. A highly metastatic clone of the murine 4T1 TNBC cells (4T1-HM) did not proliferate well under iron-rich or iron-depleted conditions by iron chelators compared to a low-metastatic clone (4T1-LM). Bulk RNA-seq analysis of RNA from 4T1-HM and 4T1-LM cells suggested that the PI3K-AKT pathway might be responsible for this difference. Indeed, DFX suppressed the proliferation via the AKT-mTOR pathway in 4T1-HM and the human MDA-MB-231 cells, a human mesenchymal-like TNBC cell line. DFX also suppressed the growth of 4T1-HM tumors in comparison to 4T1-LM tumors, and reduced lung metastases after surgical resection of primary 4T1 tumors. These results indicated, for the first time, that highly metastatic TNBC cells have limited iron metabolism, and they can be more effectively targeted by iron chelators. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2306-5354 9 11 2022 Functional Blockage of S100A8/A9 Ameliorates Ischemia-Reperfusion Injury in the Lung 673 EN Kentaro Nakata Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mikio Okazaki Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomohisa Sakaue Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuhei Komoda Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine Dai Shimizu Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Haruchika Yamamoto Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network Shin Tanaka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken Suzawa Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Shien Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaroh Miyoshi Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiaki Ohara Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichiro Sugimoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masaomi Yamane Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (1) Background: Lung ischemia-reperfusion (IR) injury increases the mortality and morbidity of patients undergoing lung transplantation. The objective of this study was to identify the key initiator of lung IR injury and to evaluate pharmacological therapeutic approaches using a functional inhibitor against the identified molecule. (2) Methods: Using a mouse hilar clamp model, the combination of RNA sequencing and histological investigations revealed that neutrophil-derived S100A8/A9 plays a central role in inflammatory reactions during lung IR injury. Mice were assigned to sham and IR groups with or without the injection of anti-S100A8/A9 neutralizing monoclonal antibody (mAb). (3) Results: Anti-S100A8/A9 mAb treatment significantly attenuated plasma S100A8/A9 levels compared with control IgG. As evaluated by oxygenation capacity and neutrophil infiltration, the antibody treatment dramatically ameliorated the IR injury. The gene expression levels of cytokines and chemokines induced by IR injury were significantly reduced by the neutralizing antibody. Furthermore, the antibody treatment significantly reduced TUNEL-positive cells, indicating the presence of apoptotic cells. (4) Conclusions: We identified S100A8/A9 as a novel therapeutic target against lung IR injury. No potential conflict of interest relevant to this article was reported.

BMC Acta Medica Okayama 1465-5411 24 1 2022 Exosomal Wnt7a from a low metastatic subclone promotes lung metastasis of a highly metastatic subclone in the murine 4t1 breast cancer 60 EN Chunning Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Miao Tian Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuze Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takamasa Kondo Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ken-Ichi Yamamoto Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Background Patients with triple-negative breast cancer (TNBC) often have poorer prognosis than those with other subtypes because of its aggressive behaviors. Cancer cells are heterogeneous, and only a few highly metastatic subclones metastasize. Although the majority of subclones may not metastasize, they could contribute by releasing factors that increase the capacity of highly metastatic cells and/or provide a favorable tumor microenvironment (TME). Here, we analyzed the interclonal communication in TNBC which leads to efficient cancer progression, particularly lung metastasis, using the polyclonal murine 4T1 BC model. Methods We isolated two 4T1 subclones, LM.4T1 and HM.4T1 cells with a low and a high metastatic potential, respectively, and examined the effects of LM.4T1 cells on the behaviors of HM.4T1 cells using the cell scratch assay, sphere-forming assay, sphere invasion assay, RT-qPCR, and western blotting in vitro. We also examined the contribution of LM.4T1 cells to the lung metastasis of HM.4T1 cells and TME in vivo. To identify a critical factor which may be responsible for the effects by LM.4T1 cells, we analyzed the data obtained from the GEO database. Results Co-injection of LM.4T1 cells significantly augmented lung metastases by HM.4T1 cells. LM.4T1-derived exosomes promoted the migration and invasion of HM.4T1 cells in vitro, and blocking the secretion of exosome abrogated their effects on HM.4T1 cells. Analyses of data obtained from the GEO database suggested that Wnt7a might be a critical factor responsible for the enhancing effects. In fact, a higher level of Wnt7a was detected in LM.4T1 cells, especially in exosomes, than in HM.4T1 cells, and deletion of Wnt7a in LM.4T1 cells significantly decreased the lung metastasis of HM.4T1 cells. Further, treatment with Wnt7a increased the spheroid formation by HM.4T1 cells via activation of the PI3K/Akt/mTOR signaling pathway. Finally, infiltration of alpha SMA-positive fibroblasts and angiogenesis was more prominent in tumors of LM.4T1 cells and deletion of Wnt7a in LM.4T1 cells markedly reduced angiogenesis. Conclusions We demonstrated, for the first time, that a low metastatic subclone can enhance lung metastasis of highly metastatic subclone via exosomal Wnt7a and propose Wnt7a as a molecular target to treat TNBC patients. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 12 1 2022 Thioredoxin interacting protein protects mice from fasting induced liver steatosis by activating ER stress and its downstream signaling pathways 4819 EN Hiroyuki Miyahara Department of Pediatrics, Okayama University Hospital Kosei Hasegawa Department of Pediatrics, Okayama University Hospital Masato Yashiro Department of Pediatrics, Okayama University Hospital Toshiaki Ohara Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Teizo Yoshimura Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hirokazu Tsukahara Department of Pediatrics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Under normal conditions, fasting results in decreased protein disulfide isomerase (PDI) activity and accumulation of unfolded proteins, leading to the subsequent activation of the unfolded protein response (UPR)/autophagy signaling pathway to eliminate damaged mitochondria. Fasting also induces upregulation of thioredoxin-interacting protein (TXNIP) expression and mice deficient of this protein (TXNIP-KO mice) was shown to develop severe hypoglycemia, hyperlipidemia and liver steatosis (LS). In the present study, we aimed to determine the role of TXNIP in fasting-induced LS by using male TXNIP-KO mice that developed LS without severe hypoglycemia. In TXNIP-KO mice, fasting induced severe microvesicular LS. Examinations by transmission electron microscopy revealed mitochondria with smaller size and deformities and the presence of few autophagosomes. The expression of beta-oxidation-associated genes remained at the same level and the level of LC3-II was low. PDI activity level stayed at the original level and the levels of p-IRE1 and X-box binding protein 1 spliced form (sXBP1) were lower. Interestingly, treatment of TXNIP-KO mice with bacitracin, a PDI inhibitor, restored the level of LC3-II after fasting. These results suggest that TXNIP regulates PDI activity and subsequent activation of the UPR/autophagy pathway and plays a protective role in fasting-induced LS. No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 16 11 2021 Expression of Spred2 in the urothelial tumorigenesis of the urinary bladder e0254289 EN Shinsuke Oda Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chunning Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshihiro Ito Department of Immunology, Nara Medical University Takahiro Yamaguchi Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Aberrant activation of the Ras/Raf/ERK (extracellular-signal-regulated kinase)-MAPK (mitogen-activated protein kinase) pathway is involved in the progression of cancer, including urothelial carcinoma; but the negative regulation remains unclear. In the present study, we investigated pathological expression of Spred2 (Sprouty-related EVH1 domain-containing protein 2), a negative regulator of the Ras/Raf/ERK-MAPK pathway, and the relation to ERK activation and Ki67 index in various categories of 275 urothelial tumors obtained from clinical patients. In situ hybridization demonstrated that Spred2 mRNA was highly expressed in high-grade non-invasive papillary urothelial carcinoma (HGPUC), and the expression was decreased in carcinoma in situ (CIS) and infiltrating urothelial carcinoma (IUC). Immunohistochemically, membranous Spred2 expression, important to interact with Ras/Raf, was preferentially found in HGPUC. Interestingly, membranous Spred2 expression was decreased in CIS and IUC relative to HGPUC, while ERK activation and the expression of the cell proliferation marker Ki67 index were increased. HGPUC with membranous Spred2 expression correlated significantly with lower levels of ERK activation and Ki67 index as compared to those with negative Spred2 expression. Thus, our pathological findings suggest that Spred2 counters cancer progression in non-invasive papillary carcinoma possibly through inhibiting the Ras/Raf/ERK-MAPK pathway, but this regulatory mechanism is lost in cancers with high malignancy. Spred2 appears to be a key regulator in the progression of non-invasive bladder carcinoma. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 2090-1232 35 2022 Spred2 controls the severity of Concanavalin A-induced liver damage by limiting interferon-gamma production by CD4(+) and CD8(+) T cells 71 86 EN Cuiming Sun Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Qiuying Liu Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chen Cao Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Xu Yang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Steven L. Kunkel Department of Pathology, University of Michigan Medical School Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Introduction: Mitogen-activated protein kinases (MAPKs) are involved in T cell-mediated liver damage. However, the inhibitory mechanism(s) that controls T cell-mediated liver damage remains unknown. Objectives: We investigated whether Spred2 (Sprouty-related, EVH1 domain-containing protein 2) that negatively regulates ERK-MAPK pathway has a biological impact on T cell-mediated liver damage by using a murine model. Methods: We induced hepatotoxicity in genetically engineered mice by intravenously injecting Concanavalin A (Con A) and analyzed the mechanisms using serum chemistry, histology, ELISA, qRT-PCR, Western blotting and flow cytometry. Results: Spred2-deficient mice (Spred2(-/-)) developed more sever liver damage than wild-type (WT) mice with increased interferon-gamma (IFNy) production. Hepatic ERK phosphorylation was enhanced in Spred2(-/-) mice, and pretreatment of Spred2(-/-) mice with the MAPK/ERK inhibitor U0126 markedly inhibited the liver damage and reduced IFN gamma production. Neutralization of IFNy abolished the damage with decreased hepatic Stat1 activation in Spred2(-/-) mice. IFN gamma was mainly produced from CD4(+) and CD8(+) T cells, and their depletion decreased liver damage and IFN gamma production. Transplantation of CD4(+) and/or CD8(+) T cells from Spred2(-/-) mice into RAG1(-/-) mice deficient in both T and B cells caused more severe liver damage than those from WT mice. Hepatic expression of T cell attractants, CXCL9 and CXCL10, was augmented in Spred2(-/-) mice as compared to WT mice. Conversely, liver damage, IFN gamma production and the recruitment of CD4(+) and CD8(+) T cells in livers after Con A challenge were lower in Spred2 transgenic mice, and Spred2-overexpressing CD4(+) and CD8(+) T cells produced lower levels of IFN gamma than WT cells upon stimulation with Con A in vitro. Conclusion: We demonstrated, for the first time, that Spred2 functions as an endogenous regulator of T cell IFNy production and Spred2-mediated inhibition of ERK-MAPK pathway may be an effective remedy for T cell-dependent liver damage. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1467-3037 43 3 2021 Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer 1726 1740 EN Mayu Imamura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tiantian Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chunning Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naofumi Mukaida Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) is shown to promote the progression of breast cancer. We previously identified cancer cell-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential regulator of MCP-1 production in the murine 4T1 breast cancer, but it played a minimum role in overall MCP-1 production. Here, we evaluated the crosstalk between 4T1 cells and fibroblasts. When fibroblasts were co-cultured with 4T1 cells or stimulated with the culture supernatants of 4T1 cells (4T1-sup), MCP-1 production by fibroblasts markedly increased. 4T1 cells expressed mRNA for platelet-derived growth factor (PDGF)-a, b and c, and the PDGF receptor inhibitor crenolanib almost completely inhibited 4T1-sup-induced MCP-1 production by fibroblasts. However, PDGF receptor antagonists failed to reduce MCP-1 production in tumor-bearing mice. Histologically, 4T1 tumors contained a small number of alpha SMA-positive fibroblasts, and Mcp-1 mRNA was mainly associated with macrophages, especially those surrounding necrotic lesions on day 14, by in situ hybridization. Thus, although cancer cells have the capacity to crosstalk with fibroblasts via PDGFs, this crosstalk does not play a major role in MCP-1 production or cancer progression in this model. Unraveling complex crosstalk between cancer cells and stromal cells will help us identify new targets to help treat breast cancer patients. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0301-4851 2021 PolyI:C suppresses TGF-ƒÀ1-induced Akt phosphorylation and reduces the motility of A549 lung carcinoma cells EN Takahiro Yamaguchi Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Gao Tong Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Backgrounds: Epithelial mesenchymal transition (EMT) is a critical process involved in the invasion and metastasis of cancer, including lung cancer (LC). Transforming growth factor (TGF)-ƒÀ is one of factors capable of inducing EMT. Polyinosinic-polycytidylic acid (polyI:C), a synthetic agonist for toll-like receptor (TLR) 3, can enhance immune responses and has been used as an adjuvant for cancer vaccines; however, it remains unclear whether it influences other process, such as EMT. In the present study, we examined the effects of polyI:C on TGF-ƒÀ-treated A549 human LC cells.<br> Methods and results: By in vitro cell proliferation assay, polyI:C showed no effect on the growth of A549 cells treated with TGF-ƒÀ1 at the concentration range up to 10 ƒÊg/ml; however, it markedly suppressed the motility in a cell scratch and a cell invasion assay. By Western blotting, polyI:C dramatically decreased TGF-ƒÀ1-induced Ak strain transforming (Akt) phosphorylation and increased phosphatase and tensin homologue (PTEN) expression without affecting the Son of mothers against decapentaplegic (Smad) 3 phosphorylation or the expression level of E-cadherin, N-cadherin or Snail, indicating that polyI:C suppressed cell motility independently of the �ecadherin switching�f. The Akt inhibitor perifosine inhibited TGF-ƒÀ1-induced cell invasion, and the PTEN-specific inhibitor VO-OHpic appeared to reverse the inhibitory effect of polyI:C.<br> Conclusion: PolyI:C has a novel function to suppress the motility of LC cells undergoing EMT by targeting the phosphatidylinositol 3-kinase /Akt pathway partly via PTEN and may prevent or reduce the metastasis of LC cells. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1320-5463 71 2 2020 Utility of gastric biopsy in diagnosing IgG4�]related gastrointestinal disease 124 134 EN Kaori Uchino Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kenji Notohara Department of Anatomic Pathology, Kurashiki Central Hospital Takeshi Uehara Department of Laboratory Medicine, Shinshu University School of Medicine Yasuhiro Kuraishi Department of Gastroenterology, Shinshu University School of Medicine Junya Itakura Department of Anatomic Pathology, Kurashiki Central Hospital Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The utility of gastric biopsy for diagnosing immunoglobulin (Ig)G4�]related gastrointestinal disease (IgG4�]GID) remains unclear. Bottom�]heavy plasmacytosis (BHP) is a distinct feature of IgG4�]GID. To clarify the feasibility of using gastric biopsies to diagnose BHP in IgG4�]GID, we analyzed the histological features and immunostaining of gastric biopsy specimens from 31 known IgG4�]related disease (IgG4�]RD) patients and we assessed the presence of BHP in 1696 consecutive routine gastric biopsies. Cases with both >10 IgG4�]positive plasma cells per high�]power field and an IgG4/IgG�]positive ratio >40% were defined as IgG4�]high. Ten of the 31 IgG4�]RD patients were concluded to have IgG4�]GID, in which IgG4�]positive plasma cells were notably detected at the deeper part of the mucosa. Six cases displayed BHP whereas the remaining four cases showed transmural infiltration with concomitant Helicobacter pylori�]associated gastritis. In addition to BHP, we identified two unique histologic features for IgG4�]GID: plasmacytic aggregation in the muscularis mucosae and permeative plasmacytic infiltration between fundic glands in the non�]atrophic mucosa. Six of the routine cases (0.35%) displayed BHP, including a case with IgG4�]RD. IgG4�]GID can be suspected by the presence of gastric biopsy specimens with characteristic histological features. Such cases are recommended to undergo further examinations to determine whether IgG4�]RD is present. No potential conflict of interest relevant to this article was reported.

Nature Acta Medica Okayama 2045-2322 10 1 2020 Spred2-deficiency enhances the proliferation of lung epithelial cells and alleviates pulmonary fibrosis induced by bleomycin 16490 EN Akina Kawara Department of Pathology and Experimental Medicine, Graduated School of Medicine, Dentistry and Pharmaceutical Science, Okayama University Ryo Mizuta Department of Pathology and Experimental Medicine, Graduated School of Medicine, Dentistry and Pharmaceutical Science, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduated School of Medicine, Dentistry and Pharmaceutical Science, Okayama University Toshihiro Ito Department of Immunology, Nara Medical University Chunning Li Department of Pathology and Experimental Medicine, Graduated School of Medicine, Dentistry and Pharmaceutical Science, Okayama University Kaoru Nakamura Department of Pathology and Experimental Medicine, Graduated School of Medicine, Dentistry and Pharmaceutical Science, Okayama University Cuiming Sun Department of Pathology and Experimental Medicine, Graduated School of Medicine, Dentistry and Pharmaceutical Science, Okayama University Masaki Kuwabara Department of Pathology and Experimental Medicine, Graduated School of Medicine, Dentistry and Pharmaceutical Science, Okayama University Masahiro Kitabatake Department of Immunology, Nara Medical University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduated School of Medicine, Dentistry and Pharmaceutical Science, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduated School of Medicine, Dentistry and Pharmaceutical Science, Okayama University The mitogen-activated protein kinase (MAPK) pathways are involved in many cellular processes, including the development of fibrosis. Here, we examined the role of Sprouty-related EVH-1-domain-containing protein (Spred) 2, a negative regulator of the MAPK-ERK pathway, in the development of bleomycin (BLM)-induced pulmonary fibrosis (PF). Compared to WT mice, Spred2�|/�| mice developed milder PF with increased proliferation of bronchial epithelial cells. Spred2�|/�| lung epithelial cells or MLE-12 cells treated with spred2 siRNA proliferated faster than control cells in vitro. Spred2�|/�| and WT macrophages produced similar levels of TNFƒ¿ and MCP-1 in response to BLM or lipopolysaccharide and myeloid cell-specific deletion of Spred2 in mice had no effect. Spred2�|/�| fibroblasts proliferated faster and produced similar levels of MCP-1 compared to WT fibroblasts. Spred2 mRNA was almost exclusively detected in bronchial epithelial cells of naïve WT mice and it accumulated in approximately 50% of cells with a characteristic of Clara cells, 14 days after BLM treatment. These results suggest that Spred2 is involved in the regulation of tissue repair after BLM-induced lung injury and increased proliferation of lung bronchial cells in Spred2�|/�| mice may contribute to faster tissue repair. Thus, Spred2 may present a new therapeutic target for the treatment of PF. No potential conflict of interest relevant to this article was reported.

Nature Acta Medica Okayama 2045-2322 10 1 2020 Decreased miR-200b-3p in cancer cells leads to angiogenesis in HCC by enhancing endothelial ERG expression 10418 EN Aye Moh-Moh-Aung Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sachio Ito Department of Molecular Oncology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hiroshi Katayama Department of Molecular Oncology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yoko Ota Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Transcription factor ERG (erythroblast transformation-specific (ETS)-related gene) is essential in endothelial differentiation and angiogenesis, in which microRNA (miR)-200b-3p targeting site is expected by miRNA target prediction database. miR-200b is known decreased in hepatocellular carcinoma (HCC), however, the functional relation between ERG and miR-200b-3p, originating from pre-miR-200b, in HCC angiogenesis remains unclear. We investigated whether hepatocyte-derived miR-200b-3p governs angiogenesis in HCC by targeting endothelial ERG. Levels of miR-200b-3p in HCC tissues were significantly lower than those in adjacent non-HCC tissues. Poorly differentiated HCC cell line expressed lower level of miR-200b-3p compared to well-differentiated HCC cell lines. The numbers of ERG-positive endothelial cells were higher in HCC tissues than in adjacent non-HCC tissues. There was a negative correlation between the number of ERG-positive cells and miR-200b-3p expression in HCC tissues. Culture supernatants of HCC cell lines with miR-200b-3p-overexpression reduced cell migration, proliferation and tube forming capacity in endothelial cells relative to the control, while those with miR-200b-3p-inhibition augmented the responses. Exosomes isolated from HCC culture supernatants with miR-200b-3p overexpression suppressed endothelial ERG expression. These results suggest that exosomal miR-200b-3p from hepatocytes suppresses endothelial ERG expression, and decreased miR-200b-3p in cancer cells promotes angiogenesis in HCC tissues by enhancing endothelial ERG expression. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 2 2020 Juvenile Granulosa Cell Tumor with an Unusual Clinical Course: A Late-onset and Late Recurrent Case 159 163 EN Thar Htet San Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoko Ota Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Soichiro Fushimi Department of Pathology, Himeji Red Cross Hospital Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroyuki Yanai Department of Pathology, Okayama University Hospital Hiroko Toda Section of Diagnostic Pathology, Fukuyama Medical Association Tadayoshi Kunitomo Department of Pathology, Kurashiki Medical Center Keisuke Kodama Department of Gynecology, Kurashiki Medical Center Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Case Report 10.18926/AMO/58275 Juvenile granulosa cell tumors (JGCTs) are rare ovarian tumors with overall good prognoses. They differ from adult granulosa cell tumors (AGCTs), which are well known for late recurrence. Most JGCTs (�`97%) occur in individuals <30 years old. We report a recurrent JGCT in a 40-year-old woman 5 years after initial presentation. The histological appearance and lack of 402C>G missense point mutation of FOXL2 gene (characteristic of AGCT but absent in JGCT) allowed differentiation from AGCT. This is the first comprehensive report of JGCT with late recurrence. Although rare, late recurrence of JGCT can occur; long-term surveillance is suggested. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 1863-6705 68 1 2019 Prolonged warm ischemia exacerbated acute rejection after lung transplantation from donation after cardiac death in a mouse 57 62 EN Yutaka Hirano Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichiro Sugimoto Department of General Thoracic Surgery, Okayama University Hospital Sumiharu Yamamoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masanori Okada Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinji Otani Department of Organ Transplant Center, Okayama University Hospital Toshiaki Ohara Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masaomi Yamane Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takahiro Oto Department of Organ Transplant Center, Okayama University Hospital Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Objective: In lung transplantation (LTx) from donation after cardiac death (DCD), the donor lungs are inevitably exposed to warm ischemic time (WIT) between the cardiac arrest and the initiation of cold preservation. We conducted this study to examine the effect of prolonged WIT on lung allograft rejection in a murine model of LTx from DCD.<br/> Methods: Allogeneic BALB/c �¨ B6 LTx from DCD was performed with a WIT of 15 min (WIT15 group, n = 5) or 60 min (WIT60 group, n = 5). Recipients were immunosuppressed by perioperative costimulatory blockade. The lung allografts were analyzed by histology and flow cytometry on day 7 after the LTx.<br/> Results: Histologically, the rejection grade in the WIT60 group was significantly higher than that in the WIT15 group (3.4 �} 0.4 vs. 2.2 �} 0.2, P = 0.0278). Moreover, the intragraft CD8+ to CD4+ T cell ratio in the WIT60 group was significantly higher than that in the WIT15 group (2.3 �} 0.12 vs. 1.2 �} 0.11, P < 0.0001).<br/> Conclusions: Prolonged WIT could exacerbate the severity of lung allograft rejection after LTx from DCD. Minimization of the WIT could improve the outcomes after LTx from DCD. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 20 24 2019 Cancer Cell-Derived Granulocyte-Macrophage Colony-Stimulating Factor Is Dispensable for the Progression of 4T1 Murine Breast Cancer EN Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kaoru Nakamura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chunning Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tsuyoshi Shiina Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mayu Imamura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tiantian Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naofumi Mukaida Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University We previously reported that 4T1 murine breast cancer cells produce GM-CSF that up-regulates macrophage expression of several cancer promoting genes, including Mcp-1/Ccl2, Ccl17 and Rankl, suggesting a critical role of cancer cell-derived GM-CSF in cancer progression. Here, we attempted to define whether 4T1 cell-derived GM-CSF contributes to the expression of these genes by 4T1tumors, and their subsequent progression. Intraperitoneal injection of anti-GM-CSF neutralizing antibody did not decrease the expression of Mcp-1, Ccl17 or Rankl mRNA by 4T1 tumors. To further examine the role of cancer cell-derived GM-CSF, we generated GM-CSF-deficient 4T1 cells by using the Crisper-Cas9 system. As previously demonstrated, 4T1 cells are a mixture of cells and cloning of cells by itself significantly reduced tumor growth and lung metastasis. By contrast, GM-CSF-deficiency did not affect tumor growth, lung metastasis or the expression of these chemokine and cytokine genes in tumor tissues. By in-situ hybridization, the expression of Mcp-1 mRNA was detected in both F4/80-expressing and non-expressing cells in tumors of GM-CSF-deficient cells. These results indicate that cancer cell-derived GM-CSF is dispensable for the tuning of the 4T1 tumor microenvironment and the production of MCP-1, CCL17 or RANKL in the 4T1 tumor microenvironment is likely regulated by redundant mechanisms. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 20 23 2019 Multipotent Neurotrophic Effects of Hepatocyte Growth Factor in Spinal Cord Injury 6078 EN Kentaro Yamane Department of Intelligent Orthopaedic System Development, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Haruo Misawa Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomoyuki Takigawa Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshihiro Ito Nano Medical Engineering Laboratory, RIKEN Toshifumi Ozaki Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Spinal cord injury (SCI) results in neural tissue loss and so far untreatable functional impairment. In addition, at the initial injury site, inflammation induces secondary damage, and glial scar formation occurs to limit inflammation-mediated tissue damage. Consequently, it obstructs neural regeneration. Many studies have been conducted in the field of SCI; however, no satisfactory treatment has been established to date. Hepatocyte growth factor (HGF) is one of the neurotrophic growth factors and has been listed as a candidate medicine for SCI treatment. The highlighted effects of HGF on neural regeneration are associated with its anti-inflammatory and anti-fibrotic activities. Moreover, HGF exerts positive effects on transplanted stem cell differentiation into neurons. This paper reviews the mechanisms underlying the therapeutic effects of HGF in SCI recovery, and introduces recent advances in the clinical applications of HGF therapy. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 1 2020 A Surgical Instructor Training Course for the Next Generation 73 76 EN Masaomi Yamane Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuhiro Mandai Center for Education in Medicine and Health Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideo Ino Center for Education in Medicine and Health Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Short Communication 10.18926/AMO/57956 In 2016, Gunma University Hospital�fs Medical Accident Investigation Committee released a report reiterating the necessity of medical education and the need for surgeons to master non-technical skills. We designed a 17-h training course for surgical instructors, designed to teach participants how to sufficiently educate surgeon trainees and encourage their professional identity formation. A post-training survey showed that participants improved their awareness, and their behavioral changes led to favorable team performances. We then began offering a 3-h workshop focusing on the participants�f experiences. We propose that the training course using participant narratives is required and effective to establish surgeons�f self-reflection and professional identity as surgeons. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 1 2020 A Promising New Anti-Cancer Strategy: Iron Chelators Targeting CSCs 1 6 EN Yuehua Chen Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiaki Ohara Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Boyi Xing Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jiping Qi Department of Pathology, the First Affiliated Hospital of Harbin Medical University Kazuhiro Noma Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Review 10.18926/AMO/57946 Iron is a trace but vital element in the human body and is necessary for a multitude of crucial processes in life. However, iron overload is known to induce carcinogenesis via oxidative stress. Cancer cells require large amounts of iron for their rapid division and cell growth. Iron was recently found to play a role in cancer stem cells (CSCs); it maintains stemness during development. Iron also plays an important role in stemness by moderating reactive oxygen species. Thus, iron metabolism in CSCs is a promising therapeutic target. In this review, we summarize the roles of iron in cancer cells and CSCs. We also summarize anti-cancer therapeutic studies with iron chelators and describe our expectation of a new therapeutic strategy for CSCs on the basis of our findings. No potential conflict of interest relevant to this article was reported.

Frontiers Media S.A Acta Medica Okayama 1664-3224 10 2019 Spred2 Regulates High Fat Diet-Induced Adipose Tissue Inflammation, and Metabolic Abnormalities in Mice 17 EN Takahiro Ohkura Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Teizo Yoshimura Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiaki Ohara Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Marutani Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kaya Usami Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Chronic low-grade inflammation in visceral adipose tissues triggers the development of obesity-related insulin resistance, leading to the metabolic syndrome, a serious health condition with higher risk of cardiovascular disease, diabetes, and stroke. In the present study, we investigated whether Sprouty-related EVH1-domain-containing protein 2 (Spred2), a negative regulator of the Ras/Raf/ERK/MAPK pathway, plays a role in the development of high fat diet (HFD)-induced obesity, adipose tissue inflammation, metabolic abnormalities, and insulin resistance. Spred2 knockout (KO) mice, fed with HFD, exhibited an augmented body weight gain, which was associated with enhanced adipocyte hypertrophy in mesenteric white adipose tissue (mWAT) and deteriorated dyslipidemia, compared with wild-type (WT) controls. The number of infiltrating macrophages with a M1 phenotype, and the crown-like structures, composed of macrophages surrounding dead or dying adipocytes, were more abundant in Spred2 KO-mWAT compared to in WT-mWAT. Exacerbated adipose tissue inflammation in Spred2 KO mice led to aggravated insulin resistance and fatty liver disease. To analyze the mechanism(s) that caused adipose tissue inflammation, cytokine response in mWAT was investigated. Stromal vascular fraction that contained macrophages from Spred2 KO-mWAT showed elevated levels of tumor necrosis factor ƒ¿ (TNFƒ¿) and monocyte chemoattractant protein-1 (MCP-1/CCL2) compared with those from WT-mWAT. Upon stimulation with palmitate acid (PA), bone marrow-derived macrophages (BMDMs) derived from Spred2 KO mice secreted higher levels of TNFƒ¿ and MCP-1 than those from WT mice with enhanced ERK activation. U0126, a MEK inhibitor, reduced the PA-induced cytokine response. Taken together, these results suggested that Spred2, in macrophages, negatively regulates high fat diet-induced obesity, adipose tissue inflammation, metabolic abnormalities, and insulin resistance by inhibiting the ERK/MAPK pathway. Thus, Spred2 represents a potential therapeutic tool for the prevention of insulin resistance and resultant metabolic syndrome. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 11 2 2019 A Novel Combination Cancer Therapy with Iron Chelator Targeting Cancer Stem Cells via Suppressing Stemness 177 EN Yuki Katsura Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiaki Ohara Department of Pathology and Experimental MedicineOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kazuhiro Noma Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takayuki Ninomiya Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hajime Kashima Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takuya Kato Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroaki Sato Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Komoto Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toru Narusaka Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuko Tomono Shigei Medical Research Institute Boyi Xing Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuehua Chen Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Tazawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shunsuke Kagawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuhiro Shirakawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomonari Kasai School of Bioscience and Biotechnology, Tokyo University of Technology Masaharu Seno Laboratory of Nano-Biotechnology, Okayama University Graduate School of Interdisciplinary Science and Engineering in Health Systems Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiyoshi Fujiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Excess iron causes cancer and is thought to be related to carcinogenesis and cancer progression including stemness, but the details remain unclear. Here, we hypothesized that stemness in cancer is related to iron metabolism and that regulating iron metabolism in cancer stem cells (CSCs) may be a novel therapy. In this study, we used murine induced pluripotent stem cells that expressed specific stem cell genes such as Nanog, Oct3/4, Sox2, Klf4, and c-Myc, and two human cancer cell lines with similar stem cell gene expression. Deferasirox, an orally available iron chelator, suppressed expression of stemness markers and spherogenesis of cells with high stemness status in vitro. Combination therapy had a marked antitumor effect compared with deferasirox or cisplatin alone. Iron metabolism appears important for maintenance of stemness in CSCs. An iron chelator combined with chemotherapy may be a novel approach via suppressing stemness for CSC targeted therapy. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 09441174 54 11 2019 The relationship between the PD-L1 expression of surgically resected and fine-needle aspiration specimens for patients with pancreatic cancer 1019 1028 EN Kazuyuki Matsumoto Department of Gastroenterology and HepatologyOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Toshiaki Ohara Department of Pathology and Experimental MedicineOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Masayoshi Fujisawa Department of Pathology and Experimental MedicineOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Akinobu Takaki Department of Gastroenterology and HepatologyOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Masahiro Takahara Noriyuki Tanaka Department of PathologyOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hironari Kato Department of Gastroenterology and HepatologyOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Shigeru Horiguchi Department of Gastroenterology and HepatologyOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Ryuichi Yoshida Department of Gastroenterological Surgery, Transplant and Surgical OncologyOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yuzo Umeda Department of Gastroenterological Surgery, Transplant and Surgical OncologyOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Soichiro Fushimi Department of PathologyHimeji Red Cross Hospital Takahito Yagi Department of Gastroenterological Surgery, Transplant and Surgical OncologyOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental MedicineOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hiroyuki Okada Department of Gastroenterology and HepatologyOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences BACKGROUND:</br> Recently, therapeutic antibodies against programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) have shown promising clinical results for several solid tumors, including pancreatic cancer. In this study, we evaluated the relationship between the PD-L1 expression of surgical resected and fine-needle aspiration (FNA) specimens for patients with pancreatic cancer. </br> METHODS:</br> Of 121 patients who underwent endoscopic ultrasound-guided (EUS)-FNA before surgery for pancreatic cancer in an academic center, the 94 (78%) with adequate FNA specimens for a histological evaluation were retrospectively analyzed. All the patients had undergone upfront surgery without any chemotherapy or radiotherapy. We performed immunohistochemistry (IHC) staining to investigate the PD-L1 expression in both resected and FNA specimens. The positive-stained cells were counted, and their percentage was used for the investigation. </br> RESULTS:</br> Of the 94 patients, 16 (17%) and 11 (10%) were defined as positive on resected cancer specimens using cutoff points of 5% and 10% positively stained cancer cell counts, respectively. The concordance rates for the positive frequency of PD-L1 expression between resected and FNA specimens were 44% (7/16) and 55% (6/11) when the positivity was set to ≥ 5% and ≥ 10%, respectively. The concordance rates for the negative frequency of PD-L1 expression between two specimens were 97% (76/78) and 99% (82/83) when the positivity was set to ≥ 5% and ≥ 10%, respectively. </br> CONCLUSIONS:</br> Approximately, half of the patients with PD-L1 expression positive and almost all the patients with PD-L1 expression negative could be diagnosed on FNA specimens. No potential conflict of interest relevant to this article was reported.

PUBLIC LIBRARY SCIENCE Acta Medica Okayama 1932-6203 8 3 2013 Monocyte chemoattractant protein-1/CCL2 produced by stromal cells promotes lung metastasis of 4T1 murine breast cancer cells e58791 EN Teizo Yoshimura O. M. Zack Howard Toshihiro Ito Masaki Kuwabara Akihiro Matsukawa Keqiang Chen Ying Liu Mingyong Liu Joost J. Oppenheim Ji Ming Wang MCP-1/CCL2 plays an important role in the initiation and progression of cancer. Since tumor cells produce MCP-1, they are considered to be the main source of this chemokine. Here, we examined whether MCP-1 produced by non-tumor cells affects the growth and lung metastasis of 4T1 breast cancer cells by transplanting them into the mammary pad of WT or MCP-1�|/�| mice. Primary tumors at the injected site grew similarly in both mice; however, lung metastases were markedly reduced in MCP-1�|/�| mice, with significantly longer mouse survival. High levels of MCP-1 mRNA were detected in tumors growing in WT, but not MCP-1�|/�| mice. Serum MCP-1 levels were increased in tumor-bearing WT, but not MCP-1�|/�| mice. Transplantation of MCP-1�|/�| bone marrow cells into WT mice did not alter the incidence of lung metastasis, whereas transplantation of WT bone marrow cells into MCP-1�|/�| mice increased lung metastasis. The primary tumors of MCP-1�|/�| mice consistently developed necrosis earlier than those of WT mice and showed decreased infiltration by macrophages and reduced angiogenesis. Interestingly, 4T1 cells that metastasized to the lung constitutively expressed elevated levels of MCP-1, and intravenous injection of 4T1 cells producing a high level of MCP-1 resulted in increased tumor foci in the lung of WT and MCP-1�|/�| mice. Thus, stromal cell-derived MCP-1 in the primary tumors promotes lung metastasis of 4T1 cells, but tumor cell-derived MCP-1 can also contribute once tumor cells enter the circulation. A greater understanding of the source and role of this chemokine may lead to novel strategies for cancer treatment. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2045-2322 4 2014 A novel, visible light-induced, rapidly cross-linkable gelatin scaffold for osteochondral tissue engineering EN Tetsuro Mazaki Yasuyuki Shiozaki Kentaro Yamane Aki Yoshida Mariko Nakamura Yasuhiro Yoshida Di Zhou Takashi Kitajima Masato Tanaka Yoshihiro Ito Toshifumi Ozaki Akihiro Matsukawa Osteochondral injuries remain difficult to repair. We developed a novel photo-cross-linkable furfurylamine-conjugated gelatin (gelatin-FA). Gelatin-FA was rapidly cross-linked by visible light with Rose Bengal, a light sensitizer, and was kept gelled for 3 weeks submerged in saline at 37 degrees C. When bone marrow-derived stromal cells (BMSCs) were suspended in gelatin-FA with 0.05% Rose Bengal, approximately 87% of the cells were viable in the hydrogel at 24 h after photo-cross-linking, and the chondrogenic differentiation of BMSCs was maintained for up to 3 weeks. BMP4 fusion protein with a collagen binding domain (CBD) was retained in the hydrogels at higher levels than unmodified BMP4. Gelatin-FA was subsequently employed as a scaffold for BMSCs and CBD-BMP4 in a rabbit osteochondral defect model. In both cases, the defect was repaired with articular cartilage-like tissue and regenerated subchondral bone. This novel, photo-cross-linkable gelatin appears to be a promising scaffold for the treatment of osteochondral injury. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1178-2013 8 1 2013 Enhanced in vivo osteogenesis by nanocarrier-fused bone morphogenetic protein-4 1349 1360 EN Yasuyuki Shiozaki Takashi Kitajima Tetsuro Mazaki Aki Yoshida Masato Tanaka Akihiro Umezawa Mariko Nakamura Yasuhiro Yoshida Yoshihiro Ito Toshifumi Ozaki Akihiro Matsukawa Purpose: Bone defects and nonunions are major clinical skeletal problems. Growth factors are commonly used to promote bone regeneration; however, the clinical impact is limited because the factors do not last long at a given site. The introduction of tissue engineering aimed to deter the diffusion of these factors is a promising therapeutic strategy. The purpose of the present study was to evaluate the in vivo osteogenic capability of an engineered bone morphogenetic protein-4 (BMP4) fusion protein. Methods: BMP4 was fused with a nanosized carrier, collagen-binding domain (CBD), derived from fibronectin. The stability of the CBD-BMP4 fusion protein was examined in vitro and in vivo. Osteogenic effects of CBD-BMP4 were evaluated by computer tomography after intramedullary injection without a collagen-sponge scaffold. Recombinant BMP-4, CBD, or vehicle were used as controls. Expressions of bone-related genes and growth factors were compared among the groups. Osteogenesis induced by CBD-BMP4, BMP4, and CBD was also assessed in a bone-defect model. Results: In vitro, CBD-BMP4 was retained in a collagen gel for at least 7 days while BMP4 alone was released within 3 hours. In vivo, CBD-BMP4 remained at the given site for at least 2 weeks, both with or without a collagen-sponge scaffold, while BMP4 disappeared from the site within 3 days after injection. CBD-BMP4 induced better bone formation than BMP4 did alone, CBD alone, and vehicle after the intramedullary injection into the mouse femur. -Bone-related genes and growth factors were expressed at higher levels in CBD-BMP4-treated mice than in all other groups, including BMP4-treated mice. Finally, CBD-BMP4 potentiated more bone formation than did controls, including BMP4 alone, when applied to cranial bone defects without a collagen scaffold. Conclusion: Altogether, nanocarrier-CBD enhanced the retention of BMP4 in the bone, thereby promoting augmented osteogenic responses in the absence of a scaffold. These results suggest that CBD-BMP4 may be clinically useful in facilitating bone formation. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 67 5 2013 Occult Compression Fracture of Metacarpal Head without Evidence of Avascular Necrosis 311 317 EN Keiichiro Nishida Hiroyuki Hashizume Akihiro Matsukawa Kenzo Hashizume Yasunori Shimamura Yasuyuki Torigoe Toshifumi Ozaki Case Report 10.18926/AMO/51867 We report a case of 4th metacarpal head collapse of a 19-year-old healthy man. MRI revealed T1 low and T2 high regions in the collapsed 4th metacarpal head, as well as in the right 3rd and left 4th metacarpal head. Our initial diagnosis was occult compression fracture due to avascular necrosis, known as Dieterich's disease. However, pathological findings of surgically resected right 4th metacarpal head were compatible with transient osteoporosis and metacarpal head fracture followed by active tissue repair. The autologous osteochondral transplants from costchondral junction survived and maintained their size and shape even at 10-year follow-up. No potential conflict of interest relevant to this article was reported.

Academic Press Inc Elsevier Science Acta Medica Okayama 1521-6616 144 3 2012 Spred-2 deficiency exacerbates acetaminophen-induced hepatotoxicity in mice 272 282 EN Hiroshi Wakabayashi Toshihiro Ito Soichiro Fushimi Yuki Nakashima Jyunya Itakura Qiuying Liu Min Min Win Cuiming Sun Cao Chen Miwa Sato Megumi Mino Tetsuya Ogino Hirofumi Makino Akihiko Yoshimura Akihiro Matsukawa MAPKs are involved in acetaminophen (APAP)-hepatotoxicity, but the regulatory mechanism remains unknown. Here, we explored the role of Spred-2 that negatively regulates Ras/ERK pathway in APAP-hepatotoxicity. Spred-2 knockout (KO) mice demonstrated exacerbated liver injury, an event that was associated with increased numbers of CD4(+) T, CD8(+) T and NK cells in the liver compared to the control. Levels of CXCL9/CXCL10 that attract and activate these cells were increased in Spred-2 KO-liver. Kupffer cells isolated from Spred-2 KO mice after APAP challenge expressed higher levels of CXCL9/CXCL10 than those from the control. Upon stimulation with APAP or IFN gamma, naive Kupffer cells from Spred-2 KO mice expressed higher levels of CXCL9/CXCL10. NK cell-depletion attenuated APAP-hepatotoxicity with lowered hepatic IFN gamma and decreased numbers of not only NK cells but also CD4(+) T and CD8(+) T cells in the liver. These results suggest that Spred-2 negatively regulates APAP-hepatotoxicity under the control of Kupffer cells and NK cells. No potential conflict of interest relevant to this article was reported.

‰ªŽRˆãŠw‰ï Acta Medica Okayama 0030-1558 125 2 2013 ƒ}ƒEƒXƒCƒ“ƒtƒ‹ƒGƒ“ƒU”x‰Š‚É‚¨‚¯‚郌ƒhƒbƒNƒX�§Œä’`”’ƒ`ƒIƒŒƒhƒLƒVƒ“�iTRX-1�j‚ÌŽ¡—ÓIŒø‰Ê 109 112 EN Masato Yashiro Hirokazu Tsukahara Akihiro Matsukawa Mutsuko Yamada Yosuke Fujii Yoshiharu Nagaoka Mitsuru Tsuge Nobuko Yamashita Toshihiro Ito Masao Yamada Hiroshi Masutani Junji Yodoi Tsuneo Morishima No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0090-3493 41 1 2013 Redox-Active Protein Thioredoxin-1 Administration Ameliorates Influenza A Virus (H1N1)-Induced Acute Lung Injury in Mice 171 181 EN Masato Yashiro Hirokazu Tsukahara Akihiro Matsukawa Mutsuko Yamada Yosuke Fujii Yoshiharu Nagaoka Mitsuru Tsuge Nobuko Yamashita Toshihiro Ito Masao Yamada Hiroshi Masutani Objectives: Influenza virus infections can cause severe acute lung injury leading to significant morbidity and mortality. Thioredoxin-1 is a redox-active defensive protein induced in response to stress conditions. Animal experiments have revealed that thioredoxin-1 has protective effects against various severe disorders. This study was undertaken to evaluate the protective effects of recombinant human thioredoxin-1 administration on influenza A virus (H1N1)-induced acute lung injury in mice. Design: Prospective animal trial. Setting: Research laboratory. Subjects: Nine-week-old male C57BL/6 mice inoculated with H1N1. Intervention: The mice were divided into a vehicle-treated group and recombinant human thioredoxin-1-treated group. For survival rate analysis, the vehicle or recombinant human thioredoxin-1 was administered intraperitoneally every second day from day -1 to day 13. For lung lavage and pathological analyses, vehicle or recombinant human thioredoxin-1 was administered intraperitoneally on days 1, 1, and 3. Measurements and Main Results: Lung lavage and pathological analyses were performed at 24, 72, and 120 hrs after inoculation. The recombinant human thioredoxin-1 treatment significantly improved the survival rate of H1N1-inoculated mice, although the treatment did not affect virus propagation in the lung. The treatment significantly attenuated the histological changes and neutrophil infiltration in the lung of H1N1-inoculated mice. The treatment significantly attenuated the production of tumor necrosis factor-a and chemokine (C-X-C motif) ligand 1 in the lung and oxidative stress enhancement, which were observed in H1N1-inoculated mice. H1N1 induced expressions of tumor necrosis factor-a and chemokine (C-X-C motif) ligand 1 in murine lung epithelial cells MLE-12, which were inhibited by the addition of recombinant human thioredoxin-1. The recombinant human thioredoxin-1 treatment started 30 mins after H1N1 inoculation also significantly improved the survival of the mice. Conclusions: Exogenous administration of recombinant human thioredoxin-1 significantly improved the survival rate and attenuated lung histological changes in the murine model of influenza pneumonia. The protective mechanism of thioredoxin-1 might be explained by its potent antioxidative and anti-inflammatory actions. Consequently, recombinant human thioredoxin-1 might be a possible pharmacological strategy for severe influenza virus infection in humans. (Crit Care Med 2013; 41:171-181) No potential conflict of interest relevant to this article was reported.

‰ªŽRŽÀŒ±“®•¨Œ¤‹†‰ï Acta Medica Okayama 27 2011 ‰ªŽR‘åŠwŽ©‘R�¶–½‰ÈŠwŒ¤‹†Žx‰‡ƒZƒ“ƒ^�[“®•¨Ž‘Œ¹•”–å’Ó‡–kŽ{�Ý 52 55 EN Katsumi Mominoki Rumi Takashima Akihiro Matsukawa No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 61 5 2007 STAT Proteins in Innate Immunity during Sepsis: Lessons from Gene Knockout Mice 239 245 EN Akihiro Matsukawa Review 10.18926/AMO/32897 The innate immune system provides immediate defense against infection and serves as the first line of host defense during infection. In innate immunity, leukocytes such as neutrophils and macrophages recognize and respond to pathogens in a non-specific manner. Therefore, the recruitment and activation of leukocytes are essential in innate immunity, and are governed by a variety of chemical mediators including cytokines. Cytokines are generally divided into 2 types, termed type-1 and type-2 cytokines. Type-1 cytokines are important in local host defense, while type-2 cytokines play a protective role when inflammatory response spreads to the body. These cytokines exert their biological functions through the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. STAT1/3/4/6 are transcription factors that mediate IFNgamma/IL-10/IL-12/IL-13 cytokine signaling, respectively. Evidence indicates that STAT proteins have a significant impact on innate immunity during sepsis. This review focuses on recent understandings in the regulation of innate immunity by STAT proteins during sepsis and septic shock. The suppressor of cytokine signaling (SOCS) proteins are a family of SH2 domain-containing cytoplasmic proteins that complete a negative feedback loop to attenuate signal transduction from cytokines that act through the JAK/STAT pathway. The participation of SOCS proteins in sepsis is also discussed. No potential conflict of interest relevant to this article was reported.

‰ªŽRˆãŠw‰ï Acta Medica Okayama 0030-1558 118 2 2006 Ž©‘R–Ɖu/‰Š�ǂƃTƒCƒgƒJƒCƒ“�EƒVƒOƒiƒ‹“`’B 109 112 EN No potential conflict of interest relevant to this article was reported.