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Although choledochojejunostomy is the standard technique for biliary reconstruction, there are various associated problems that need to be solved such as reflux cholangitis. Interposition with an artificial bile duct (ABD) to replace the resected bile duct maintains a physiological conduit for bile and may solve this problem. This study investigated the usefulness of an ABD made of gelatin hydrogel nonwoven fabric (GHNF). GHNF was prepared by the solution blow spinning method. The migration and activity of murine fibroblast L929 cells were examined in GHNF sheets. L929 cells migrated into GHNF sheets, where they proliferated and synthesized collagen, suggesting GHNF is a promising scaffold for bile duct regeneration. ABDs made of GHNF were implanted in place of resected bile duct segments in rats. The rats were killed at 2, 6, and 12 weeks postimplantation. The implantation site was histologically evaluated for bile duct regeneration. At postoperative 2 weeks, migrating cells were observed in the ABD pores. The implanted ABD was mostly degraded and replaced by collagen fibers at 6 weeks. Ki67-positive bile duct epithelial cells appeared within the implanted ABD. These were most abundant within the central part of the ABD after 6 weeks. The percentages of Ki67-positive cells were 31.7 ± 9.1% in the experimental group and 0.8 ± 0.6% in the sham operation group at 6 weeks (p

Purpose:Few studies have investigated prognostic biomarkers in patients with intrahepatic cholangiocarcinoma (ICC). Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has been suggested to play important roles in inflammation and several cancer types. We herein examined the clinical significance and biological function of NRDC in ICC.Experimental Design: We measured serum NRDC levels in 98 patients with ICC who underwent surgical resection in two independent cohorts to assess its prognostic impact. We also analyzed NRDC mRNA levels in cancerous tissue specimens from 43 patients with ICC. We investigated the roles of NRDC in cell proliferation, migration, gemcitabine sensitivity, and gene expression in ICC cell lines using gene silencing.Results:High serum NRDC levels were associated with shorter overall survival and disease-free survival in the primary (n = 79) and validation (n = 19) cohorts. A correlation was observed between serum protein levels and cancerous tissue mRNA levels of NRDC (Spearman ρ = 0.413; P = 0.006). The gene knockdown of NRDC in ICC cell lines attenuated cell proliferation, migration, and tumor growth in xenografts, and increased sensitivity to gemcitabine. The gene knockdown of NRDC was also accompanied by significant changes in the expression of several epithelial–mesenchymal transition (EMT)-related genes. Strong correlations were observed between the mRNA levels of NRDC and EMT-inducing transcription factors, ZEB1 and SNAI1, in surgical specimens from patients with ICC.Conclusions:Serum NRDC, a possible surrogate marker reflecting the EMT state in primary tumors, predicts the outcome of ICC after surgical resection.

Supplementary Tables S1-S7; Supplementary Figures S1-S7

Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment has become a focus of intense research; however, there are few reports on the dynamics of the tumor immune environment in leukemia. Here, we analyzed the tumor immune environment in pediatric B cell precursor acute lymphoblastic leukemia by analyzing serial bone marrow samples from nine patients with primary and recurrent disease by mass cytometry using 39 immunophenotype markers, and transcriptome analysis. High‐dimensional single‐cell mass cytometry analysis elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that, during relapse, the tumor immune environment comprised a T helper 1‐polarized immune profile, together with an increased number of effector regulatory T cells. These results were confirmed in a validation cohort using conventional flow cytometry. Furthermore, RNA transcriptome analysis identified the upregulation of immune‐related pathways in B cell precursor acute lymphoblastic leukemia cells during relapse, suggesting interaction with the surrounding environment. In conclusion, a tumor immune environment characterized by a T helper 1‐polarized immune profile, with an increased number of effector regulatory T cells, could contribute to the pathophysiology of recurrent B cell precursor acute lymphoblastic leukemia. This information could contribute to the development of effective immunotherapeutic approaches against B cell precursor acute lymphoblastic leukemia relapse., Tumor immune environment characterized by a T helper 1‐polarized immune profile was observed in recurrent B cell precursor acute lymphoblastic leukemia. Regulatory T cells were activated in recurrent B cell precursor acute lymphoblastic leukemia.

Sinusoidal obstruction syndrome (SOS) induced by oxaliplatin-including chemotherapies (OXCx) is associated with impaired hepatic reserve and higher morbidity after hepatic resection. However, in the absence of an appropriate animal experimental model, little is known about its pathophysiology. This study aimed to establish a clinically relevant reproducible model of FOLFOX-induced SOS and to compare the clinical/histopathological features between the clinical and animal SOS settings.We performed clinical/pathological analyses of colorectal liver metastasis (CRLM) patients who underwent hepatectomy with/without preoperative treatment of FOLFOX (n = 22/18). Male micro-minipigs were treated with 50% of the standard human dosage of the FOLFOX regimen.In contrast to the monocrotaline-induced SOS model in rats, hepatomegaly, ascites, congestion, and coagulative necrosis of hepatocytes were absent in patients with CRLM with OXCx pretreatment and OXCx-treated micro-minipigs. In parallel to CRLM cases with OXCx pretreatment, OXCx-challenged micro-minipigs exhibited deteriorated indocyanine green clearance, morphological alteration of liver sinusoidal endothelial cells, and upregulated matrix metalloproteinase-9. Using our novel porcine SOS model, we identified the hepatoprotective influence of recombinant human soluble thrombomodulin in OXCx-SOS.With distinct differences between monocrotaline-induced rat SOS and human/pig OXCx-SOS, our pig OXCx-SOS model serves as a preclinical platform for future investigations to dissect the pathophysiology of OXCx-SOS and seek preventive strategies.

Background/aim Sinusoidal obstruction syndrome (SOS) after neoadjuvant chemotherapy with oxaliplatin for colorectal liver metastases (CRLM) has been reported to lead to early recurrence. This study investigated the effects of SOS on the development of CRLM in a rat model. Materials and methods RCN-H4 cells were injected into the spleen or liver of ten monocrotaline-treated (SOS group) and ten untreated (control group) rats. The number and size of liver tumors were compared between the groups. Results The number of liver tumors in the splenic RCN-H4 injection model was significantly higher in the SOS group than in the control group (332±213 vs. 16±5, p=0.029); however, the largest tumor diameter in the hepatic model was similar between groups (6.2±1.8 vs. 6.4±2.4 mm, p=0.87). Conclusion SOS promotes CRLM development by splenic RCN-H4 cell injection. This might be due to the higher incidence of cancer cell implantation into the liver.

Background and aim Studies on the resolution of liver fibrosis are becoming more important in this era of etiologic eradication. In contrast to the extensive research on the recovery of liver fibrosis induced by hepatotoxic injuries, regression of cholestatic liver fibrosis has been insufficiently examined owing to the limited availability of animal models. Methods We examined our novel recanalization mice model of biliary obstruction, involving anastomosis between the gallbladder and jejunum (G-J anastomosis) by invagination. Transgenic mice expressing green fluorescent protein (GFP) under the collagen 1(α)1 promoter underwent G-J anastomosis 14 days after bile duct ligation (BDL) and were sacrificed 14 days later. Results Transaminase and total bilirubin levels decreased to almost normal values on day 14 after G-J anastomosis. G-J anastomosis resulted in dramatic reversal of liver fibrosis induced by BDL. Activated portal fibroblasts (PFs) double-positive for GFP and Thy-1 on immunofluorescence in the liver of BDL-injured mice became less noticeable following G-J anastomosis. Messenger RNA expression of markers for activated PFs in the liver was downregulated after anastomosis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were induced by BDL. After anastomosis, expressions of MMP-3, 8 as well as hepatocyte growth factor were further upregulated, whereas those of TIMP-1 and -3 were markedly downregulated. Conclusions Our established G-J anastomosis model is associated with fibrosis resolution and reduced PF activation through reopening of bile duct obstruction and will be valuable for studying the recovery process of cholestatic liver fibrosis.

The mechanism underlying liver regeneration following partial hepatectomy (PH) is not fully elucidated. We aimed to characterize collagen gene expressing hepatic cells following PH and examine their contribution to liver regeneration.Col-GFP mice, which express GFP under the control of the collagen gene promoter, were used to detect collagen gene expressing cells following PH. The GFP-expressing cells were analyzed via single-cell RNA sequencing (scRNA-seq). Additionally, Col-ER Cre/RFP and Col-ER Cre/DTA mice were utilized to examine the cell fates and functional roles of collagen gene expressing cells in liver regeneration, respectively. The number of collagen gene expressing cells was found to be increased on day 3 and subsequently decreased on day 7 following PH. ScRNA-seq analysis of sorted collagen gene expressing cells showed that the regenerating liver was characterized by three distinct hepatic stellate cell (HSC) clusters, including one representing classic myofibroblasts. The other HSC clusters included an intermediately activated HSC cluster and a proliferating HSC cluster. Of these, the latter cluster was absent in the CClHeterogeneous HSC clusters, one of which was a unique proliferating cluster, were found to appear in the liver following PH. Collagen gene expressing cells, including HSCs, were found to promote liver regeneration.

This study aimed to evaluate postoperative long-term liver restoration and splenic enlargement and their clinical significance in living donor liver transplantation. One hundred and sixteen donors who had donated livers more than 5 years previously accepted the invitation to participate in this study. The liver restoration rate and the splenic enlargement rate were calculated as the rate with respect to the original volume. The mean liver restoration rate was 0.99 ± 0.12 and older age was associated with a higher incidence for liver restoration rate

Background The fatty acid (FA) composition of phosphatidylinositols (PIs) is tightly regulated in mammalian tissue since its disruption impairs normal cellular functions. We previously found its significant alteration in breast cancer by using matrix-assisted laser desorption and ionisation imaging mass spectrometry (MALDI-IMS). Methods We visualised the histological distribution of PIs containing different FAs in 65 primary breast cancer tissues using MALDI-IMS and investigated its association with clinicopathological features and gene expression profiles. Results Normal ductal cells (n = 7) predominantly accumulated a PI containing polyunsaturated FA (PI-PUFA), PI(18:0/20:4). PI(18:0/20:4) was replaced by PIs containing monounsaturated FA (PIs-MUFA) in all non-invasive cancer cells (n = 12). While 54% of invasive cancer cells (n = 27) also accumulated PIs-MUFA, 46% of invasive cancer cells (n = 23) accumulated the PIs-PUFA, PI(18:0/20:3) and PI(18:0/20:4). The accumulation of PI(18:0/20:3) was associated with higher incidence of lymph node metastasis and activation of the PD-1-related immune checkpoint pathway. Fatty acid-binding protein 7 was identified as a putative molecule controlling PI composition. Conclusions MALDI-IMS identified PI composition associated with invasion and nodal metastasis of breast cancer. The accumulation of PI(18:0/20:3) could affect the PD-1-related immune checkpoint pathway, although its precise mechanism should be further validated.

We investigated the role of mesothelin (Msln) and thymocyte differentiation antigen 1 (Thy1) in the activation of fibroblasts across multiple organs and demonstrated that Msln(−/−) mice are protected from cholestatic fibrosis caused by Mdr2 (multidrug resistance gene 2) deficiency, bleomycin-induced lung fibrosis, and UUO (unilateral urinary obstruction)-induced kidney fibrosis. On the contrary, Thy1(−/−) mice are more susceptible to fibrosis, suggesting that a Msln–Thy1 signaling complex is critical for tissue fibroblast activation. A similar mechanism was observed in human activated portal fibroblasts (aPFs). Targeting of human MSLN(+) aPFs with two anti-MSLN immunotoxins killed fibroblasts engineered to express human mesothelin and reduced collagen deposition in livers of bile duct ligation (BDL)–injured mice. We provide evidence that antimesothelin-based therapy may be a strategy for treatment of parenchymal organ fibrosis.

We investigated the role of mesothelin (Msln) and thymocyte differentiation antigen 1 (Thy1) in the activation of fibroblasts across multiple organs and demonstrated that Msln-/- mice are protected from cholestatic fibrosis caused by Mdr2 (multidrug resistance gene 2) deficiency, bleomycin-induced lung fibrosis, and UUO (unilateral urinary obstruction)-induced kidney fibrosis. On the contrary, Thy1-/- mice are more susceptible to fibrosis, suggesting that a Msln-Thy1 signaling complex is critical for tissue fibroblast activation. A similar mechanism was observed in human activated portal fibroblasts (aPFs). Targeting of human MSLN+ aPFs with two anti-MSLN immunotoxins killed fibroblasts engineered to express human mesothelin and reduced collagen deposition in livers of bile duct ligation (BDL)-injured mice. We provide evidence that antimesothelin-based therapy may be a strategy for treatment of parenchymal organ fibrosis.

Purpose: Few studies have investigated prognostic biomarkers in patients with intrahepatic cholangiocarcinoma (ICC). Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has been suggested to play important roles in inflammation and several cancer types. We herein examined the clinical significance and biological function of NRDC in ICC. Experimental Design: We measured serum NRDC levels in 98 patients with ICC who underwent surgical resection in two independent cohorts to assess its prognostic impact. We also analyzed NRDC mRNA levels in cancerous tissue specimens from 43 patients with ICC. We investigated the roles of NRDC in cell proliferation, migration, gemcitabine sensitivity, and gene expression in ICC cell lines using gene silencing. Results: High serum NRDC levels were associated with shorter overall survival and disease-free survival in the primary (n = 79) and validation (n = 19) cohorts. A correlation was observed between serum protein levels and cancerous tissue mRNA levels of NRDC (Spearman ρ = 0.413; P = 0.006). The gene knockdown of NRDC in ICC cell lines attenuated cell proliferation, migration, and tumor growth in xenografts, and increased sensitivity to gemcitabine. The gene knockdown of NRDC was also accompanied by significant changes in the expression of several epithelial–mesenchymal transition (EMT)-related genes. Strong correlations were observed between the mRNA levels of NRDC and EMT-inducing transcription factors, ZEB1 and SNAI1, in surgical specimens from patients with ICC. Conclusions: Serum NRDC, a possible surrogate marker reflecting the EMT state in primary tumors, predicts the outcome of ICC after surgical resection.

Chemosensitivity to cisplatin derivatives varies among individual patients with intractable malignancies including ovarian cancer, while how to unlock the resistance remain unknown. Ovarian cancer tissues were collected the debulking surgery in discovery- (n = 135) and validation- (n = 47) cohorts, to be analyzed with high-throughput automated immunohistochemistry which identified cystathionine γ-lyase (CSE) as an independent marker distinguishing non-responders from responders to post-operative platinum-based chemotherapy. We aimed to identify CSE-derived metabolites responsible for chemoresistant mechanisms: gold-nanoparticle (AuN)-based surface-enhanced Raman spectroscopy (SERS) was used to enhance electromagnetic fields which enabled to visualize multiple sulfur-containing metabolites through detecting scattering light from Au–S vibration two-dimensionally. Clear cell carcinoma (CCC) who turned out less sensitive to cisplatin than serous adenocarcinoma was classified into two groups by the intensities of SERS intensities at 480 cm−1; patients with greater intensities displayed the shorter overall survival after the debulking surgery. The SERS signals were eliminated by topically applied monobromobimane that breaks sulfane-sulfur bonds of polysulfides to result in formation of sulfodibimane which was detected at 580 cm−1, manifesting the presence of polysulfides in cancer tissues. CCC-derived cancer cell lines in culture were resistant against cisplatin, but treatment with ambroxol, an expectorant degrading polysulfides, renders the cells CDDP-susceptible. Co-administration of ambroxol with cisplatin significantly suppressed growth of cancer xenografts in nude mice. Furthermore, polysulfides, but neither glutathione nor hypotaurine, attenuated cisplatin-induced disturbance of DNA supercoiling. Polysulfide detection by on-tissue SERS thus enables to predict prognosis of cisplatin-based chemotherapy. The current findings suggest polysulfide degradation as a stratagem unlocking cisplatin chemoresistance., Graphical abstract Image 1, Highlights • Cystathionine-γ-lyase predicts post-operative chemoresistance in ovarian cancer. • SERS visualizes on-tissue polysulfides in non-responders against chemotherapy. • Polysulfides ameliorate cisplatin-induced DNA damages to block cancer cell death. • Ambroxol degrades polysulfides to unlock chemoresistance in vitro and in vivo.

Cancer cell lines are widely used in basic research to study cancer development, growth, invasion, or metastasis. They are also used for the development and screening of anticancer drugs. However, there are no clear criteria for choosing the most suitable cell lines among the wide variety of cancer cell lines commercially available for research, and the choice is often based on previously published reports. Here, we investigated the characteristics of liver cancer cell lines by analyzing the gene expression data available in the Cancer Cell Line Encyclopedia. Unsupervised clustering analysis of 28 liver cancer cell lines yielded two main clusters. One cluster showed a gene expression pattern similar to that of hepatocytes, and the other showed a pattern similar to that of fibroblasts. Analysis of hepatocellular carcinoma gene expression profiles available in The Cancer Genome Atlas showed that the gene expression patterns in most hepatoma tissues were similar to those in the hepatocyte-like cluster. With respect to liver cancer research, our findings may be useful for selecting an appropriate cell line for a specific study objective. Furthermore, our approach of utilizing a public database for comparing the properties of cell lines could be an attractive cell line selection strategy that can be applied to other fields of research.

Aim: To investigate the therapeutic effect of FZHY on hepatic fibrosis in mice and to determine the mechanism of its action. Methods: Wild type mice were subjected to toxic (carbon tetrachloride, CCl4) or cholestatic (bile duct ligation, BDL). Upon induction of liver fibrosis, mice were treated with FZHY (4.0g/kg, 2w, oral gavage) or vehicle (PBS). Livers were analyzed by Sirius Red staining, immunostaining and RT-PCR for pro-fibrogenic and pro-inflammatory genes. The effect of FZHY on hepatocytes, inflammatory responses, activation of fibrogenic myofibroblasts, and ROS production was assessed. Results: FZHY strongly inhibited the development of CCl4- and BDL-induced liver fibrosis in mice. Liver fibrosis was significantly improved in FZHY-treated mice, as demonstrated by reduced content of hepatic hydroxyproline and Sirius Red positive area. Moreover, the number of SMA +and Desmin+ myofibroblasts was significantly reduced in the livers of FZHY-treated mice, and correlated with downregulation of the mRNA levels of α-SMA, collagen-α1(I), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), TGF-β1 and its receptor TGF-βRI, and platelet-derived growth factor-β (PDGF-β), suggesting that FZHY inhibits activation of fibrogenic myofibroblasts. Furthermore, administration of FZHY markedly decreased recruitment of F4/80+ inflammatory macrophages to the livers of CCl4- and BDL-injured mice, and this effect was associated with downregulation of monocyte chemoattractant protein-1(MCP-1) and macrophage inflammatory protein-1 (MIP-1) mRNA. In addition, the lipid peroxidation products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) were reduced, demonstrating that treatment with FZHY can effectively block ROS production in livers of CCl4- and BDL-injured mice. Conclusions: Traditional Chinese Medicine FZHY has a variety of anti-fibrotic effects, including strong anti-oxidant, anti-inflammatory and anti-fibrotic effects on myeloid cells and hepatocytes. Although FZHY compound does not seem to directly affect HSCs, it regulates HSC activation via inhibition of macrophage recruitment to fibrotic liver.

Mac-2 binding protein glycosylation isomer (M2BPGi) is a novel liver fibrosis biomarker, but there are few studies on M2BPGi in liver transplantation (LT) recipients. This study aimed to evaluate the utility of M2BPGi measurement in LT recipients. We collected the clinicopathological data of 233 patients who underwent a liver biopsy at Kyoto University Hospital after LT between August 2015 and June 2019. The median values of M2BPGi in patients with METAVIR fibrosis stages F0, F1, F2, and ≥F3 were 0.61, 0.76, 1.16, and 1.47, respectively, whereas those in patients with METAVIR necroinflammatory indexes A0, A1, and ≥A2 were 0.53, 1.145, and 2.24, respectively. Spearman rank correlation test suggested that the necroinflammatory index had a stronger correlation to the M2BPGi value than the fibrosis stage. The area under the receiver operating characteristic curve of M2BPGi to predict ≥A1 was 0.75, which was significantly higher than that of any other liver fibrosis and inflammation marker. Patients with a rejection activity index (RAI) of ≥3 had a higher M2BPGi value than those with RAI ≤ 2 (P = 0.001). Patients with hepatitis C virus viremia had a higher M2BPGi value than sustained virological responders or those with other etiologies. In conclusion, the present study demonstrated that M2BPGi values are more strongly influenced by necroinflammatory activity and revealed M2BPGi, which has been thought to be a so-called fibrosis marker, as a disease activity marker in transplant recipients. M2BPGi measurement may be useful to detect early stage liver inflammation that cannot be detected by routine blood examination of LT recipients.

Background Little is known about the clinical significance and risk factors for incomplete liver restoration after partial hepatectomy, which is defined by a liver volume restoration of less than 100% of the original volume. Methods We retrospectively analyzed patients who underwent hepatic resection for liver tumors at the Kyoto University Hospital between January 2011 and October 2015 and survived without recurrence for more than 3 years. The preoperative and postoperative data, as well as liver and splenic volume after 3 postoperative years, were assessed. Results The percentage of resected liver was higher in the incomplete liver restoration group (n = 52, 41.6%) than in the complete liver restoration group (n = 73, 58.4%) (28 [3–78]% vs 14.5 [2–63]%, P = .0226). The percentage of resected liver was also higher in the splenomegaly group (defined by spleen volume increases of more than 35% of the original volume) than in the nonsplenomegaly group (40 [4–63]% vs 16.5 [2–78]%, P = .0002). Multivariate analysis demonstrated that the percentage of resected liver was a significant predictor of incomplete liver restoration (odds ratio = 9.75, P = .0043) and splenomegaly (odds ratio = 74.4, P = .0006). Incomplete liver restoration 3 years after hepatectomy was associated with lower serum albumin levels (4.0 [2.4–4.7] g/dL compared with 4.2 [2.6–4.8] g/dL in the complete liver restoration group, P = .0032). Splenomegaly was associated with a lower platelet count (109.9 ± 49.8 x103/μL vs 163 ± 58.1 × 103/μL, P = .0007) and lower serum albumin level (3.6 [2.6–4.4] g/dL vs 4.1 [2.4–4.8] g/dL, P = .0002). Conclusion An extensive resection of the liver parenchyma results in an increased risk for incomplete liver restoration and splenomegaly long after hepatectomy, which is associated with the clinical consequences of hypoalbuminemia and thrombocytopenia.

Ischemia/reperfusion injury (IRI) is one of the main causes of liver dysfunction after liver surgery. Involvement of endoplasmic reticulum (ER) stress in various diseases has been demonstrated, and CAAT/enhancer binding protein-homologous protein (CHOP) is a transcriptional regulator that is induced by ER stress. It is also a key regulator of ER stress-mediated apoptosis. The aim of this study was to investigate the role of CHOP in liver IRI. Wild type (WT) and CAAT/enhancer binding protein-homologous protein knockout (CHOP-/-) mice were subjected to 70% liver warm ischemia/reperfusion for 60 minutes. At different times after reperfusion, liver tissues and blood samples were collected for evaluation. Induction of ER stress including CHOP expression was ascertained. Liver damage was evaluated based on serum liver enzymes, liver histology, and neutrophil infiltration. Hepatocyte death including apoptosis was assessed. Liver warm IRI induced ER stress in both WT and CHOP-/- mice. In addition, CHOP expression was up-regulated in WT mice. At 6 hours after reperfusion, liver damage was attenuated in CHOP-/- mice. On the basis of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, apoptotic and necrotic cells were significantly reduced in CHOP-/- mice. CHOP deficiency also reduced the cleavage of caspase 3 and expression of the proapoptotic protein B cell lymphoma 2-associated X protein. Liver IRI induces CHOP expression, and CHOP deficiency attenuates liver IRI by inhibiting apoptosis. Elucidation of the function of CHOP in liver IRI may contribute to further investigation for a therapy against liver IRI associated with the ER stress pathway. Liver Transplantation 24 645-654 2018 AASLD.

Background Surgical resection remains the mainstay of curative treatment for intrahepatic cholangiocarcinoma (ICC). Prognosis after surgery is unsatisfactory despite improvements in treatment and post-operative clinical management. Despite developments in the molecular profiling of ICC, the preoperative prediction of prognosis remains a challenge. This study aimed to identify clinical prognostic indicators by investigating the molecular profiles of ICC and evaluating the preoperative imaging data of 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET). Methods A retrospective analysis was performed on 50 consecutive patients with ICC who underwent curative hepatectomy after 18F-FDG-PET examination. To evaluate the molecular profiles of ICC, KRAS mutation status was assessed in resected specimens. For the assessment of glucose uptake, we observed the expression of glucose transporter-1 (GLUT-1) by immunohistochemistry. The data of 18F-FDG-PET were re-evaluated as follows: maximum standardized uptake value, metabolic tumor volume (MTV), and total lesion glycolysis (TLG). Cut-off values were determined using receiver operating characteristic (ROC) curve analysis. Cumulative overall survival (OS) was analyzed using the Kaplan–Meier analysis. Results Overall, 16 (32.0%) patients had mutations in KRAS. Patients with mutated KRAS exhibited shorter OS than those with wild-type KRAS (5-year OS, 0% vs. 35.1%, P

Introduction Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment (TIE) has become a focus of intense research; however, there are few reports on the dynamics of the TIE in leukemia, especially in bone marrow (BM), the primary site of leukemia. Mass cytometry, which allows high-dimensional analysis with single-cell resolution, is a powerful tool for the characterization of the TIE, and enables us to examine pediatric BM samples containing only a few numbers of immune cells. Methods Primary and recurrent BM samples were collected serially from pediatric patients with BCP-ALL at Kyoto University Hospital from 2006 to 2013. Mononuclear cells were isolated by density centrifugation and viably preserved until they were used. We examined the TIE of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) by analyzing serial BM samples from patients in primary and recurrent disease phases by mass cytometry, using 39 immunophenotype markers, and transcriptome analysis. Results and Discussion The proportion of BCP-ALL cells in the samples was 87.3-97.8% (mean: 94.0%) at onset, and 81.3-98.8% (mean: 92.2%) at relapse. High-dimensional single-cell analysis by mass cytometry elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that the TIE during relapse comprised a T helper 1 (Th1)-polarized immune profile, together with the increase of effector regulatory T cells (Tregs). Th1 cells are typically known as supporters of cytotoxic T lymphocytes which would eliminate leukemia cells and work against relapse, but recently, it is reported that Th1 cells directly support ALL proliferation in vitro (Traxel et al. Oncogene. 2019; 38:2420-2431), and that the concentrations of pro-inflammatory cytokines and Th1 cytokines (IFN-γ and IL-12) were elevated in patients with ALL, which could create favorable conditions for ALL (Perez-Figueroa et al. Oncol Rep. 2016; 35: 2699-2706, Vilchis-Ordonez et al. Biomed Res Int. 2015; 2015: 386165). Therefore, there is a possibility that the Th1-polarized immune profile would work in favor of leukemia survival for relapse. Furthermore, Gene set enrichment analysis based on RNA expression identified the enrichment of six immune-related pathways were enriched at the time of relapse; chemokine activity, chemokine production, complement activation, positive regulation of cytokine production involved in immune response, positive regulation of lymphocyte chemotaxis, and positive regulation of lymphocyte migration. These expression signatures suggest that BCP-ALL cells attract lymphocytes, and upregulate immune activities at relapse. Conclusion In summary, a TIE characterized by a Th1-polarized immune profile, with the increase of effector Tregs, may be involved in the pathophysiology of recurrent ALL, and BCP-ALL cells at relapse were enriched with gene expressions related to lymphocyte attraction and activities. This information could contribute to the development of effective immunotherapeutic approaches against BCP-ALL relapse. Disclosures Ogawa: Eisai Co., Ltd.: Research Funding; Kan Research Laboratory, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding.

Chemosensitivity to cisplatin derivatives varies among individual patients with intractable malignancies including ovarian cancer, while how to unlock the resistance remain unknown. Ovarian cancer tissues were collected the debulking surgery in discovery- (n = 135) and validation- (n = 47) cohorts, to be analyzed with high-throughput automated immunohistochemistry which identified cystathionine γ-lyase (CSE) as an independent marker distinguishing non-responders from responders to post-operative platinum-based chemotherapy. We aimed to identify CSE-derived metabolites responsible for chemoresistant mechanisms: gold-nanoparticle (AuN)-based surface-enhanced Raman spectroscopy (SERS) was used to enhance electromagnetic fields which enabled to visualize multiple sulfur-containing metabolites through detecting scattering light from Au-S vibration two-dimensionally. Clear cell carcinoma (CCC) who turned out less sensitive to cisplatin than serous adenocarcinoma was classified into two groups by the intensities of SERS intensities at 480 cm

The origin of collagen-producing myofibroblasts in pancreatic fibrosis is still controversial. Pancreatic stellate cells (PSCs), which have been recognized as the pancreatic counterparts of hepatic stellate cells (HSCs), are thought to play an important role in the development of pancreatic fibrosis. However, sources of myofibroblasts other than PSCs may exist because extensive studies of liver fibrosis have uncovered myofibroblasts that did not originate from HSCs. This study aimed to characterize myofibroblasts in an experimental pancreatic fibrosis model in mice.We used transgenic mice expressing green fluorescent protein via the collagen type I α1 promoter and induced pancreatic fibrosis with repetitive injections of cerulein.Collagen-producing cells that are negative for glial fibrillary acidic protein (ie, not derived from PSCs) exist in the pancreas. Pancreatic stellate cells had different characteristics from those of HSCs in a very small possession of vitamin A using mass spectrometry and a low expression of lecithin retinol acyltransferase. The microstructure of PSCs was entirely different from that of HSCs using flow cytometry and electron microscopy.Our study showed that characteristics of PSCs are different from those of HSCs, and myofibroblasts in the pancreas might be derived not only from PSCs but also from other fibrogenic cells.

The commensal microbiota within the gastrointestinal tract is essential in maintaining homeostasis. Indeed, dysregulation in the repertoire of microbiota can result in the development of intestinal immune-inflammatory diseases. Further, this immune regulation by gut microbiota is important systemically, impacting health and disease of organ systems beyond the local environment of the gut. What has not been explored is how distant organs might in turn shape the microbiota via microbe-targeted molecules. Here, we provide evidence that surfactant protein D (SP-D) synthesized in the gallbladder and delivered into intestinal lumen binds selectively to species of gut commensal bacteria. SP-D-deficient mice manifest intestinal dysbiosis and show a susceptibility to dextran sulfate sodium-induced colitis. Further, fecal transfer from SP-D-deficient mice to wild-type, germ-free mice conveyed colitis susceptibility. Interestingly, colitis caused a notable increase in Sftpd gene expression in the gallbladder, but not in the lung, via the activity of glucocorticoids produced in the liver. These findings describe a unique mechanism of interorgan regulation of intestinal immune homeostasis by SP-D with potential clinical implications such as cholecystectomy.

Nardilysin (NRDC) is a metalloendopeptidase of the M16 family. We previously showed that NRDC activates inflammatory cytokine signaling, including interleukin‐6‐signal transducer and activator of transcription 3 (STAT3) signaling. NRDC has been implicated in the promotion of breast, gastric and esophageal cancer, as well as the development of liver fibrosis. In this study, we investigated the role of NRDC in the promotion of hepatocellular carcinoma (HCC), both clinically and experimentally. We found that NRDC expression was upregulated threefold in HCC tissue compared to the adjacent non‐tumor liver tissue, which was confirmed by immunohistochemistry and western blotting. We also found that high serum NRDC was associated with large tumor size (>3 cm, P = 0.016) and poor prognosis after hepatectomy (median survival time 32.0 vs 73.9 months, P = 0.003) in patients with hepatitis C (n = 120). Diethylnitrosamine‐induced hepatocarcinogenesis was suppressed in heterozygous NRDC‐deficient mice compared to their wild‐type littermates. Gene silencing of NRDC with miRNA diminished the growth of Huh‐7 and Hep3B spheroids in vitro. Notably, phosphorylation of STAT3 was decreased in NRDC‐depleted Huh‐7 spheroids compared to control spheroids. The effect of a STAT3 inhibitor (S3I‐201) on the growth of Huh‐7 spheroids was reduced in NRDC‐depleted cells relative to controls. Our results show that NRDC is a promising prognostic marker for HCC in patients with hepatitis C, and that NRDC promotes tumor growth through activation of STAT3.

Neuropilin-1 (NRP-1)-expressing macrophages are engaged in antitumor immune functions via various mechanisms. In this study, we investigated the role of NRP-1 on macrophages in antibody-mediated tumoricidal activity. Treatment of macrophages with NRP-1 knockdown or an anti-NRP-1-neutralizing antibody significantly suppressed antibody-dependent cellular cytotoxicity and modulated cytokine secretion from macrophages in vitro. Furthermore, in vivo studies using a humanized mouse model bearing human epidermal growth factor receptor-2 (HER2)-positive breast cancer xenografts showed that antibody-mediated antitumor activity and tumor infiltration of CD4+ T lymphocytes were significantly downregulated when peripheral blood mononuclear cells in which NRP-1 was knocked down were co-administered with an anti-HER2 antibody. These results revealed that NRP-1 expressed on macrophages plays an important role in antibody-mediated antitumor immunity. Taken together, the induction of NRP-1 on macrophages may be a therapeutic indicator for antibody treatments that exert antibody-dependent cellular cytotoxicity activity, although further studies are needed in order to support this hypothesis.

Cholestatic liver fibrosis is caused by obstruction of the biliary tract and is associated with early activation of portal fibroblasts (PFs) that express Thy-1, fibulin 2, and the recently identified marker mesothelin (MSLN). Here, we have demonstrated that activated PFs (aPFs) and myofibroblasts play a critical role in the pathogenesis of liver fibrosis induced by bile duct ligation (BDL). Conditional ablation of MSLN+ aPFs in BDL-injured mice attenuated liver fibrosis by approximately 50%. Similar results were observed in MSLN-deficient mice (Msln-/- mice) or mice deficient in the MSLN ligand mucin 16 (Muc16-/- mice). In vitro analysis revealed that MSLN regulates TGF-β1-inducible activation of WT PFs by disrupting the formation of an inhibitory Thy-1-TGFβRI complex. MSLN also facilitated the FGF-mediated proliferation of WT aPFs. Therapeutic administration of anti-MSLN-blocking Abs attenuated BDL-induced fibrosis in WT mice. Liver specimens from patients with cholestatic liver fibrosis had increased numbers of MSLN+ aPFs/myofibroblasts, suggesting that MSLN may be a potential target for antifibrotic therapy.

Nonalcoholic fatty liver disease ranges from simple steatosis to nonalcoholic steatohepatitis (NASH). Kupffer cells play a central role in promoting hepatic inflammation, which leads to the development of NASH. We investigated the anti-inflammatory effect of hepatic vagus-mediated stimulation of the α7 nicotinic acetylcholine receptor (α7nAChR) on Kupffer cells in NASH pathogenesis. Wild-type (WT) mice undergoing hepatic vagotomy (HV) were fed a methionine- and choline-deficient (MCD) diet for 1 week. α7nAChR knockout (α7KO) chimeric mice were generated by transplanting α7KO bone marrow cells into irradiated and Kupffer cell-deleted WT recipients. Kupffer cells were isolated from WT mice and treated with α7nAChR agonist under stimulation by lipopolysaccharide and/or palmitic acid. HV aggravated MCD diet-induced NASH in both steatosis and inflammation. The hepatic inflammatory response, including the upregulation of tumor necrosis factor alpha (TNFα), interleukin (IL)-12, and monocyte chemoattractant protein 1 (MCP-1), was accelerated in HV mice, accompanied by the downregulation of PPARα pathway genes. Kupffer cells were highly activated via the phosphorylation and nuclear translocation of nuclear factor-kappa B (NF-κB) in MCD diet-fed HV mice. The α7nAchR agonist suppressed the inflammatory response of primary Kupffer cells induced by lipopolysaccharide and palmitic acid by attenuating the NF-κB cascade. α7KO chimeric mice fed an MCD diet for 1 week developed advanced NASH with highly activated Kupffer cells. The hepatic expression of TNFα, IL-12, and MCP-1 was upregulated in α7KO chimeric mice, accompanied by abnormal lipid metabolism. Hepatic vagus activity regulates the inflammatory response of Kupffer cells via α7nAChR in NASH development.

As a novel type of anticancer reagent, imatinib inhibits not only BCR-ABL oncogenic protein but also LCK in T cells as an off-target, being able to selectively deplete mature T reg cells and thereby evoke effective immune responses to various cancers., This report addresses whether small molecules can deplete FoxP3-expressing regulatory T (T reg) cells, thereby augmenting antitumor immunity. Imatinib, a tyrosine kinase inhibitor of oncogenic BCR-ABL protein expressed by chronic myelogenous leukemia (CML) cells, possesses off-targets including LCK expressed in T cells. We showed that imatinib-treated CML patients in complete molecular remission (CMR) exhibited selective depletion of effector T reg (eT reg) cells and significant increase in effector/memory CD8+ T cells while non-CMR patients did not. Imatinib at CML-therapeutic concentrations indeed induced apoptosis specifically in eT reg cells and expanded tumor antigen–specific CD8+ T cells in vitro in healthy individuals and melanoma patients, and suppressed colon tumor growth in vivo in mice. Mechanistically, because of FoxP3-dependent much lower expression of LCK and ZAP-70 in T reg cells compared with other T cells, imatinib inhibition of LCK further reduced their TCR signal intensity, rendering them selectively susceptible to signal-deprived apoptotis. Taken together, eT reg cell depletion by imatinib is instrumental in evoking effective immune responses to various cancers.

Background & Aims Chronic liver injury often results in the activation of hepatic myofibroblasts and the development of liver fibrosis. Hepatic myofibroblasts may originate from 3 major sources: hepatic stellate cells (HSCs), portal fibroblasts (PFs), and fibrocytes, with varying contributions depending on the etiology of liver injury. Here, we assessed the composition of hepatic myofibroblasts in multidrug resistance gene 2 knockout (Mdr2−/−) mice, a genetic model that resembles primary sclerosing cholangitis in patients. Methods Mdr2−/− mice expressing a collagen-GFP reporter were analyzed at different ages. Hepatic non-parenchymal cells isolated from collagen-GFP Mdr2−/− mice were sorted based on collagen-GFP and vitamin A. An NADPH oxidase (NOX) 1/4 inhibitor was administrated to Mdr2−/− mice aged 12–16 weeks old to assess the therapeutic approach of targeting oxidative stress in cholestatic injury. Results Thy1+ activated PFs accounted for 26%, 51%, and 54% of collagen-GFP+ myofibroblasts in Mdr2−/− mice at 4, 8, and 16 weeks of age, respectively. The remaining collagen-GFP+ myofibroblasts were composed of activated HSCs, suggesting that PFs and HSCs are both activated in Mdr2−/− mice. Bone-marrow-derived fibrocytes minimally contributed to liver fibrosis in Mdr2−/− mice. The development of cholestatic liver fibrosis in Mdr2−/− mice was associated with early recruitment of Gr1+ myeloid cells and upregulation of pro-inflammatory cytokines (4 weeks). Administration of a NOX inhibitor to 12-week-old Mdr2−/− mice suppressed the activation of myofibroblasts and attenuated the development of cholestatic fibrosis. Conclusions Activated PFs and activated HSCs contribute to cholestatic fibrosis in Mdr2−/− mice, and serve as targets for antifibrotic therapy. Lay summary Activated portal fibroblasts and hepatic stellate cells, but not fibrocytes, contributed to the production of the fibrous scar in livers of Mdr2−/− mice, and these cells can serve as targets for antifibrotic therapy in cholestatic injury. Therapeutic inhibition of the enzyme NADPH oxidase (NOX) in Mdr2−/− mice reversed cholestatic fibrosis, suggesting that targeting NOXs may be an effective strategy for the treatment of cholestatic fibrosis.

Osteoclasts play a crucial role in osteolytic bone diseases, such as osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease of bone and bone metastatic tumors. Therefore, controlling osteoclast differentiation and function has been considered a promising therapeutic strategy. Here, we show that necrostatin (Nec)-7, an inhibitor of programmed necrosis, strongly suppressed receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption, without compromising macrophage colony-stimulating factor (M-CSF)-supported survival and growth of osteoclast precursor cells. Accordingly, Nec-7 significantly decreased the levels of RANKL-induced osteoclastogenic marker genes, such as cathepsin K. Mechanistically, Nec-7 neither affected MAPK nor NF-κB activation; however, it strongly inhibited the RANKL receptor (RANK) to nuclear factor of activated T cells c1 (NFATc1) signaling. Lentiviral expression of RANK in bone marrow-derived macrophages significantly restored osteoclastogenesis and NFATc1 amplification in Nec-7-treated cells. In this study, we revealed that Nec-7-sensitive pathways are crucially involved in osteoclast formation and function. Investigation of the molecular mechanism(s) through which Nec-7 inhibits RANK-NFATc1 signaling axis may lead to the development of new therapeutic strategies for bone disease.

We prospectively compared the diagnostic accuracies of PET/CT and BS in patients with suspected bone metastases from breast cancer. This single-institution prospective study included consecutive patients with suspected bone metastases from biopsy-proven breast cancer seen at Tokai University Hospital between September 2011 and March 2014. Inclusion criteria included suspicions for bone metastases (bone pain, elevated alkaline phosphatase, elevated tumor markers, or suspected bone metastases by BS). Two nuclear medicine physicians evaluated PET/CT and BS images. Thirty patients were initially enrolled in this study. Two were excluded from the analyses because they declined to undergo imaging during follow-up. PET/CT successfully detected bone metastases in all 10 patients finally diagnosed with the condition, whereas BS identified 2. The two methods were not highly concordant in detecting osseous metastases. In 19 of 28 paired studies (68 %), 2 (10 %) were positive for metastasis, and 17 (90 %) were negative. Nine occurrences (32 %) were discordant; of these, 2 were PET/CT positive and BS negative; 5 were PET/CT positive and BS equivocal; one was PET/CT negative and BS equivocal, and one was PET/CT equivocal and BS negative. Our results indicated that PET/CT was superior to BS for the diagnosis of bone metastases. On the basis of the results of previous studies as well as ours, PET/CT could replace BS as the initial modality to detect bone metastases in patients suspected for the condition.

Background: Stimulation with antigen and IgE is known to activate NF-κB in mast cells. In the present research, we studied the role of NF-κB on cellular migration in mast cell-like RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) using the NF-κB inhibitor (-)-DHMEQ. Methods: A Matrigel invasion chamber was used to evaluate cell migration. A PCR array was used to screen the expression of 84 key genes involved in cell migration. Results: (-)-DHMEQ inhibited antigen/IgE-induced NF-κB activation and expressions of its target genes such as IL-6 and TNF-α. (-)-DHMEQ was found to inhibit in vitro invasion toward the antigen without any toxicity. We then looked for NF-κB-dependent genes that would be important for mast cell invasion using the PCR array. (-)-DHMEQ was found to lower the expression of matrix metalloproteinase (MMP)-2. The MMP inhibitor GM6001 also inhibited cellular invasion toward the antigen. These effects of (-)-DHMEQ were obtained in both RBL-2H3 cells and BMMCs. Conclusions: These findings indicate that (-)-DHMEQ suppressed mast cell migration via the inhibition of NF-κB-regulated MMP-2 expression.

Preconditioning by brief ischemia protects not only the concerned organ but also other distant organs against subsequent lethal damage; this is called remote ischemic preconditioning (RIPC). This study was designed to investigate the impact of intestinal RIPC on hepatic ischemia/reperfusion injury (IRI) with a special interest in heme oxygenase 1 (HO-1) induction in the second window of protection (SWOP). Male Wistar rats were randomly assigned to 1 of 2 groups: an RIPC group or a sham group. Before hepatic IRI, either intestinal RIPC, consisting of 2 cycles of 4-minute superior mesenteric artery clamping separated by 11 minutes of declamping (RIPC group), or a sham procedure (sham group) was performed. After 48 hours of recovery, the rats were exposed to 30 minutes of total hepatic IRI. Transaminase releases and proinflammatory cytokines were determined at several time points after reperfusion. Histopathological analysis and animal survival were also investigated. Intestinal RIPC significantly lowered transaminase release (alanine aminotransferase at 2 hours: 873.3 ± 176.4 IU/L for the RIPC group versus 3378.7 ± 871.1 IU/L for the sham group, P