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We previously showed that human hepatic intrasinusoidal (HI) natural killer (NK) T cells selectively eliminate hepatocellular carcinoma (HCC) cell lines. In this study, we investigated the underlying mechanisms on how HI γδ T cells, expanded with zoledronate, exhibit a superior cytotoxic effect on HI NK-resistant Huh7 HCC cells.γδ T cells were obtained from living liver transplant donors or from peripheral blood mononuclear cells (PBMC) of healthy volunteers and were expanded in the presence of IL-2, IL-15, and zoledronate for 2 weeks. Cytotoxicity was measured using the lactate dehydrogenase (LDH) assay in vitro and by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) in vivo.The cytotoxicity of expanded HI γδ T cells against Huh7 cells was associated with a higher pyrophosphate expression in Huh7 cells compared to SNU398 cells. In contrast, the cytotoxicity of HI γδ T cells against SNU398 cells depended on NKG2D. HI γδ T cells expressed less PD-1 than PB γδ T cells. The cytotoxicity of HI γδ T cells against Du145 and PC3 prostate cancer cells was also associated with pyrophosphate expression in these cells, as well as NKG2D and DNAM-1.The expression levels of phospho-antigen in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules also contribute to their cytotoxicity. γδ T cells are attractive candidates for cancer immune cell therapy.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) aggregates in the brain. Clusterin (CLU), also known as apolipoprotein J, is a potent risk factor associated with AD pathogenesis, in which Aβ aggregation is essentially involved. We observed close colocalization of CLU and Aβ(1-42) (Aβ42) in parenchymal amyloid plaques or vascular amyloid deposits in the brains of human amyloid precursor protein (hAPP)-transgenic Tg2576 mice. Therefore, to elucidate the binding interaction between CLU and Aβ42 and its impact on amyloid aggregation and toxicity, the two synthetic proteins were incubated together under physiological conditions, and their structural and morphological variations were investigated using biochemical, biophysical, and microscopic analyses. Synthetic CLU spontaneously bound to different possible variants of Aβ42 aggregates with very high affinity (Kd = 2.647 nM) in vitro to form solid CLU-Aβ42 complexes. This CLU binding prevented further aggregation of Aβ42 into larger oligomers or fibrils, enriching the population of smaller Aβ42 oligomers and protofibrils and monomers. CLU either alleviated or augmented Aβ42-induced cytotoxicity and apoptosis in the neuroblastoma-derived SH-SY5Y and N2a cells, depending on the incubation period and the molar ratio of CLU:Aβ42 involved in the reaction before addition to the cells. Thus, the effects of CLU on Aβ42-induced cytotoxicity were likely determined by the extent to which it bound and sequestered toxic Aβ42 oligomers or protofibrils. These findings suggest that CLU could influence amyloid neurotoxicity and pathogenesis by modulating Aβ aggregation.

Recurrent gene mutations and copy number alterations in cancer patients are presumably associated with resistance to targeted therapy. In the present study, we assessed the gene mutations and copy number alterations that recurrently occurred in cetuximab-treated patients with metastatic colorectal cancer (mCRC). Targeted next-generation sequencing was performed in the tumor samples obtained pre- and postcetuximab treatment to assess the variations that occurred during cetuximab treatment. Moreover, we identified the emergent gene mutations (CDK6, EPHA3, ERCC2, MYC, PCMTD1, PIK3CA, PRIM2, RICTOR, and ZNRF3) and copy number alterations (ARAF, BCL2, BRCA2, EGFR, MYC, and SMAD4) that were recurrently observed only in postprogression samples and not in pretreatment or posttreatment samples from patients revealing clinical response. Furthermore, to identify the feasible candidate variations implicated in treatment resistance, we examined the variants with clonal expansion during treatment and discovered PCBP1 as a variant associated with posttreatment progression. Various recurrent mutations were enriched in the TGF-beta signaling pathway. Collectively, we identified recurrent variations in mCRC samples exhibiting post-cetuximab progression. Additionally, future studies are required to evaluate the therapeutic potential of these variations.

The anatomy of the left hepatic vein (LHV) is variable; thus, it should be considered for graft hepatic vein (GHV) venoplasty for left lateral section (LLS) and left liver grafts. This study assessed the incidence of superficial LHV (sLHV) branches according to LHV anatomy and its usability for GHV venoplasty in pediatric liver transplantation (LT).This study consisted of three parts: (1) anatomical classification of LHV variations and the incidence of sLHV branches; (2) morphometric simulative analysis of GHV reconstruction and (3) clinical application based on LHV anatomy.The LHV anatomy of 248 potential LLS graft donors was classified into four types according to the number and location of GHV openings: one single opening (type 1; n = 186 [75.0%]), two large openings (type 2; n = 35 [14.1%]), one large and one small adjacent opening (type 3; n = 14 [5.6%]), and two large widely-separated openings (type 4; n = 13 [5.2%]). An sLHV branch was identified in 87 of 248 (35.1%) donor livers. Morphometric analysis of simulative GHV venoplasty with an sLHV branch increased GHV diameters by 30% in type 1 LLS grafts and 20% in type 2/3 LLS grafts. An analysis of 50 consecutive patients who underwent pediatric LT showed that the 2-year rates of GHV obstruction were 2.0% with LLS grafts and 0% with left liver grafts.The GHV orifice can be enlarged through LHV anatomy-based unification venoplasty. Unification venoplasty with an sLHV branch provided sufficient enlargement of the GHV orifice.

Locoregional treatments for hepatocellular carcinoma (HCC) induce immunogenic cell death and a tumor-specific immune response, but infiltration and activation of immune cells in the liver have not been clearly described. Transarterial chemoembolization (TACE) or transarterial chemotherapy (TAC) without embolization have been used to treat intermediate or advanced stage HCC patients. The identification of intrahepatic immune cell changes after locoregional therapy provides a theoretical basis for the combination with immune checkpoint inhibitors (ICIs) in HCC. This study aimed to determine the anticancer effect and changes in the liver immune cell population and function after direct injection of polymerized phenylboronic acid-conjugated doxorubicin (pPBA-Dox) nanocomplexes into the liver through TAC.pPBA-Dox nanocomplexes were delivered directly to the liver cancer in a rat model by transarterial methods. Anticancer effect was confirmed by magnetic resonance imaging (MRI), and the immune cell population and functional changes were confirmed by flow cytometry (FACS).We first established a rat liver cancer model by implanting McA-RH7777 into rats and confirmed the formation of liver cancer through MRI, pathological examinations, and biochemical tests. Transarterial injection of pPBA-Dox nanocomplexes had a stronger anticancer effect than conventional Dox alone. Higher numbers of CD8This study provides a theoretical basis for TAC combined with ICIs and insight into novel targeted therapies using nanocomplexes for the treatment of HCC.

Coronavirus disease 2019 (COVID-19) vaccination in immunocompromised, especially transplant recipients, may induce a weaker immune response. But there are limited data on the immune response after COVID-19 vaccination in liver transplant (LT) recipients, especially on the comparison of Ab responses after different vaccine platforms between mRNA and adenoviral vector vaccines. Thus, we conducted a prospective study on LT recipients who received two doses of the ChAdOx1 nCoV-19 (ChAdOx1), mRNA-1273, or BNT162b2 vaccines compared with healthy healthcare workers (HCWs). SARS-CoV-2 S1-specific IgG Ab titers were measured using ELISA. Overall, 89 LT recipients (ChAdOx1, n=16 [18%]) or mRNA vaccines (mRNA-1273 vaccine, n=23 [26%]; BNT162b2 vaccine, n=50 [56%]) received 3 different vaccines. Of them, 16 (18%) had a positive Ab response after one dose of COVID-19 vaccine and 62 (73%) after 2 doses. However, the median Ab titer after two doses of mRNA vaccines was significantly higher (44.6 IU/ml) than after two doses of ChAdOx1 (19.2 IU/ml, p=0.04). The longer time interval from transplantation was significantly associated with high Ab titers after two doses of vaccine (p=0.003). However, mycophenolic acid use was not associated with Ab titers (p=0.53). In conclusion, about 3-quarters of LT recipients had a positive Ab response after 2 doses of vaccine, and the mRNA vaccines induced higher Ab responses than the ChAdOx1 vaccine.

Multiple pathogenic elements, including reactive oxygen species, amyloidogenic proteins, and metal ions, are associated with the development of neurodegenerative disorders. We report minimalistic redox-based principles for preparing compact aromatic compounds by derivatizing the phenylene moiety with various functional groups. These molecular agents display enhanced reactivities against multiple targets such as free radicals, metal-free amyloid-β (Aβ), and metal-bound Aβ that are implicated in the most common form of dementia, Alzheimer's disease (AD). Mechanistic studies reveal that the redox properties of these reagents are essential for their function. Specifically, they engage in oxidative reactions with metal-free and metal-bound Aβ, leading to chemical modifications of the Aβ peptides to form covalent adducts that alter the aggregation of Aβ. Moreover, the administration of the most promising candidate significantly attenuates the amyloid pathology in the brains of AD transgenic mice and improves their cognitive defects. Our studies demonstrate an efficient and effective redox-based strategy for incorporating multiple functions into simple molecular reagents.

Neurofibromin 1 (NF1) is a tumor suppressor that has been previously reported to regulate RAS-MAPK signaling. The present study investigated the possible relationship between NF1 expression and anti-EGFR antibody (cetuximab) sensitivity in colorectal cancer cell lines. In addition, primary or metastatic colorectal cancer samples from patients treated with cetuximab were assessed for the association of cetuximab sensitivity. The quantities of the NF1 transcript, NF1-related pathway enrichment and NF1 mutation profile were measured and investigated using RNA sequencing and targeted DNA sequencing. Based on growth inhibition and colony formation assay results, cell lines were designated to be cetuximab-sensitive (NCI-H508 and Caco2) or cetuximab-resistant (KM12C and SM480). Western blotting revealed NF1 was highly expressed in cetuximab-sensitive cell lines whilst there was little expression in their cetuximab-resistant counterparts. Knocking down NF1 expression using small interfering RNA in the cetuximab-sensitive cell lines enhanced the phosphorylation of MEK and ERK according to western blotting. NF1 knockdown also reduced apoptosis, as observed by the decreased number of apoptotic bodies by DAPI nuclear staining and reduced cleavage of caspase and poly-(ADP ribose) polymerase. NF1 overexpression by transfection with GTPase-activating protein-related domain subunit rendered the cetuximab-resistant cell lines, KM12C and SW480, more susceptible to cetuximab-induced apoptosis. RNA sequencing of 111 RAS and BRAFV600 wild-type tumor samples collected from cetuximab-treated patients with metastatic colorectal cancer revealed that the pre-treatment NF1 expression levels were not associated with the cetuximab response. However, tumor samples obtained after cetuximab treatment displayed slightly lower NF1 transcript levels compared with those in the pre-treatment samples, suggesting that exposure to the anti-EGFR antibody may be associated with reduced NF1 expression levels. Next-generation sequencing revealed that the frequency of inactivating mutations in NF1 were rare (1.8%) in patients with colorectal cancer and were not associated with the protein expression levels of NF1 except for in a small number of cases (0.5%), where the biallelic inactivation of NF1 was observed. To conclude, the present study showed that modification of NF1 expression can affect sensitivity to cetuximab in colorectal cancer cell lines, though a limitation exists in terms of its potential application as a biomarker for RAS and BRAF V600 wild-type tumors.

Background: Anticancer strategies using nanocarrier systems via the enhanced permeability and retention (EPR) effect and tumor targeting have been explored in various cancers. In previous studies, the anticancer effect of polymerized phenylboronic acid-conjugated doxorubicin (pPBA-Dox) nanocomplexes was confirmed in various cancers, and their anticancer effect and tumor targeting ability was confirmed in hepatocellular carcinoma (HCC).This study aimed to determine the anticancer effect and changes in the liver immune cell population and function after pPBA-Dox nanocomplex infusion through transcatheter arterial chemoembolization (TACE), which is a locoregional therapy (LRT), in HCC. TACE was performed in a rat liver cancer model, and the anticancer effects, immune cell populations and functional changes were confirmed after 1 week. Magnetic resonance imaging (MRI) and flow cytometry (FACS) were performed to analyze the anticancer effect and immune cell population and function. Results: In HCC, the infusion of pPBA-Dox nanocomplexes through TACE had a stronger anticancer effect than conventional doxorubicin (Dox) and it promoted the infiltration and activation of CD4+ and CD8+ T cells in the liver. Conclusions: This study provides insight into novel targeted therapies using nanocomplexes for the treatment of HCC.

Hepatic ischemia and reperfusion (IR) injury contributes to the morbidity and mortality associated with liver transplantation. microRNAs (miRNAs) constitute a family of noncoding RNAs that regulate gene expression at the posttranslational level through the repression of specific target genes. Here, we hypothesized that miRNAs could be targeted to enhance hepatic ischemia tolerance. A miRNA screen in a murine model of hepatic IR injury pointed us toward the liver-specific miRNA miR122. Subsequent studies in mice with hepatocyte-specific deletion of miR122 (miR122loxP/loxP Alb-Cre+ mice) during hepatic ischemia and reperfusion revealed exacerbated liver injury. Transcriptional studies implicated hypoxia-inducible factor-1α (HIF1α) in the induction of miR122 and identified the oxygen-sensing prolyl hydroxylase domain 1 (PHD1) as a miR122 target. Further studies indicated that HIF1α-dependent induction of miR122 participated in a feed-forward pathway for liver protection via the enhancement of hepatic HIF responses through PHD1 repression. Moreover, pharmacologic studies utilizing nanoparticle-mediated miR122 overexpression demonstrated attenuated liver injury. Finally, proof-of-principle studies in patients undergoing orthotopic liver transplantation showed elevated miR122 levels in conjunction with the repression of PHD1 in post-ischemic liver biopsies. Taken together, the present findings provide molecular insight into the functional role of miR122 in enhancing hepatic ischemia tolerance and suggest the potential utility of pharmacologic interventions targeting miR122 to dampen hepatic injury during liver transplantation. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:131 issue:7 ispartof: location:United States status: published

Human liver-derived stem cells (hLD-SCs) have been proposed as a possible resource for stem cell therapy in patients with irreversible liver diseases. However, it is not known whether liver resident hLD-SCs can differentiate toward a hepatic fate better than mesenchymal stem cells (MSCs) obtained from other origins. In this study, we compared the differentiation ability and regeneration potency of hLD-SCs with those of human umbilical cord matrix-derived stem cells (hUC-MSCs) by inducing hepatic differentiation. Undifferentiated hLD-SCs expressed relatively high levels of endoderm-related markers (GATA4 and FOXA1). During directed hepatic differentiation supported by two small molecules (Fasudil and 5-azacytidine), hLD-SCs presented more advanced mitochondrial respiration compared to hUC-MSCs. Moreover, hLD-SCs featured higher numbers of hepatic progenitor cell markers on day 14 of differentiation (CPM and CD133) and matured into hepatocyte-like cells by day 7 through 21 with increased hepatocyte markers (ALB, HNF4A, and AFP). During in vivo cell transplantation, hLD-SCs migrated into the liver of ischemia-reperfusion injury-induced mice within 2 h and relieved liver injury. In the thioacetamide (TAA)-induced liver injury mouse model, transplanted hLD-SCs trafficked into the liver and spontaneously matured into hepatocyte-like cells within 14 days. These results collectively suggest that hLD-SCs hold greater hepatogenic potential, and hepatic differentiation-induced hLD-SCs may be a promising source of stem cells for liver regeneration.

While clusterin is reportedly involved in Alzheimer's disease (AD) pathogenesis, how clusterin interacts with amyloid-β (As) to cause As neurotoxicity remains unclear in vivo. Using 5×FAD transgenic mice, which develop robust AD pathology and memory deficits when very young, we detected interactions between clusterin and As in the mouse brains. The two proteins were concurrently upregulated and bound or colocalized with each other in the same complexes or in amyloid plaques. Neuropathology and cognitive performance were assessed in the progeny of clusterin-null mice crossed with 5×FAD mice, yielding clu-/- ;5×FAD and clu+/+ ;5×FAD. We found far less of the various pools of As proteins, most strikingly soluble As oligomers and amyloid plaques in clu-/- ;5×FAD mice at 5 months of age. At that age, those mice also had higher levels of neuronal and synaptic proteins and better motor coordination, spatial learning and memory than age-matched clu+/+ ;5×FAD mice. However, at 10 months of age, these differences disappeared, with As and plaque deposition, neuronal and synaptic proteins and impairment of behavioral and cognitive performance similar in both groups. These findings demonstrate that clusterin is necessarily involved in early stages of AD pathogenesis by enhancing toxic As pools to cause As-directed neurodegeneration and behavioral and cognitive impairments, but not in late stage.

Label-free optical diffraction tomography (ODT), an imaging technology that does not require fluorescent labeling or other pre-processing, can overcome the limitations of conventional cell imaging technologies, such as fluorescence and electron microscopy. In this study, we used ODT to characterize the cellular organelles of three different stem cells-namely, human liver derived stem cell, human umbilical cord matrix derived mesenchymal stem cell, and human induced pluripotent stem cell-based on their refractive index and volume of organelles. The physical property of each stem cell was compared with that of fibroblast. Based on our findings, the characteristic physical properties of specific stem cells can be quantitatively distinguished based on their refractive index and volume of cellular organelles. Altogether, the method employed herein could aid in the distinction of living stem cells from normal cells without the use of fluorescence or specific biomarkers.

Anti-cancer strategies using nanocarrier systems have been explored in a variety of cancers; these systems can easily be incorporated into tumors via the enhanced permeability and retention (EPR) effect leading to enhanced anti-tumor activity while reducing systemic toxicity by specific tumor-targeting. The prognosis of hepatocellular carcinoma (HCC) is extremely poor when the condition is diagnosed at the unresectable stage as treatment options are limited. In order to improve the treatment of cancer and the overall anti-cancer effect, polymerized phenylboronic acid conjugated doxorubicin (pPBA-Dox) nanocomplexes were generated, and conjugated doxorubicin, which is conventionally used in HCC. The nanocomplexes exhibited enhanced anti-tumor activity via tumor-specific targeting in the subcutaneous and orthotopic HCC syngeneic mice tumor model, implying that the nanocomplexes facilitate the targeted Dox delivery to liver cancer in which the sialic acid is over-expressed. Therefore, this study provides insight into the novel targeted therapy using the nanocomplexes for the treatment of HCC.

As a central feature of neuroinflammation, microglial dysfunction has been increasingly considered a causative factor of neurodegeneration implicating an intertwined pathology with amyloidogenic proteins. Herein, we report the smallest synthetic molecule ( N , N ′-diacetyl- p -phenylenediamine [DAPPD]), simply composed of a benzene ring with 2 acetamide groups at the para position, known to date as a chemical reagent that is able to promote the phagocytic aptitude of microglia and subsequently ameliorate cognitive defects. Based on our mechanistic investigations in vitro and in vivo, 1) the capability of DAPPD to restore microglial phagocytosis is responsible for diminishing the accumulation of amyloid-β (Aβ) species and significantly improving cognitive function in the brains of 2 types of Alzheimer’s disease (AD) transgenic mice, and 2) the rectification of microglial function by DAPPD is a result of its ability to suppress the expression of NLRP3 inflammasome-associated proteins through its impact on the NF-κB pathway. Overall, our in vitro and in vivo investigations on efficacies and molecular-level mechanisms demonstrate the ability of DAPPD to regulate microglial function, suppress neuroinflammation, foster cerebral Aβ clearance, and attenuate cognitive deficits in AD transgenic mouse models. Discovery of such antineuroinflammatory compounds signifies the potential in discovering effective therapeutic molecules against AD-associated neurodegeneration.

Remote ischemic preconditioning (RIPC) is known to have a protective effect against hepatic ischemia-reperfusion (IR) injury in animal models. However, the underlying mechanism of action is not clearly understood. This study examined the effectiveness of RIPC in a mouse model of hepatic IR and aimed to clarify the mechanism and relationship of the ATP-sensitive potassium channel (KATP) and HMGB1-induced TLR4/MyD88/NF-&kappa, B signaling. C57BL/6 male mice were separated into six groups: (i) sham-operated control, (ii) IR, (iii) RIPC+IR, (iv) RIPC+IR+glyburide (KATP blocker), (v) RIPC+IR+diazoxide (KATP opener), and (vi) RIPC+IR+diazoxide+glyburide groups. Histological changes, including hepatic ischemia injury, were assessed. The levels of circulating liver enzymes and inflammatory cytokines were measured. Levels of apoptotic proteins, proinflammatory factors (TLR4, HMGB1, MyD88, and NF-&kappa, B), and I&kappa, B&alpha, were measured by Western blot and mRNA levels of proinflammatory cytokine factors were determined by RT-PCR. RIPC significantly decreased hepatic ischemic injury, inflammatory cytokine levels, and liver enzymes compared to the corresponding values observed in the IR mouse model. The KATP opener diazoxide + RIPC significantly reduced hepatic IR injury demonstrating an additive effect on protection against hepatic IR injury. The protective effect appeared to be related to the opening of KATP, which inhibited HMGB1-induced TRL4/MyD88/NF-kB signaling.

Liver cancer exhibits the fourth most common cause of cancer-associated mortality worldwide. Due to the rapid growth, solid tumors undergo severe hypoxia and produce high levels of extracellular adenosine to maintain homeostasis. A previous study indicated that the hypoxic condition in liver cancer increased hepatic adenosine, which is known to facilitate cancer survival and proliferation. Extracellular adenosine has been revealed to regulate pathological and physiological processes in cells and tissues. However, its pathophysiological role in liver cancer remains undetermined. Emerging evidence has indicated that the adenosine A2B receptor promotes the progression of liver cancer. Therefore, it was hypothesized that HIF-1α is a transcriptional regulator of A2B in human liver cancer. The current study determined A2B expression of a number of liver cancer cell lines and performed functional studies of HIF-1α as a master transcriptional regulator of hepatic A2B signaling during hypoxic conditions. The current study aimed to identify the promoter region of A2B, which has a hypoxia response element, by performing luciferase assays. The present study demonstrated that reduced HIF-1α expression is associated with low expression of A2B, and HIF-1α overexpression is associated with A2B induction. Furthermore, the siRNA-mediated downregulation of A2B inhibited the growth and proliferation of HepG2, which is a liver cancer cell line. The relationship between HIF-1α and A2B expression was also identified in human liver cancer specimens. In conclusion, the current study indicated that A2B is induced by the HIF-1α transcriptional regulator during hypoxia, and it may be a potential pharmacologic and therapeutic target for the treatment of patients with liver cancer.

We report a minimalistic redox-based design strategy for engineering compact molecules based on the simplest aromatic framework, benzene, with multi-reactivity against free radicals, metal-free amyloid-β, and metal-bound amyloid-β, implicated in the most common form of dementia, Alzheimer’s disease.

Hepatic intrasinusoidal (HI) natural killer (NK) cells from liver perfusate have unique features that are similar to those of liver-resident NK cells. Previously, we have reported that HI CD56bright NK cells effectively degranulate against SNU398 hepatocellular carcinoma (HCC) cells. Thus, the aim of this study was to further investigate the phenotype and function of HI NK cells. We found that HI CD56bright NK cells degranulated much less to Huh7 cells. HI CD56bright NK cells expressed NKG2D, NKp46, TNF-related apoptosis-inducing ligand (TRAIL), and FAS ligand (FASL) at higher levels than CD56dim cells. SNU398 cells expressed more NKG2D ligands and FAS and less PD-L1 than Huh7 cells. Blockade of NKG2D, TRAIL, and FASL significantly reduced the cytotoxicity of HI NK cells against SNU398 cells, but blockade of PD-L1 did not lead to any significant change. However, HI NK cells produced IFN-γ well in response to Huh7 cells. In conclusion, the cytotoxicity of HI CD56bright NK cells was attributed to the expression of NKG2D, TRAIL, and FASL. The results suggest the possible use of HI NK cells for cancer immunotherapy and prescreening of HCC cells to help identify the most effective NK cell therapy recipients.

Currently, liver transplantation is the only available remedy for patients with end-stage liver disease. Conservation of transplanted liver graft is the most important issue as it directly related to patient survival. Carbonyl reductase 1 (CBR1) protects cells against oxidative stress and cell death by inactivating cellular membrane-derived lipid aldehydes. Ischemia-reperfusion (I/R) injury during living-donor liver transplantation is known to form reactive oxygen species. Thus, the objective of this study was to investigate whether CBR1 transcription might be increased during liver I/R injury and whether such increase might protect liver against I/R injury. Our results revealed that transcription factor Nrf2 could induce CBR1 transcription in liver of mice during I/R. Pre-treatment with sulforaphane, an activator of Nrf2, increased CBR1 expression, decreased liver enzymes such as aspartate aminotransferase and alanine transaminase, and reduced I/R-related pathological changes. Using oxygen-glucose deprivation and recovery model of human normal liver cell line, it was found that oxidative stress markers and lipid peroxidation products were significantly lowered in cells overexpressing CBR1. Conversely, CBR1 knockdown cells expressed elevated levels of oxidative stress proteins compared to the parental cell line. We also observed that Nrf2 and CBR1 were overexpressed during liver transplantation in clinical samples. These results suggest that CBR1 expression during liver I/R injury is regulated by transcription factor Nrf2. In addition, CBR1 can reduce free radicals and prevent lipid peroxidation. Taken together, CBR1 induction might be a therapeutic strategy for relieving liver I/R injury during liver transplantation.

Programmed death protein 1 (PD-1) pathway is one of the most critical mechanisms in tumor biology of hepatocellular carcinoma (HCC). The study aimed to assess the prognostic influence of pretransplant serum soluble PD-1 (sPD-1) in patients undergoing liver transplantation for treatment of HCC. Data from 229 patients with HCC who underwent living donor liver transplantation between January 2010 and December 2015 were retrospectively evaluated. Stored serum samples were used to measure sPD-1 concentrations. Overall survival (OS) and disease-free survival (DFS) rates were 94.3% and 74.5% at 1 year; 78.2% and 59.2% at 3 years; and 75.4% and 55.5% at 5 years, respectively. Prognostic analysis using pretransplant serum sPD-1 with a cut-off of 93.6 μg/mL (median value of the study cohort) did not have significant prognostic influence on OS (P = .69) and DFS (P = .26). Prognostic analysis using sPD-1 with a cut-off of 300 μg/mL showed similar OS (P = .46) and marginally lower DFS (P = .070). Combination of Milan criteria and sPD-1 with a cutoff of 300 μg/mL showed similar outcomes of OS and DFS in patients within and beyond Milan criteria. Multivariate analysis revealed that only Milan criteria was an independent prognostic for OS and DFS, but pretransplant sPD1 with a cut-off of 300 μg/mL did not become a prognostic factor. The results of this study demonstrate that pretransplant serum sPD-1 did not show significant influences on post-transplant outcomes in patients with HCC. Further large-scale, multicenter studies are necessary to clarify the role of serum sPD-1 in liver transplantation recipients.

Background: The study aimed to assess the prognostic influence of pretransplant serum soluble programmed death protein 1 (sPD-1) in patients undergoing liver transplantation (LT) for treatment of hepatocellular carcinoma (HCC). Methods: Data from 229 patients with HCC who underwent living donor LT between January 2010 and December 2015 were ret rospectively evaluated. Stored serum samples were used to evaluate sPD-1 concentrations. Results: Tumor recurrence, overall survival, and HCC-specific survival rates were 25.5%, 94.3%, and 96.0% at 1 year; 40.8%, 78.2%, and 80.7% at 3 years; and 44.5%, 75.4%, and 77.9% at 5 years, respectively. Prognostic analysis using pretransplant serum sPD-1 with a cutoff of 93.6 µg/mL (median value of the study cohort) did not have significant prognostic influence on HCC recur rence, HCC-specific patient survival and post-recurrence patient survival (P≥0.26). Prognostic analysis using sPD-1 with a cutoff of 300 µg/mL showed marginally higher tumor recurrence (P=0.069), similar HCC-specific patient survival (P=0.25) and higher post-recurrence patient survival (P=0.045). Multivariate analysis revealed that Milan criteria were prognostic for HCC recurrence and HCC-specific patient survival, but pretransplant sPD1 with a cutoff of 300 μg/mL did not become an independent prognostic factor. Conclusions: The results of this study demonstrate that pretransplant serum sPD-1 may not have significant influences on post transplant outcomes in patients with HCC, although there might be some potential prognostic influence from very high expres sion of serum sPD-1. Additional large-scale, multicenter studies and detailed mechanism studies are required to clarify the role of serum sPD-1 in LT recipients.

Objective To examine if hypoxia inducible factor-1α (HIF-1α) can induce the upregulation of the purinergic receptor P2Y2 (P2Y2) and thereby promote the viability of human hepatocellular carcinoma (HCC) cells under hypoxic conditions. Methods Archival HCC tumour specimens and corresponding non-cancerous tissues were examined immunohistochemically for P2Y2 protein. A series of in vitro experiments were undertaken using HCC cell lines to determine the effect of hypoxia on HIF-1α and P2Y2 levels, the effect of HIF-1α upregulation on P2Y2 levels, and the effect of P2Y2 upregulation on cell viability under hypoxic conditions. Results Human HCC specimens were positive for P2Y2. Hypoxia and upregulated HIF-1α both upregulated the P2Y2 levels in HCC cell lines. P2Y2 upregulation using plasmid transfection resulted in enhanced cell viability under hypoxia. Treatment of HepG2 cells with the selective P2Y2 antagonist MRS2312 downregulated P2Y2 and reduced cell viability in five HCC cell lines. P2Y2 knockdown reduced HepG2 cell viability under hypoxia. Conclusions These present results suggest that HCC cells upregulate P2Y2 levels during hypoxia, which in turn promotes their growth. P2Y2 could be a potential therapeutic target for treating HCC.

Objective To investigate the protective effects of epigallocatechin-3-gallate (EGCG), a major polyphenol source in green tea, against hepatic ischaemia–reperfusion injury in mice. Methods The partial hepatic ischaemia–reperfusion injury model was created by employing the hanging-weight method in C57BL/6 male mice. EGCG (50 mg/kg) was administered via an intraperitoneal injection 45 min before performing the reperfusion. A number of markers of inflammation, oxidative stress, apoptosis and liver injury were measured after the ischaemia–reperfusion injury had been induced. Results The treatment groups were: sham-operated (Sham, n = 10), hepatic ischaemia–reperfusion injury (IR, n = 10), and EGCG with ischaemia–reperfusion injury (EGCG-treated IR, n = 10). Hepatic ischaemia–reperfusion injury increased the levels of biochemical and histological markers of liver injury, increased the levels of malondialdehyde, reduced the glutathione/oxidized glutathione ratio, increased the levels of oxidative stress and lipid peroxidation markers, decreased B-cell lymphoma 2 levels, and increased the levels of Bax, cytochrome c, cleaved caspase-3, and cleaved caspase-9. Pretreatment with EGCG ameliorated all of these changes. Conclusion The antioxidant and antiapoptotic effects of EGCG protected against hepatic ischaemia–reperfusion injury in mice.

Background/aim We investigated the expression of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and HBV X protein (HBx) in human hepatocellular carcinoma (HCC) and evaluated the effect of high-concentration nucleos(t)ide analogs (NUCs) on liver tumor cell lines. Materials and methods This study consisted of three parts: part I used human blood and non-tumor liver tissues; part II used human HCC and adjacent liver tissues; and part III used an HBV-expressing liver tumor cell line. Results There were close correlations among blood and liver HBV DNA and liver cccDNA. HBV cccDNA and HBx were highly up-regulated in HCC compared to adjacent liver tissues despite NUC therapy. HBV cccDNA and HBx were highly up-regulated in the cccDNA-expressing HepG2.2.15 cell line. Their expression was down-regulated and apoptosis was induced by a very high concentration of NUCs in dose- and time-dependent manner. Conclusion Very high concentrations of NUCs may have a novel potential to kill replicating HBV-expressing liver tumor cells.

Discovery of non-invasive diagnostic and predictive biomarkers for acute rejection in liver transplant patients would help to ensure the preservation of liver function in the graft, eventually contributing to improved graft and patient survival. We evaluated selected cytokines and chemokines in the sera from liver transplant patients as potential biomarkers for acute rejection, and found that the combined detection of IL-10, IL-17, and CXCL10 at 1-2 weeks post-operation could predict acute rejection following adult liver transplantation with 97% specificity and 94% sensitivity.

Retrospective studies on the association between metformin and clinical outcomes have mainly been performed on patients with non-resectable pancreatic ductal adenocarcinoma and may have been affected by time-related bias. To avoid this bias, recent studies have used time-varying analysis, however, they have only considered the start date of metformin use and not the stop date. We studied 283 patients with type 2 diabetes and pancreatic ductal adenocarcinoma following pancreaticoduodenectomy, and performed analysis using a Cox model with time-varying covariates, while considering both start and stop dates of metformin use. When start and stop dates were not considered, the metformin group showed significantly better survival. Compared with previous studies, adjusted analysis based on Cox models with time-varying covariates only considering the start date of postoperative metformin use showed no significant differences in survival. However, although adjusted analysis considering both start and stop dates showed no significant difference in recurrence-free survival, the overall survival was significantly better in the metformin group (Hazard ratio (HR), 0.747, 95% Confidence interval (CI), 0.562&ndash, 0.993, p = 0.045). Time-varying analysis incorporating both start and stop dates thus revealed that metformin use is associated with a higher overall survival following pancreaticoduodenectomy in patients with type 2 diabetes and pancreatic ductal adenocarcinoma.

The study of the liver microenvironment and hepatocyte’s response to this environment in the setting of healthy liver, cirrhotic liver or cultured primary human hepatocytes (PHHs) addresses key questions for the development of novel liver therapies and predicts relevance of ex vivo PHHs models in liver biology. This study compared quantitative gene and protein expression of the inflammatory profile, oxidative stress response, angiogenesis and homing mechanisms in the biopsies of healthy and cirrhotic human livers and isolated PHHs. These profiles were correlated with the metabolic health of liver and PHHs defined by albumin production. The analysis demonstrated that cirrhotic liver and PHHs exhibited a distinct upregulation of the pro-inflammatory, oxidative stress and homing mechanism markers when compared to normal liver. The upregulation of the oxidative stress markers in PHHs inversely correlated with the albumin production. PHHs had diverse secretion of matrix metalloproteinases and their inhibitors, reflective of the cellular response to non-physiological culture conditions. The current study suggests that ex vivo PHHs manifest adaptive behavior by upregulating stress mechanisms (similar to the cirrhotic liver), downregulating normal metabolic function and upregulating matrix turnover. The ex vivo profile of PHHs may limit their therapeutic functionality and metabolic capacity to serve as in vitro metabolism and toxicology models.

Rationale and Objectives Variation in tissue damage after cerebral ischemia/reperfusion (I/R) can cause uncertainty in stroke-related studies, which can be reduced if the damage can be predicted early after ischemia by measuring the apparent diffusion coefficient (ADC). We investigated whether ADC measurement in the acute phase can predict permanent cerebral I/R injury. Materials and Methods The middle cerebral artery occlusion model was established using the intraluminal suture method to induce 60 minutes of ischemia followed by reperfusion in rats. T2-weighted images and diffusion-weighted images were obtained at 30 minutes and 24 hours after ischemia. Neuronal cell survival was assessed by neuronal nuclei (NeuN) immunofluorescence staining. The correlation between relative ADC (rADC) values at 30 minutes and I/R injury at 24 hours after ischemia was analyzed. Magnetic resonance imaging results were confirmed by histologic analysis. Results The correlation between rADC values at 30 minutes and 24 hours was strong in the ischemic core and peri-infarct region but moderate in the anterior choroidal and hypothalamic region. Histologic analysis revealed that the correlation between rADC values at 30 minutes and the number of NeuN-positive cells at 24 hours was strong in the ischemic core and peri-infarct region but moderate in the anterior choroidal and hypothalamic region. Furthermore, there was a strong positive correlation between the sum of rADC values of three regions at 30 minutes and the infarct volume at 24 hours. Conclusion ADC measurement in the acute phase can predict permanent cerebral I/R injury and provide important information for the evaluation of ischemic stroke.

Backgrounds/Aims Hepatocellular carcinoma (HCC) recurrence remains a great concern following hepatic resection and liver transplantation. We investigated the metformin-induced cytotoxic effects on sorafenib in an in vitro study using HCC cell lines. Methods This research was conducted through an in vitro study using one HepG2.2.15 liver tumor and two patient-derived graft HCC cell lines. Results An in vitro study revealed noticeable cytotoxic effects of metformin as well as noticeable synergistic cytotoxic effects of metformin and sorafenib on cell viability. Assays for the mechanisms of action of antitumor effects revealed that alpha-fetoprotein expression was suppressed by both metformin and sorafenib, but no synergistic effect was observed. LC3-I and LC3-II assays revealed the synergistic upregulation of autophagy and assays for IL-1β, IL-6, p53, and TNF-α revealed the synergistic upregulation of cell damage and apoptosis. In contrast, metformin did not affect HBx expression, thus no noticeable synergistic effect was considered to be present. Conclusions Our in vitro study demonstrated cytotoxic effects of metformin and synergistic antitumor effects of sorafenib. These results should be verified in further clinical studies with patients of advanced HCC.

Purpose OncoHepa test is a multigene expression profile test developed for assessment of hepatocellular carcinoma (HCC) prognosis. Multiplication of α-FP, des-γ-carboxy prothrombin (DCP) and tumor volume (TV) gives the α-FP-DCP-volume (ADV) score, which is also developed for assessment of HCC prognosis. Methods The predictive powers of OncoHepa test and ADV score were validated in 35 patients who underwent curative hepatic resection for naïve solitary HCCs ≤5 cm. Results Median tumor diameter was 3.0 cm. Tumor recurrence and patient survival rates were 28.6% and 100% at 1 year, 48.6% and 82.9% at 3 years, and 54.3% and 71.4% at 5 years, respectively. The site of first tumor recurrence was the remnant liver in 18, lung in 1, and the peritoneum in 1. All patients with HCC recurrence received locoregional treatment. OncoHepa test showed marginal prognostic significance for tumor recurrence and patient survival. ADV score at 4log also showed marginal prognostic difference with respect to tumor recurrence and patient survival. Combination of these 2 tests resulted in greater prognostic significance for both tumor recurrence (P = 0.046) and patient survival (P = 0.048). Conclusion Both OncoHepa test and ADV score have considerably strong prognostic power, thus individual and combined findings of OncoHepa test and ADV score will be helpful to guide postresection surveillance in patients with solitary HCCs ≤5 cm.

Background/aim We investigated metformin-induced cytotoxic effects in vitro and assessed the chemopreventive effects of metformin in patients undergoing hepatic resection (HR) for hepatocellular carcinoma (HCC). Materials and methods This study consisted of laboratory and clinical studies. Results In vitro study using HCC cell lines revealed noticeable cytotoxic effects of metformin, that were largely weaker than those of sorafenib. In the clinical study, no statistical differences were found in tumor recurrence or overall survival between metformin and control groups. In contrast, there was a non-significant difference in tumor recurrence between metformin and propensity score-matched control groups, but there was a significant difference in overall patient survival. Metformin administration was an independent risk factor for patient survival. Conclusion In conclusion, our in vitro laboratory study demonstrated the presence of cytotoxic effects of metformin. Metformin administration was associated with reduced tumor recurrence and helped induce significant improvements in overall patient survival in patients who underwent HR for HCC.

We investigated metformin-induced cytotoxic effects in vitro and assessed the chemopreventive effects of metformin in patients undergoing hepatic resection (HR) for hepatocellular carcinoma (HCC).This study consisted of laboratory and clinical studies.In vitro study using HCC cell lines revealed noticeable cytotoxic effects of metformin, that were largely weaker than those of sorafenib. In the clinical study, no statistical differences were found in tumor recurrence or overall survival between metformin and control groups. In contrast, there was a non-significant difference in tumor recurrence between metformin and propensity score-matched control groups, but there was a significant difference in overall patient survival. Metformin administration was an independent risk factor for patient survival.In conclusion, our in vitro laboratory study demonstrated the presence of cytotoxic effects of metformin. Metformin administration was associated with reduced tumor recurrence and helped induce significant improvements in overall patient survival in patients who underwent HR for HCC.