Publications by authors named "Moto Fukai"

42 Publications

Genomewide transcriptomic profiling identifies a gene signature for predicting recurrence in early-stage hepatocellular carcinoma.

Clin Transl Med 2021 06;11(6):e405

Center for Gastrointestinal Research, Center from Translational Genomics and Oncology, Baylor Scott and White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, Texas, USA.

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http://dx.doi.org/10.1002/ctm2.405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8181200PMC
June 2021

The Phenolic Antioxidant 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) Prevents Enterocyte Cell Death under Oxygen-Dissolving Cold Conditions through Polyphyletic Antioxidant Actions.

J Clin Med 2021 May 4;10(9). Epub 2021 May 4.

Department of Gastroenterological Surgery I, Graduate School of Medicine, Hokkaido University, Nishi 7, Kita 15, Kita-ku, Sapporo 060-8638, Hokkaido, Japan.

Cold preservation in University of Wisconsin (UW) solution is not enough to maintain the viability of the small intestine, due to the oxidative stress. The novel phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has dual properties to reduce oxidative stress, radical scavenging, and antioxidant protein induction, in other cells. This study was designed to determine whether DHMBA reduces cold preservation injury of enterocytes, and to identify the effector site. Enterocytes were subjected to 48-h cold preservation under atmosphere in UW solution (±DHMBA), and then returned to normal culture to replicate reperfusion of the small intestine after cold preservation. At the end of cold preservation (ECP) and at 1, 3, 6, and 72 h after rewarming (R1h, R3h, R6h, and R72h), we evaluated cell function and the injury mechanism. The results showed that DHMBA protected mitochondrial function mainly during cold preservation, and suppressed cell death after rewarming, as shown by the MTT, ATP, mitochondrial membrane potential, LDH, and lipid peroxidation assays, together with enhanced survival signals (PI3K, Akt, p70S6K) and induction of antioxidant proteins (HO-1, NQO-1, TRX-1). We found that DHMBA mitigates the cold-induced injury of enterocytes by protecting the mitochondria through direct and indirect antioxidative activities.
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http://dx.doi.org/10.3390/jcm10091972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124816PMC
May 2021

Myostatin as a fibroblast-activating factor impacts on postoperative outcome in patients with hepatocellular carcinoma.

Hepatol Res 2021 Jul 25;51(7):803-812. Epub 2021 May 25.

Department of Liver Disease, The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan.

Aim: In patients with liver cirrhosis, high levels of serum myostatin are associated with poor prognosis. We aimed to clarify the influence of myostatin on the prognosis of patients with non-alcoholic fatty liver disease-hepatocellular carcinoma (NAFLD-HCC) without cirrhosis and on the progression of liver fibrosis.

Methods: Serum myostatin levels were evaluated in 234 patients who underwent primary surgical resection for single HCC. To clarify the impact of myostatin on liver fibrosis, we established human primary liver fibroblasts from resected livers, and cultured them in the presence of myostatin.

Results: The median age was 67.4 years, the median L3 skeletal muscle mass index was 44.4 cm /m , and the median body mass index was 23.4 kg/m . Eighty-two (35.0%) patients had sarcopenia (L3 skeletal muscle mass index: men <42, women <38 cm /m ). The etiologies of liver disease were hepatitis B virus (n = 61), hepatitis C virus (n = 86), and non-B non-C hepatitis (n = 87) including NAFLD (n = 74). High preoperative serum myostatin and vascular invasion were independent predictors of poor overall survival (OS). High serum myostatin was associated with poor OS in patients with no sarcopenia (n = 152). In patients without advanced liver fibrosis (Fibrosis stage, 0-2; n = 58), high levels of serum myostatin were also associated with poor OS, regardless of sarcopenia. Serum myostatin levels were increased with the progression of liver fibrosis. Liver fibroblasts were activated and produced collagen following stimulation with myostatin.

Conclusions: In patients with NAFLD-HCC without advanced liver fibrosis, high levels of serum myostatin were associated with poor OS. Myostatin activated primary fibroblasts and stimulated collagen production.
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http://dx.doi.org/10.1111/hepr.13667DOI Listing
July 2021

Early administration of amatuximab, a chimeric high-affinity anti-mesothelin monoclonal antibody, suppresses liver metastasis of mesothelin-expressing pancreatic cancer cells and enhances gemcitabine sensitivity in a xenograft mouse model.

Invest New Drugs 2021 Oct 27;39(5):1256-1266. Epub 2021 Apr 27.

Department of Gastroenterological Surgery 1, Hokkaido University Graduate School of Medicine, N15W7, Kitaku, Sapporo, Hokkaido, 060-8638, Japan.

Amatuximab is a promising therapeutic antibody targeting mesothelin, a 40-kDa glycoprotein that is highly expressed in pancreatic cancer. We investigated the effectiveness of early amatuximab treatment, imitating an adjuvant chemotherapy setting, and combination therapy with amatuximab and gemcitabine in liver metastasis of pancreatic cancer. Liver metastasis mouse models were established in 8-week-old male BALB/c nu/nu mice using the hemisplenic injection method. Tridaily amatuximab monotherapy or combination with gemcitabine was administered to the liver metastasis mouse model before metastatic lesions had formed huge masses. Gaussia luciferase-transfected AsPC-1 was used as a mesothelin-overexpressing pancreatic cancer cell line. The amount of liver metastases and the serum luciferase activity were significantly lower in the treatment groups than those in the control IgG group. Notably, the anti-tumor activity of gemcitabine was synergically enhanced by combination therapy with amatuximab. Furthermore, western blotting revealed that the high expression of phosphorylated c-Met and AKT in liver metastatic lesions treated with gemcitabine monotherapy was canceled by its combination with amatuximab. This result indicated that the observed synergic therapeutic effect may have occurred as a result of the inhibitory effect of amatuximab on the phosphorylation of c-Met and AKT, which were promoted by exposure to GEM. In conclusion, our study revealed that early administration of amatuximab alone or in combination with GEM significantly suppressed the liver metastases of mesothelin-expressing pancreatic cancer cells. A phase II clinical trial of amatuximab as part of an adjuvant chemotherapy regimen for resected pancreatic cancer is expected.
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http://dx.doi.org/10.1007/s10637-021-01118-1DOI Listing
October 2021

Establishment of a Cell Culture Model Permissive for Infection by Hepatitis B and C Viruses.

Hepatol Commun 2021 04 19;5(4):634-649. Epub 2020 Dec 19.

Department of Microbiology Graduate School of Medical Science University of Yamanashi Yamanashi Japan.

Compared with each monoinfection, coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is well known to increase the risks of developing liver cirrhosis and hepatocellular carcinoma. However, the mechanism by which HBV/HCV coinfection is established in hepatocytes is not well understood. Common cell culture models for coinfection are required to examine viral propagation. In this study, we aimed to establish a cell line permissive for both HBV and HCV infection. We first prepared a HepG2 cell line expressing sodium taurocholate cotransporting polypeptide, an HBV receptor, and then selected a cell line highly permissive for HBV infection, G2/NT18-B. After transduction with a lentivirus-encoding microRNA-122, the cell line harboring the highest level of replicon RNA was selected and then treated with anti-HCV compounds to eliminate the replicon RNA. The resulting cured cell line was transduced with a plasmid-encoding CD81. The cell line permissive for HCV infection was cloned and then designated the G2BC-C2 cell line, which exhibited permissiveness for HBV and HCV propagation. JAK inhibitor I potentiated the HCV superinfection of HBV-infected cells, and fluorescence-activated cell-sorting analysis indicated that HBV/HCV double-positive cells accounted for approximately 30% of the coinfected cells. Among several host genes tested, cyclooxygenase-2 showed synergistic induction by coinfection compared with each monoinfection. These data indicate that our HBV/HCV coinfection system provides an easy-to-use platform for the study of host and viral responses against coinfection and the development of antiviral agents targeting HBV and HCV.
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http://dx.doi.org/10.1002/hep4.1653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034569PMC
April 2021

Serum fatty acid-binding protein 5 is a significant factor in hepatocellular carcinoma progression independent of tissue expression level.

Carcinogenesis 2021 Jun;42(6):794-803

Department of Gastroenterological Surgery I, Graduate School of Medicine, Hokkaido University, Sapporo, Japan.

Fatty acid-binding protein 5 (FABP5) is highly expressed in hepatocellular carcinoma (HCC) tissues and is related to HCC progression. In this study, we analyzed the potential of serum FABP5 (sFABP5) as a tumor marker in HCC and its clinical significance in HCC progression. We compared the sFABP5 concentration in patients with HCC (HCC group) with that of patients with hepatitis without HCC (hepatitis group). Moreover, we measured the FABP5 expression levels in resected HCC tissues (tFABP5) and analyzed their relationship with sFABP5. We also performed cell-based assays using FABP5 knockout and overexpressing HCC cell lines to analyze the effect of extrinsic FABP5 (exFABP5) on HCC cells. We showed that sFABP5 was not a useful tumor marker for HCC, as HCC and sFABP5 were not correlated. However, sFABP5 and tFABP5 significantly correlated with survival after surgery for HCC, while sFABP5 and tFABP5 were independent of each other. In cell-based assays, exFABP5 was taken up by HCC cell lines and positively affected cell survival under glucose-depleted conditions by complementing the endogenous FABP5 function. In conclusion, sFABP5 had a significant impact on HCC progression irrespective of tFABP5 by augmenting cell viability under glucose-depleted conditions. As tFABP5 and sFABP5 are important factors that are independent of each other in HCC progression, both of them should be considered independently in improving the prognosis of patients with HCC.
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http://dx.doi.org/10.1093/carcin/bgab025DOI Listing
June 2021

Mesothelin blockage by Amatuximab suppresses cell invasiveness, enhances gemcitabine sensitivity and regulates cancer cell stemness in mesothelin-positive pancreatic cancer cells.

BMC Cancer 2021 Feb 26;21(1):200. Epub 2021 Feb 26.

Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-Ku, Sapporo, Hokkaido, 060-8638, Japan.

Background: Mesothelin is a 40-kDa glycoprotein that is highly overexpressed in various types of cancers, however molecular mechanism of mesothelin has not been well-known. Amatuximab is a chimeric monoclonal IgG1/k antibody targeting mesothelin. We recently demonstrated that the combine therapy of Amatuximab and gemcitabine was effective for peritonitis of pancreatic cancer in mouse model.

Methods: We discover the role and potential mechanism of mesothelin blockage by Amatuximab in human pancreatic cells both expressing high or low level of mesothelin in vitro experiment and peritonitis mouse model of pancreatic cancer.

Results: Mesothelin blockage by Amatuximab lead to suppression of invasiveness and migration capacity in AsPC-1 and Capan-2 (high mesothelin expression) and reduce levels of pMET expression. The combination of Amatuximab and gemcitabine suppressed proliferation of AsPC-1 and Capan-2 more strongly than gemcitabine alone. These phenomena were not observed in Panc-1 and MIA Paca-2 (Mesothelin low expression). We previously demonstrated that Amatuximab reduced the peritoneal mass in mouse AsPC-1 peritonitis model and induced sherbet-like cancer cell aggregates, which were vanished by gemcitabine. In this study, we showed that the cancer stem cell related molecule such as ALDH1, CD44, c-MET, as well as proliferation related molecules, were suppressed in sherbet-like aggregates, but once sherbet-like aggregates attached to peritoneum, they expressed these molecules strongly without the morphological changes.

Conclusions: Our work suggested that Amatuximab inhibits the adhesion of cancer cells to peritoneum and suppresses the stemness and viability of those, that lead to enhance the sensitivity for gemcitabine.
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http://dx.doi.org/10.1186/s12885-020-07722-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912898PMC
February 2021

Analysis of the correlation between alterations in N‑glycans and invasiveness in liver cancer cell lines.

Oncol Rep 2020 Dec 8;44(6):2757-2769. Epub 2020 Oct 8.

Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060‑8638, Japan.

The N‑glycoforms of glycoproteins modify protein function and control a number of biological pathways. The aim of the present study was to investigate the correlation between alterations in N‑glycans and cancer aggressiveness in terms of cancer cell invasion ability. The expression of urokinase‑type plasminogen activator (uPA) and N‑acetylglucosaminyltransferase V (GnT‑V) in liver cancer cell lines was analyzed by western blotting. Cell invasiveness was analyzed by Matrigel invasion assays. uPA and GnT‑V expression in liver cancer cell lines was knocked down by RNA interference. Furthermore, uPA was overexpressed in liver cancer cells using lentiviral vectors, and a mutant strain of HepG2 cells overexpressing uPA deficient in N‑glycans was established. A glycoblotting‑assisted matrix‑assisted laser desorption/ionization‑time‑of‑flight/mass spectrometry‑based quantitative analysis of liver cancer cell lines was performed, in which invasiveness was altered by modifying the expression of uPA and GnT‑V. N‑glycan profiles were found to differ between the highly invasive liver cancer cell line HLE and the less invasive cell line HepG2. The expression of several N‑glycans, including a form with m/z=1892, was changed according to invasiveness controlled by knockdown and overexpression of uPA. The invasiveness of HepG2 cells with mutant uPA did not increase regardless of the level of expression of uPA. Following GnT‑V knockdown and N‑glycan alteration, uPA expression did not change, whereas cell invasiveness decreased. One N‑glycan (m/z=1892) was common among N‑glycans in the comparative analysis between HLE and HepG2, HLE and uPA knockdown HLE, HepG2 and uPA‑overexpressing HepG2, and HLE and GnT‑V knockdown HLE cells and among N‑glycan profiles in human uPA. Therefore, N‑glycosylation is an important factor controlling invasiveness of liver cancer cells, and a specific N‑glycan (m/z=1892) associated with the invasion of liver cancer cells via uPA was identified in the present study.
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http://dx.doi.org/10.3892/or.2020.7795DOI Listing
December 2020

Triazolopyrimidine derivative NK026680 and donor-specific transfusion induces CD4CD25Foxp3 T cells and ameliorates allograft rejection in an antigen-specific manner.

Transpl Immunol 2021 04 3;65:101338. Epub 2020 Oct 3.

Department of Transplant Surgery, Hokkaido University Graduate School of Medicine, Hokkaido University, Sapporo, Japan. Electronic address:

We have previously demonstrated the unique properties of a new triazolopyrimidine derivative, NK026680, which exerts immunosuppressive effects in rat heart transplant model and confers tolerogeneic properties on ex vivo-conditioned dendritic cells in mice. We herein demonstrate that NK026680 promotes the expansion of regulatory T cells (Tregs) with potent immunoregulatory effects when used in combination with donor-specific transfusion (DST). BALB/c (H-2) heart graft were transplanted into C57BL/6 (H-2) mice following intravenous injection of donor splenocytes (DST) and oral administration of NK026680. The NK026680 plus DST treatment markedly prolonged the survival time of the donor-graft, but not that of the 3rd party-graft (C3H; H-2). Treg cells in the recipient spleen on day 0 expanded when stimulated with donor-antigens in vivo and in vitro. After heart transplantation, Treg cells accumulated into the graft and increased in the spleen. NK026680 plus DST also decreased activated CD8 T cells in the spleen and inhibited infiltration of CD8 T cells into the graft. Depletion of CD25 cells inhibited the graft prolonging effect of the NK026680 plus DST treatment. NK026680 administration together with DST induces potent immunoregulatory effects in an antigen-specific manner, likely due to the in vivo generation of donor-specific Tregs.
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http://dx.doi.org/10.1016/j.trim.2020.101338DOI Listing
April 2021

Role of Wnt5a in suppressing invasiveness of hepatocellular carcinoma via epithelial-mesenchymal transition.

Oncol Lett 2020 Nov 21;20(5):268. Epub 2020 Sep 21.

Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638, Japan.

Inappropriate activation of the canonical Wnt signaling pathway is associated with progression of hepatocellular carcinoma (HCC). However, the association between the non-canonical pathway activated by Wnt5a and HCC is not well known. The present study investigated the significance of Wnt5a expression in HCC. Immunohistochemical staining of Wnt5a was performed on specimens from 243 patients who underwent hepatic resection for HCC. The present study investigated whether Wnt5a expression was associated with clinical and pathological factors and prognosis. Wnt5a expression in human HCC cell lines was investigated using western blotting. The effects of overexpression or knockdown of Wnt5a were evaluated using proliferation and invasion assays. Changes in epithelial-mesenchymal transition (EMT)-related molecules were investigated using western blotting. Wnt5a negativity was significantly associated with poor tumor differentiation and positive vascular invasion. In univariate analysis, Wnt5a negativity was identified as a significant prognostic factor for overall survival (OS). Multivariate analysis of OS demonstrated that Wnt5a negativity was an independent prognostic factor. Wnt5a expression was lower in HLE and HLF cells than in HepG2 and Huh7 cells. Knockdown of Wnt5a by short hairpin RNA transfection increased the proliferation and invasiveness of Huh7 cells, and decreased the expression levels of E-cadherin. In HLF cells, overexpression of Wnt5a inhibited invasiveness and decreased the expression levels of vimentin. Wnt5a negativity was associated with poor tumor differentiation and positive vascular invasion, and was an independent poor prognostic factor in patients with HCC. Wnt5a may be a tumor suppressor involved in EMT-mediated changes in invasiveness.
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http://dx.doi.org/10.3892/ol.2020.12131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7517569PMC
November 2020

Adenomatous polyposis coli-binding protein end-binding 1 promotes hepatocellular carcinoma growth and metastasis.

PLoS One 2020 21;15(9):e0239462. Epub 2020 Sep 21.

Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.

This study was performed to determine the clinical significance of adenomatous polyposis coli (APC)-binding protein end-binding 1 (EB1) in hepatocellular carcinoma (HCC) and to characterize its biochemical role in comparison with previous reports. We performed immunohistochemical staining to detect EB1 expression in tissues from 235 patients with HCC and investigated its correlations with clinicopathological features and prognosis. We also investigated the roles of EB1 in cell proliferation, migration, and tumorigenesis in vitro and in vivo by siRNA- and CRISPR/Cas9-mediated modulation of EB1 expression in human HCC cell lines. The results showed that EB1 expression was significantly correlated with several important factors associated with tumor malignancy, including histological differentiation, portal vein invasion status, and intrahepatic metastasis. Patients with high EB1 expression in HCC tissue had poorer overall survival and higher recurrence rates than patients with low EB1 expression. EB1 knockdown and knockout in HCC cells reduced cell proliferation, migration, and invasion in vitro and inhibited tumor growth in vivo. Further, genes encoding Dlk1, HAMP, and SLCO1B3 that were differentially expressed in association with EB1 were identified using RNA microarray analysis. In conclusion, elevated expression of EB1 promotes tumor growth and metastasis of HCC. EB1 may serve as a new biomarker for HCC, and genes coexpressed with EB1 may represent potential targets for therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239462PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505586PMC
November 2020

Prognostic Significance of Circulating Tumor Cells with Mesenchymal Phenotypes in Patients with Gastric Cancer: A Prospective Study.

Ann Surg Oncol 2021 Feb 7;28(2):1178-1186. Epub 2020 Aug 7.

Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.

Background: Circulating tumor cells (CTCs) have been shown to be heterogeneous. Focusing on the epithelial-mesenchymal transition and perioperative kinetics, we evaluated CTCs with mesenchymal phenotypes as a potential prognostic biomarker for patients with gastric cancer.

Methods: Peripheral blood was collected from 54 patients with gastric cancer before surgery and at 1 week and 1 month after surgery. CTCs were enriched using density-gradient centrifugation and magnetic-activated cell sorting (negative selection). Cell suspensions were characterized by multi-immunofluorescence staining against cytokeratin and N-cadherin, and by 4',6'-diamidino-2-phenyldole staining.

Results: CTCs were detected in five patients (17%) with early cancer and 14 patients (56%) with advanced cancer (p < 0.05). In our system, N-cadherin, but not cytokeratin, was expressed in the CTCs of 90% (19/21) of patients. Postoperative recurrence was detected in 10 patients, all of whom had N-cadherin+/cytokeratin-/CD45- CTCs preoperatively. Regarding perioperative kinetics, we divided patients into three risk groups: a high-risk group, with one or more preoperative CTCs and increased CTCs postoperatively; an intermediate-risk group, with one or more preoperative CTCs and decreased CTCs postoperatively; and a low-risk group, with no preoperative CTCs. Recurrence rates were 57% (4/7), 33% (4/12), and 6% (2/35), respectively. The relapse-free survival rate was lower in patients at high risk versus those at intermediate or low risk, for all patients (p = 0.00024) and in patients with advanced cancer (p = 0.00103).

Conclusions: N-cadherin is a highly useful marker to detect CTCs lacking cytokeratin, and the perioperative kinetics of CTC numbers is beneficial in risk stratification for survival in patients with gastric cancer.
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http://dx.doi.org/10.1245/s10434-020-08827-6DOI Listing
February 2021

Imaging Mass Spectrometry Reveals the Changes in the Taurine Conjugates of Dihydroxycholanoic Acid During Hepatic Warm Ischemia and Reperfusion in a Rat Model.

Transplant Proc 2020 Jul - Aug;52(6):1880-1883. Epub 2020 Jun 30.

Department of Gastroenterological Surgery I, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.

Warm ischemia and reperfusion injury (IRI) is a prognostic factor in donation after cardiac death donor transplantation. However, a reliable method to predict IRI before transplantation has not been established. The aim of this study was to identify predictive markers of hepatic IRI by simultaneous measurement of endogenous molecules using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Rats were subjected to hepatic warm ischemia (70%) for 30 or 90 minutes and subsequent reperfusion. The livers were collected at the end of ischemia and 1 hour, 6 hours, and 24 hours after reperfusion. The liver tissue sections were applied to IMS (m/z 200-2000). Candidate molecules were identified by tandem mass spectrometry. Imaging mass spectrometry (IMS) revealed a significant increase in the taurine conjugates of dihydroxycholanoic acid (TDHCA) during ischemia and a tendency to return to the basal level after reperfusion. Notably, high-resolution measurements revealed focal accumulation of TDHCA in the intrahepatic bile duct with ischemic time. In conclusion, IMS is a useful method to detect minute changes provoked by ischemia, which are barely detectable in assays involving homogenization. Accordingly, focal accumulation of TDHCA during ischemia may be a candidate marker for predicting later IRI.
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http://dx.doi.org/10.1016/j.transproceed.2020.01.169DOI Listing
November 2020

Novel immunological approach to asses donor reactivity of transplant recipients using a humanized mouse model.

Hum Immunol 2020 Jul 25;81(7):342-353. Epub 2020 Apr 25.

Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo 060-8648, Japan. Electronic address:

In organ transplantation, a reproducible and robust immune-monitoring assay has not been established to determine individually tailored immunosuppressants (IS). We applied humanized mice reconstituted with human (hu-) peripheral blood mononuclear cells (PBMCs) obtained from living donor liver transplant recipients to evaluate their immune status. Engraftment of 2.5 × 10 hu-PBMCs from healthy volunteers and recipients in the NSG mice was achieved successfully. The reconstituted lymphocytes consisted mainly of hu-CD3 lymphocytes with predominant CD45RACD62L T and CCR6CXCR3CD4 Th1 cells in hu-PBMC-NSG mice. Interestingly, T cell allo-reactivity of hu-PBMC-NSG mice was amplified significantly compared with that of freshly isolated PBMCs (p < 0.05). Furthermore, magnified hu-T cell responses to donor antigens (Ag) were observed in 2/10 immunosuppressed recipients with multiple acute rejection (AR) experiences, suggesting that the immunological assay in hu-PBMC-NSG mice revealed hidden risks of allograft rejection by IS. Furthermore, donor Ag-specific hyporesponsiveness was maintained in recipients who had been completely weaned off IS (n = 4), despite homeostatic proliferation of hu-T cells in the hu-PBMC-NSG mice. The immunological assay in humanized mice provides a new tool to assess recipient immunity in the absence of IS and explore the underlying mechanisms to maintaining operational tolerance.
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http://dx.doi.org/10.1016/j.humimm.2020.04.007DOI Listing
July 2020

Prognostic impact of CD8+ T cell distribution and its association with the HLA class I expression in intrahepatic cholangiocarcinoma.

Surg Today 2020 Aug 10;50(8):931-940. Epub 2020 Feb 10.

Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Kita-ku, Kita 15, Nishi 7, Sapporo, Hokkaido, 060-8638, Japan.

Purpose: A lack of effective systemic therapy is one reason for the poor prognosis of intrahepatic cholangiocarcinoma. Newly developed immune checkpoint inhibitors function by minimizing CD8+ T cell suppression to improve tumor-specific responses. This study aimed to examine the characteristics of CD8+ T cells in intrahepatic cholangiocarcinoma.

Methods: Clinicopathological data, including the overall survival, of 69 cases of postoperative intrahepatic cholangiocarcinoma were prospectively investigated. We then immunohistochemically stained for CD8, Foxp3, CD163, PD-L1, and human leukocyte antigen (HLA) class I and counted the number of CD8+ T cells, Foxp3+ T cells, and CD163+ macrophages in different areas (outer border, interborder, and intratumor).

Results: A significant difference was found in the 5-year overall survival between the CD8+ T cell high group (45.5%) and low group (24.7%) in the outer border area (p = 0.0103). Furthermore, the number of CD8+ T cells and the high expression of HLA class I were positively correlated (p = 0.0341).

Conclusion: The number of CD8+ T cells in the outer border area of the tumor correlated with the HLA class I expression of intrahepatic cholangiocarcinoma and may therefore be a prognostic factor for patients with postoperative intrahepatic cholangiocarcinoma.
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http://dx.doi.org/10.1007/s00595-020-01967-yDOI Listing
August 2020

Heavy Water (DO) Containing Preservation Solution Reduces Hepatic Cold Preservation and Reperfusion Injury in an Isolated Perfused Rat Liver (IPRL) Model.

J Clin Med 2019 Nov 1;8(11). Epub 2019 Nov 1.

Departments of Gastroenterological Surgery I; Hokkaido University Graduate School of Medicine; Kita15-Nishi7, Kita-Ku, Sapporo, Hokkaido 060-8638, Japan.

Background: Heavy water (DO) has many biological effects due to the isotope effect of deuterium. We previously reported the efficacy of DO containing solution (Dsol) in the cold preservation of rat hearts. Here, we evaluated whether Dsol reduced hepatic cold preservation and reperfusion injury.

Methods: Rat livers were subjected to 48-hour cold storage in University of Wisconsin (UW) solution or Dsol, and subsequently reperfused on an isolated perfused rat liver. Graft function, injury, perfusion kinetics, oxidative stress, and cytoskeletal integrity were assessed.

Results: In the UW group, severe ischemia and reperfusion injury (IRI) was shown by histopathology, higher liver enzymes leakage, portal resistance, and apoptotic index, oxygen consumption, less bile production, energy charge, and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio (versus control). The Dsol group showed that these injuries were significantly ameliorated (versus the UW group). Furthermore, cytoskeletal derangement was progressed in the UW group, as shown by less degradation of α-Fodrin and by the inactivation of the actin depolymerization pathway, whereas these changes were significantly suppressed in the Dsol group.

Conclusion: Dsol reduced hepatic IRI after extended cold preservation and subsequent reperfusion. The protection was primarily due to the maintenance of mitochondrial function, cytoskeletal integrity, leading to limiting oxidative stress, apoptosis, and necrosis pathways.
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http://dx.doi.org/10.3390/jcm8111818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912838PMC
November 2019

Serum milk fat globule-EGF factor 8 (MFG-E8) as a diagnostic and prognostic biomarker in patients with hepatocellular carcinoma.

Sci Rep 2019 10 31;9(1):15788. Epub 2019 Oct 31.

Department of Liver Disease, The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan.

Current serum hepatocellular carcinoma (HCC) biomarkers are insufficient for early diagnosis. We aimed to clarify whether serum MFG-E8 can serve as a diagnostic or prognostic biomarker of HCC. Serum MFG-E8 levels of 282 HCC patients, who underwent primary hepatectomy, were examined by ELISA. We also quantified serum MFG-E8 levels in patients with chronic hepatitis (CH), liver cirrhosis (LC), as well as in healthy volunteers (HVs). Serum MFG-E8 levels were significantly lower in HCC patients than in HVs regardless of the etiology of liver disease (3.6 ± 0.1 vs 5.8 ± 0.2 ng/mL, p < 0.0001), and recovered after treatment of HCC. Serum MFG-E8 levels in CH and LC patients were comparable to those in HVs. Serum MFG-E8 could detect HCCs, even α-fetoprotein (AFP)-negative or des-γ-carboxy prothrombin (DCP)-negative HCCs, in CH and LC patients. Our new HCC prediction model using MFG-E8 and DCP (Logit(p) = 2.619 - 0.809 × serum MFG-E8 + 0.0226 × serum DCP) distinguished HCC patients from CH and LC patients with an area under the curve of 0.923, a sensitivity of 81.1%, and a specificity of 89.8%. Futhermore, low preoperative serum MFG-E8 was an independent predictor of poor overall survival. Thus, serum MFG-E8 could serve as a feasible diagnostic and prognostic biomarker for HCC.
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http://dx.doi.org/10.1038/s41598-019-52356-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823494PMC
October 2019

USP15 Participates in Hepatitis C Virus Propagation through Regulation of Viral RNA Translation and Lipid Droplet Formation.

J Virol 2019 03 5;93(6). Epub 2019 Mar 5.

Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses and We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs. Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.
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http://dx.doi.org/10.1128/JVI.01708-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401473PMC
March 2019

Post-reperfusion hydrogen gas treatment ameliorates ischemia reperfusion injury in rat livers from donors after cardiac death: a preliminary study.

Surg Today 2018 Dec 6;48(12):1081-1088. Epub 2018 Jul 6.

Department of Gastroenterological Surgery I, Graduate School of Medicine, Hokkaido University, N-15, W-7, Kita-Ku, Sapporo, 060-8638, Japan.

Background And Purpose: We reported previously that hydrogen gas (H) reduced hepatic ischemia and reperfusion injury (IRI) after prolonged cold storage (CS) of livers retrieved from heart-beating donors. The present study was designed to assess whether H reduced hepatic IRI during donation of a cardiac death (DCD) graft with subsequent CS.

Methods: Rat livers were harvested after 30-min cardiac arrest and stored for 4 h in University of Wisconsin solution. The graft was reperfused with oxygenated buffer, with or without H (H or NT groups, respectively), at 37° for 90 min on isolated perfused rat liver apparatus.

Results: In the NT group, liver enzyme leakage, apoptosis, necrosis, energy depletion, redox status, impaired microcirculation, and bile production were indicative of severe IRI, whereas in the H group these impairments were significantly suppressed. The phosphorylation of cytoplasmic MKK4 and JNK were enhanced in the NT group and suppressed in the H group. NFkB-p65 and c-Fos in the nucleus were unexpectedly unchanged by IRI regardless of H treatment, indicating the absence of inflammation in this model.

Conclusion: H was observed to ameliorate IRI in the DCD liver by maintaining microcirculation, mitochondrial functions, and redox status, as well as suppressing the cytoplasmic MKK4-JNK-mediated cellular death pathway.
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http://dx.doi.org/10.1007/s00595-018-1693-0DOI Listing
December 2018

Genome-wide association study identified new susceptible genetic variants in HLA class I region for hepatitis B virus-related hepatocellular carcinoma.

Sci Rep 2018 05 21;8(1):7958. Epub 2018 May 21.

Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.

We have performed a genome-wide association study (GWAS) including 473 Japanese HBV (hepatitis B virus)-positive HCC (hepatocellular carcinoma) patients and 516 HBV carriers including chronic hepatitis and asymptomatic carrier individuals to identify new host genetic factors associated with HBV-derived HCC in Japanese and other East Asian populations. We identified 65 SNPs with P values < 10 located within the HLA class I region and three SNPs were genotyped in three independent population-based replication sets. Meta-analysis confirmed the association of the three SNPs (rs2523961: OR = 1.73, P = 7.50 × 10; rs1110446: OR = 1.79, P = 1.66 × 10; and rs3094137: OR = 1.73, P = 7.09 × 10). We then performed two-field HLA genotype imputation for six HLA loci using genotyping data to investigate the association between HLA alleles and HCC. HLA allele association testing revealed that HLA-A 33:03 (OR = 1.97, P = 4.58 × 10) was significantly associated with disease progression to HCC. Conditioning analysis of each of the three SNPs on the HLA class I region abolished the association of HLA-A33:03 with disease progression to HCC. However, conditioning the HLA allele could not eliminate the association of the three SNPs, suggesting that additional genetic factors may exist in the HLA class I region.
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http://dx.doi.org/10.1038/s41598-018-26217-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962604PMC
May 2018

Copy number profiles of paired primary and metastatic colorectal cancers.

Oncotarget 2018 Jan 15;9(3):3394-3405. Epub 2017 Dec 15.

Conjoint Gastroenterology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia.

Liver metastasis is the major cause of death following a diagnosis of colorectal cancer (CRC). In this study, we compared the copy number profiles of paired primary and liver metastatic CRC to better understand how the genomic structure of primary CRC differs from the metastasis. Paired primary and metastatic tumors from 16 patients and their adjacent normal tissue samples were analyzed using single nucleotide polymorphism arrays. Genome-wide chromosomal copy number alterations were assessed, with particular attention to 188 genes known to be somatically altered in CRC and 24 genes that are clinically actionable in CRC. These data were analyzed with respect to the timing of primary and metastatic tissue resection and with exposure to chemotherapy. The genomic differences between the tumor and paired metastases revealed an average copy number discordance of 22.0%. The pairs of tumor samples collected prior to treatment revealed significantly higher copy number differences compared to post-therapy liver metastases ( = 0.014). Loss of heterozygosity acquired in liver metastases was significantly higher in previously treated liver metastasis samples compared to treatment naive liver metastasis samples ( = 0.003). Amplification of the clinically actionable genes , , or was observed in the metastatic tissue of 4 patients but not in the paired primary CRC. These examples highlight the intra-patient genomic discrepancies that can occur between metastases and the primary tumors from which they arose. We propose that precision medicine strategies may therefore identify different actionable targets in metastatic tissue, compared to primary tumors, due to substantial genomic differences.
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http://dx.doi.org/10.18632/oncotarget.23277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790471PMC
January 2018

Pro-angiogenic TIE-2-expressing monocytes/TEMs as a biomarker of the effect of sorafenib in patients with advanced hepatocellular carcinoma.

Int J Cancer 2017 09 8;141(5):1011-1017. Epub 2017 Jun 8.

Department of Liver Disease, The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan.

Sorafenib, a multi-kinase inhibitor, inhibits tumor angiogenesis and is the first-line systemic therapy for patients with advanced hepatocellular carcinoma (HCC). However, due to its limited effects and frequent occurrence of side effects, biomarkers are needed to predict the effects of sorafenib. We considered the possibility of using TIE-2-expressing monocytes (TEMs) to predict the response in sorafenib-treated patients with advanced HCC. TEMs serve as a diagnostic marker of HCC and are related to angiogenesis. We analyzed 25 advanced HCC patients and prospectively evaluated TEMs before (Pre TEMs) and at 1 month after initial therapy (T1m TEMs). The radiologic response was evaluated by modified Response Evaluation Criteria in Solid Tumors (mRECIST). Median survival time (MST) was significantly longer in the partial response/stable disease (PR/SD) group (21.8 months) than in the PD group (8.7 months). ΔTEMs (changes of T1m TEMs compared to Pre TEMs) were significantly lower in the PR/SD group than in the PD group. MST of the ΔTEMs low group (14.2 months) was significantly longer than that of the high group (8.7 months). Univariate and multivariate Cox regression analyses showed that ΔTEMs [hazard ratio (HR) = 8.53, 95% confidence interval (CI) = 1.51-48.16, p = 0.015] and Child-Pugh class (HR = 5.59, 95% CI = 1.06-29.63, p = 0.043) were independently associated with overall survival. Our results suggest that ΔTEMs could serve as a biomarker for predicting radiologic response and overall survival in sorafenib-treated patients with advanced HCC.
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http://dx.doi.org/10.1002/ijc.30804DOI Listing
September 2017

Fatty acid-binding protein 5 function in hepatocellular carcinoma through induction of epithelial-mesenchymal transition.

Cancer Med 2017 May 4;6(5):1049-1061. Epub 2017 Apr 4.

Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

Hepatocellular carcinoma (HCC) is a highly prevalent cancer with poor prognosis. The correlation between overexpression of fatty acid-binding protein 5 (FABP5) and malignant potential of tumor growth and metastasis in several cancers has been previously reported. However, the correlation between FABP5 expression and HCC malignant behavior remains unknown. We compared FABP5 expression and patient characteristics in paired HCC and adjacent noncancerous liver tissues from 243 patients who underwent surgical resection of primary HCC. Cell proliferation, invasion, and migration assays were performed in HCC cell lines overexpressing FABP5 or downregulated for FABP5. Tumor growths were monitored in xenograft model, and liver and lung metastasis models were established. In the 243 HCC patients, FABP5-positive staining (n = 139/243, 57.2%) was associated with poor prognosis and recurrence (P < 0.0001) and showed positive correlation with distant metastasis, tumor size and vascular invasion (P < 0.05). Cell proliferation, invasion, and migration in vitro were enhanced by upregulation of FABP5 and decreased by downregulation of FABP5 in HCC cell lines. Similar results in tumor formation and metastasis were obtained through in vivo analyses. PCR array results revealed upregulation of SNAI1 in FABP5-overexpressing HepG2 cells. Western blot analysis showed significantly increased expression of E-cadherin and ZO-1 and decreased SNAI1 expression and nuclear translocation of β-catenin by knockdown of FABP5. We revealed a significant role for FABP5 in HCC progression and metastasis through the induction of epithelial-to-mesenchymal transition. FABP5 may be a potential novel prognostic biomarker and new therapeutic target for HCC.
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http://dx.doi.org/10.1002/cam4.1020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430096PMC
May 2017

Interleukin-34 as a fibroblast-derived marker of liver fibrosis in patients with non-alcoholic fatty liver disease.

Sci Rep 2016 07 1;6:28814. Epub 2016 Jul 1.

The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba, Japan.

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic non-viral liver disease. Activation of macrophages and hepatic stellate cells is a critical step that promotes liver fibrosis. We aimed to explore the feasibility of interleukin-34 (IL-34), a key regulator of macrophages, as a fibrosis marker in patients with NAFLD. We enrolled 197 liver biopsy-proven NAFLD patients. We evaluated the serum levels of IL-34, macrophage-colony stimulating factor (M-CSF), soluble CD163 (sCD163), 40 cytokines/chemokines, hyaluronic acid, type IV collagen 7s, and clinically-approved fibrosis scores. IL-34 increased with the progression of fibrosis and was an independent marker for liver fibrosis. Immunostaining experiments, using resected liver specimens from NAFLD patients, revealed that IL-34 was mainly expressed on liver fibroblasts. IL-34 based fibrosis score (0.0387*IL-34 (pg/ml) + 0.3623*type IV collagen 7s (ng/ml) + 0.0184*age (year)-1.1850) was a practical predictive model of liver fibrosis. Using receiver-operating characteristic analyses, the area under the curve, sensitivity, and specificity of IL-34 based fibrosis score were superior or comparable to the other fibrosis biomarkers and scores. In conclusion, the IL-34 based fibrosis score, including serum IL-34, type IV collagen 7s and age, is a feasible diagnostic marker of liver fibrosis in NAFLD patients.
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http://dx.doi.org/10.1038/srep28814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929441PMC
July 2016

Hydrogen Gas Ameliorates Hepatic Reperfusion Injury After Prolonged Cold Preservation in Isolated Perfused Rat Liver.

Artif Organs 2016 Dec 2;40(12):1128-1136. Epub 2016 May 2.

Department of Gastroenterological Surgery I.

Hydrogen gas reduces ischemia and reperfusion injury (IRI) in the liver and other organs. However, the precise mechanism remains elusive. We investigated whether hydrogen gas ameliorated hepatic I/R injury after cold preservation. Rat liver was subjected to 48-h cold storage in University of Wisconsin solution. The graft was reperfused with oxygenated buffer with or without hydrogen at 37° for 90 min on an isolated perfusion apparatus, comprising the H (+) and H (-) groups, respectively. In the control group (CT), grafts were reperfused immediately without preservation. Graft function, injury, and circulatory status were assessed throughout the perfusion. Tissue samples at the end of perfusion were collected to determine histopathology, oxidative stress, and apoptosis. In the H (-) group, IRI was indicated by a higher aspartate aminotransferase (AST), alanine aminotransferase (ALT) leakage, portal resistance, 8-hydroxy-2-deoxyguanosine-positive cell rate, apoptotic index, and endothelial endothelin-1 expression, together with reduced bile production, oxygen consumption, and GSH/GSSG ratio (vs. CT). In the H (+) group, these harmful changes were significantly suppressed [vs. H (-)]. Hydrogen gas reduced hepatic reperfusion injury after prolonged cold preservation via the maintenance of portal flow, by protecting mitochondrial function during the early phase of reperfusion, and via the suppression of oxidative stress and inflammatory cascades thereafter.
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http://dx.doi.org/10.1111/aor.12710DOI Listing
December 2016

Novel anti-inflammatory agent 3-[(dodecylthiocarbonyl)-methyl]-glutarimide ameliorates murine models of inflammatory bowel disease.

Inflamm Res 2016 Mar 18;65(3):245-60. Epub 2015 Dec 18.

Department of Gastroenterological Surgery I, Graduate School of Medicine, Hokkaido University, N-15, W-7, Kita-ku, Sapporo, 060-8638, Japan.

Objective And Design: To examine the effect of 3-[(dodecylthiocarbonyl)-methyl]-glutarimide (DTCM-G), a novel anti-inflammatory agent that inhibits lipopolysaccharide (LPS) activation of RAW264.7 macrophages, on murine models of colitis and RAW264.7 cells.

Materials And Methods: Colitis was induced by rectally infusing trinitrobenzenesulfonic acid (TNBS) (1.5 mg in 50% ethanol) in BALB/c mice or orally administering 3% dextran sulfate sodium (DSS) for 5 days in C57BL/6 mice. The severity of colitis was assessed after intraperitoneally injecting DTCM-G (40 mg/kg). The anti-inflammatory properties of DTCM-G and its mechanisms were investigated in LPS-stimulated RAW264.7 cells.

Results: DTCM-G significantly ameliorated TNBS-induced colitis, according to the body weight loss, disease activity index, colonic obstruction, macroscopic colonic inflammation score, mucosal myeloperoxidase activity, and histopathology. Immunohistochemistry and isolated lamina propria mononuclear cells showed significantly reduced colonic F4/80(+) and CD11b(+) macrophage infiltration. DTCM-G significantly suppressed tumor necrosis factor (TNF)-α and interleukin (IL)-6 messenger RNA expression in the colon and attenuated DSS-induced colitis, according to the disease activity index and histopathology. In RAW264.7 cells, DTCM-G suppressed LPS-induced TNF-α/IL-6 production and enhanced glycogen synthase kinase-3β phosphorylation.

Conclusions: DTCM-G attenuated murine experimental colitis by inhibiting macrophage infiltration and inflammatory cytokine expression. Thus, DTCM-G may be a promising treatment for inflammatory bowel disease.
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http://dx.doi.org/10.1007/s00011-015-0911-0DOI Listing
March 2016

Strong cytoplasmic expression of NF-κB/p65 correlates with a good prognosis in patients with triple-negative breast cancer.

Surg Today 2016 Jul 22;46(7):843-51. Epub 2015 Oct 22.

Breast Surgery, Hokkaido University Hospital, Kita 14 Nishi 5, Kita-ku, Sapporo, 060-8648, Japan.

Purpose: Recent studies have indicated that constitutive NF-κB activity could be involved in the proliferation of triple-negative breast cancer.

Methods: The NF-κB/p65 expression and the effects of a NF-κB inhibitor, (-)-DHMEQ, were examined in triple-negative MDA-MB-231 breast cancer cells. Women with triple-negative breast cancer treated with neoadjuvant chemotherapy between 2002 and 2012 were retrospectively analyzed for their expression of NF-κB/p65, Bcl2 and Ki67 by immunohistochemistry in pre- and post-treatment specimens. The factors predicting the response to neoadjuvant chemotherapy and the prognosis were analyzed.

Results: NF-κB/p65 was predominantly expressed in the cytoplasm of MDA-MB-231 cells. Of 34 triple-negative breast cancer patients, positive staining for NF-κB/p65 expression was detected in the nuclei of a few cells in seven tumors before neoadjuvant chemotherapy, while the expression of NF-κB/p65 in the cytoplasm was detected in almost all tumor cells of 33 tumors. The expression levels of NF-κB/p65 were not associated with the response to neoadjuvant chemotherapy, although the cytoplasmic NF-κB/p65 staining intensity was significantly decreased in the post-treatment tumor samples compared with the pretreatment samples. All patients whose tumors showed strong cytoplasmic NF-κB/p65 expression before neoadjuvant chemotherapy are currently disease free.

Conclusion: Our results suggest that strong cytoplasmic NF-κB/p65 expression could be a prognostic marker for patients with triple-negative breast cancer.
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http://dx.doi.org/10.1007/s00595-015-1265-5DOI Listing
July 2016

Human Amnion-Derived Mesenchymal Stem Cell Transplantation Ameliorates Liver Fibrosis in Rats.

Transplant Direct 2015 May 26;1(4):e16. Epub 2015 May 26.

Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

Unlabelled: Mesenchymal stem cells (MSCs) are a valuable cell source in regenerative medicine. Recently, several studies have shown that MSCs can be easily isolated from human amnion. In this study, we investigated the therapeutic effect of transplantation of human amnion-derived MSCs (hAMSCs) in rats with liver fibrosis.

Methods: Liver fibrosis was induced by an intraperitoneal injection of 2 mL/kg of 50% carbon tetrachloride twice a week for 6 weeks. At 3 weeks, hAMSCs (1 × 10(6) cells) were transplanted intravenously. Rats were sacrificed at 7 weeks, and histological analyses and quantitative reverse-transcription polymerase chain reaction were performed. In vitro experiments were conducted to investigate the effect of hAMSCs on the activation of Kupffer cells.

Results: Transplantation of hAMSCs significantly reduced the fibrotic area, deposition of type-I collagen, the number of α-smooth muscle actin-positive hepatic stellate cells, and CD68-positive Kupffer cells in the livers. messenger RNA expression of α-smooth muscle actin and tissue inhibitor of metalloproteinase-1 was significantly decreased and the expression of matrix metalloproteinase-9 and hepatocyte growth factor was significantly increased in the liver of hAMSC-treated rats. Transplantation of hAMSCs at 3 weeks plus 5 weeks did not have an additive effect. In vitro experiments demonstrated that Kupffer cell activation induced by lipopolysaccharide was significantly decreased by culturing with conditioned medium obtained from hAMSCs.

Conclusions: Transplantation of hAMSCs provided significant improvement in a rat model of liver fibrosis, possibly through the inhibition of Kupffer cell and hepatic stellate cell activation. hAMSCs may be a potential new treatment for liver fibrosis.
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http://dx.doi.org/10.1097/TXD.0000000000000525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946462PMC
May 2015

Clinicopathological characteristics and diagnostic performance of Wisteria floribunda agglutinin positive Mac-2-binding protein as a preoperative serum marker of liver fibrosis in hepatocellular carcinoma.

J Gastroenterol 2015 Nov 15;50(11):1134-44. Epub 2015 Mar 15.

Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Kita 15-jo Nishi 7-chome Kita-ku, Sapporo, 060-8638, Japan.

Background: Wisteria floribunda agglutinin positive Mac-2-binding protein (WFA(+)-M2BP) is a novel serum marker of liver fibrosis identified in glycoproteomic biomarker screening studies, and its clinicopathological characteristics have yet to be elucidated sufficiently for clinical utilization.

Methods: We retrospectively analyzed the clinicopathology data and serum WFA(+)-M2BP levels in 376 hepatocellular carcinoma patients undergoing liver surgery. WFA(+)-M2BP was quantified in frozen serum samples collected at the time of surgery using the FastLec-Hepa method.

Results: Significant independent determinants of serum WFA(+)-M2BP levels included pathological diagnosis of cirrhosis, female gender, hepatitis C virus (HCV) infection, and liver dysfunction characteristics, such as abnormal indocyanine green retention rate at 15 min, platelet counts, albumin levels, alanine aminotransferase levels, and total bilirubin levels. Serum WFA(+)-M2BP levels increased with the pathological fibrosis stage and liver dysfunction severity. HCV infection significantly affected serum WFA(+)-M2BP levels throughout the pathological and functional progression of liver fibrosis, and the effect of gender was significant only in F4 stage patients with severe liver dysfunction. The diagnostic thresholds for cutoff index values for cirrhosis were 1.435 and 4.615 in HCV-negative and HCV-positive patients, respectively. Serum WFA(+)-M2BP levels at the time of operation were a significant predictor of hepatocellular carcinoma recurrence and overall survival in both HCV-negative and HCV-positive patients.

Conclusions: Serum WFA(+)-M2BP levels reflected both the pathological and functional progression of liver fibrosis comprehensively and continuously. Elevated WFA(+)-M2BP levels were a significant risk factor for tumor recurrence and decreased overall survival after liver surgery independent of HCV infection.
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http://dx.doi.org/10.1007/s00535-015-1063-2DOI Listing
November 2015

3-[(dodecylthiocarbonyl)methyl]-glutarimide attenuates graft arterial disease by suppressing alloimmune responses and vascular smooth muscle cell proliferation.

Transplantation 2015 May;99(5):948-56

1 Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Japan. 2 Department of Transplant Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan. 3 Department of Molecular Target Medicine Screening, Aichi Medical University School of Medicine, Nagakude, Japan.

Background: Graft arterial disease (GAD) is a major cause of late graft loss after organ transplantation. Alloimmune responses and vascular remodeling eventually cause the transplant organ to develop GAD. In this study, we aimed to limit the development of GAD by inhibiting alloimmune responses and vascular smooth muscle cell (VSMC) proliferation with a new compound, 3-[(dodecylthiocarbonyl)methyl]-glutarimide ([DTCM]-glutarimide), in a murine cardiac model of GAD.

Methods: The hearts from B6.CH-2 mice were transplanted into C57BL/6 mouse recipients to examine the extent of GAD. The recipients were treated with either vehicle or DTCM-glutarimide intraperitoneally (40 mg/kg per day) for 4 weeks.

Results: The administration of DTCM-glutarimide attenuated GAD formation (luminal occlusion: 37.9 ± 5.9% vs 14.8 ± 5.4%, P < 0.05) by inhibiting the number of graft-infiltrating cells and decreasing alloreactive interferon (IFN)-γ production compared with control mice, as measured by the Enzyme-linked ImmunoSpot assay. In vitro, VSMCs proliferated on stimulation with either basic fibroblast growth factor or IFN-γ and splenocytes after transplantation, but the addition of DTCM-glutarimide resulted in the inhibition of VSMC proliferation. Moreover, DTCM-glutarimide suppressed cyclin D1 expression and inhibited cell cycle progression from G1 to S in VSMCs.

Conclusions: The compound DTCM-glutarimide suppressed GAD development by inhibiting not only alloimmune responses but also VSMC proliferation in the graft.
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http://dx.doi.org/10.1097/TP.0000000000000576DOI Listing
May 2015
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