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Background: In controlled donation after circulatory determination of death (DCD), it is common to administer premortem heparin to potential donors. This practice remains controversial because there is limited evidence for it and there is the possibility of inducing hemorrhage. To our knowledge, no previous studies have assessed the effects of heparin timing and dose on graft function., Methods: We performed a multicentre cohort study of consecutive DCD donors and the recipients of their organs. Anticoagulation administration was considered early if given near the time of withdrawal of life-sustaining measures and late if delayed until the onset of donor hypoxemia (oxygen saturation < 70%) or hypotension (systolic blood pressure < 60 mm Hg or mean blood pressure < 50 mm Hg). The anticoagulation dose was considered high if it was 300 units/kg or greater., Results: Donor anticoagulation data were available for 301 kidney, 75 liver and 46 lung recipients. Heparin was administered in 92% of cases and was most commonly withheld in donors with cerebrovascular causes of death ( p = 0.01). Administration was late in 59% and the dose was low in 27%. Among kidney recipients, there were no significant differences in need for dialysis, glomerular filtration rate over the first year after transplantation or graft survival on the basis of whether or not the donor received heparin, the timing of heparin administration or the dose of heparin. Among liver recipients, alkaline phosphatase concentrations over the first year were significantly higher among recipients who received organs from donors to whom lower doses of heparin had been administered., Conclusion: Premortem heparin is widely used in DCD cases, but there is variability in timing and dose, which was not associated with kidney outcomes in this study. Donor anticoagulation may have a greater impact in preventing biliary complications following liver transplantation., Competing Interests: Competing interests: None declared., (© 2022 CMA Impact Inc. or its licensors.)

Aim: To investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions., Methods: Chimeric mice with humanized liver were treated with 20-hydroxyecdysone for 3 days, and hepatic steroidogenic pathway genes and plasma progesterone were measured by transcriptomics and GC-MS/MS, respectively. Direct effects on muscle and mesenteric arterioles were assessed by myography., Results: CYP17A1 was downregulated in 20-hydroxyecdysone-treated mice compared with untreated group (p = 0.04), with an insignificant increase in plasma progesterone. Progesterone caused vasorelaxation which was blocked by 60 mM KCl, but unaffected by nitric oxide synthase inhibition., Conclusion: In the short term, 20-hydroxyecdysone mediates CYP17A1 downregulation without a significant increase in plasma progesterone, which has a vasodilatory effect involving inhibition of voltage-dependent calcium channels, and the potential to enhance 20-hydroxyecdysone vasorelaxation., (© 2022 The Authors.)

Background & Aims: Association between sarcopenia and mortality in cirrhosis is well recognised; however, little is known about the clinical implications of adipose tissue radiodensity, indicative of biological features. This study aimed to determine an association between high subcutaneous adipose tissue (SAT) radiodensity and survival, compare the prevalence of high SAT radiodensity between healthy population and patients with cirrhosis, and identify an association between computed tomography (CT)-measured SAT radiodensity and histological characteristics., Methods: Adult patients with cirrhosis (n = 786) and healthy donors (n = 129) with CT images taken as part of the liver transplant (LT) assessment were included. Abdominal SAT biopsies (1-2 g) were harvested from the incision site at the time of LT from 12 patients with cirrhosis., Results: The majority of patients were male (67%) with a mean model for end-stage liver disease (MELD) score of 15 ± 8. SAT radiodensity above -83 HU in females (sub-distribution hazard ratio [sHR] 1.84, 95% CI 1.20-2.85, p  = 0.006) and higher than -74 HU in males (sHR 1.51, 95% CI 1.05-1.18, p  = 0.02) was associated with the highest mortality risk after adjusting for confounders in competing risk analysis. The frequency of high SAT radiodensity was 26% for those with cirrhosis, compared with 2% in healthy donors ( p <0.001). An inverse correlation was found between SAT radiodensity and the mean cross-sectional area of SAT adipocytes ( r  = -0.67, p  = 0.02). Shrunken, smaller adipocytes with expanded interstitial space were predominant in patients with high SAT radiodensity, whereas larger adipocytes with a thin rim of cytoplasm were observed in patients with low SAT radiodensity (744 ± 400 vs. 1,521 ± 1,035 μm 2 , p <0.001)., Conclusion: High SAT radiodensity frequently presents and is associated with a higher mortality in cirrhosis. SAT morphological rearrangement in patients with high SAT radiodensity might indicate diminished lipid stores and alterations in tissue characteristics., Lay Summary: Poor quality of subcutaneous adipose tissue (fat under the skin) is associated with higher mortality in patients with end-stage liver disease. Fat cells are smaller in patients with poor adipose tissue quality., Competing Interests: The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Author(s).)

Background: Hepatic artery thrombosis represents a potentially fatal complication following liver transplantation. Rates of hepatic artery thrombosis are significantly higher in children, with mortality reported up to 80 percent. Microsurgical anastomosis has been shown to decrease the rate of hepatic artery thrombosis and now represents the standard of care at the authors' institution. In this article, the authors present the largest study of its type directly comparing rates of hepatic artery thrombosis with and without microsurgical reconstruction of the hepatic artery., Methods: All pediatric patients who underwent primary orthotopic liver transplantation between 1989 and 2018 were included. Patients were divided into two cohorts: standard anastomosis with loupes, and microsurgical anastomosis under the operating microscope. The authors' primary outcome was the rate of hepatic artery thrombosis. Secondary outcomes were graft survival, patient survival, retransplantation rate, requirement for intraoperative blood products, and length of stay., Results: Two hundred thirty-one children met criteria for inclusion. One hundred eighty cases were performed with loupe magnification and 51 cases were performed under the microscope. The hepatic artery thrombosis rate was lower, but not significantly so (p = 0.114), in the microsurgical group [n = 1 (2.0 percent)] compared with the standard cohort [n = 15 (8.3 percent)]. Survival analysis revealed a significant increase in graft survival with microsurgical anastomosis (p = 0.020), but not patient survival (p = 0.196). The retransplantation rate was significantly lower with microsurgical anastomosis (p = 0.021)., Conclusions: Microsurgical anastomosis was associated with a clinically important decrease in hepatic artery thrombosis compared with standard loupe anastomosis. The graft survival rate was significantly higher in the microsurgical cohort, with a reduced retransplantation rate at 1 year. On this basis, the authors recommend microsurgical hepatic artery anastomosis in cases of pediatric liver transplantation. ., Clinical Question/level of Evidence: Therapeutic, III., (Copyright © 2021 by the American Society of Plastic Surgeons.)

Plasma low-density lipoprotein (LDL) is primarily cleared by LDL receptor (LDLR). LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into extracellular milieu. However, the proteinase responsible for LDLR cleavage is unknown. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) co-immunoprecipitates and co-localizes with LDLR and promotes LDLR cleavage. Plasma sLDLR and cholesterol levels are reduced while hepatic LDLR is increased in mice lacking hepatic MT1-MMP. Opposite effects are observed when MT1-MMP is overexpressed. MT1-MMP overexpression significantly increases atherosclerotic lesions, while MT1-MMP knockdown significantly reduces cholesteryl ester accumulation in the aortas of apolipoprotein E (apoE) knockout mice. Furthermore, sLDLR is associated with apoB and apoE-containing lipoproteins in mouse and human plasma. Plasma levels of sLDLR are significantly increased in subjects with high plasma LDL cholesterol levels. Thus, we demonstrate that MT1-MMP promotes ectodomain shedding of hepatic LDLR, thereby regulating plasma cholesterol levels and the development of atherosclerosis.

Background: Undifferentiated embryonal cell sarcoma (UESL) of the liver is the third most common malignant liver disease of childhood presenting as a rapidly enlarging intraabdominal mass. This systematic review explores the practicality of liver transplantation as a viable option in the treatment armamentarium for locally advanced undifferentiated embryonal cell sarcoma., Methods: A systematic review of the literature was performed using Medline and Embase, from inception of databases to December 31, 2018. Keywords and MeSH headings used were embryonal sarcoma, mesenchymal sarcoma, and liver transplant. Reviews and manuscripts with incomplete data were excluded., Results: Twenty-eight patients had orthotopic liver transplantation (OLT) as a curative treatment option. The median age at presentation was 8 and 27 years in the pediatric and adult population, respectively, with a similar male to female ratio. A majority of the patients presented with abdominal pain, palpable mass, and a normal alpha-feto-protein. The median tumor size was 15 cm mainly affecting the right lobe (62%) of the liver. Eighty-two percent of the patients underwent primary OLT and 5 patients had salvage OLT. One death (3.6%) was due to initial misdiagnosis and management for hepatoblastoma. Recurrence was noted in 7.1% of the population. The median follow-up was noted to be 28.5 months. The documented survival rate post-liver transplant for UESL was 96%., Conclusions: Based on available data and the very positive results therein, liver transplantation is a practical and justifiable use of a scarce resource as a treatment option for locally unresectable, undifferentiated embryonal cell sarcoma. The authors propose (accepting existence of different proposals) neoadjuvant therapy before curative resection, and if not achievable, then liver transplantation followed by adjuvant chemotherapy is an option for suitable candidates. For recurrent tumors after surgical resection, adjuvant therapy with salvage liver transplantation is an option., Competing Interests: The authors declare no funding and conflicts of interest., (Copyright © 2021 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Purpose: Donation after circulatory determination of death (DCD) has been performed in Canada since 2006. Numerous aspects of donor management remain controversial., Methods: We performed a multicentre cohort study involving potential DCD donors in western Canada (2008-2017), as well as recipients of their organs, to describe donor characteristics and critical care practices, and their relation to one-year recipient and graft survival., Results: There were 257 patients in four provinces that underwent withdrawal of life-sustaining therapies (WLST) in anticipation of possible DCD. The proportion of patients that died within two hours of WLST ranged from 67% to 88% across provinces (P = 0.06), and was predicted by deeper coma (P = 0.01), loss of pupillary light or corneal reflexes (P = 0.02), and vasopressor use (P = 0.01). There were significant differences between provinces in time intervals from onset of hypotension to death (9-11 min; P = 0.02) and death to vascular cannulation (7-10 min; P < 0.001). There was inconsistency in pre-mortem heparin administration (82-96%; P = 0.03), including timing (before vs after WLST; P < 0.001) and dose (≥ 300 vs < 300 units·kg -1 ; P < 0.001). Donation after circulatory death provided organs for 321 kidney, 81 liver, and 50 lung transplants. One-year recipient and graft survival did not differ among provinces (range 85-90%, P = 0.45). Predictors of death or graft failure included older recipient age (odds ratio [OR] per year, 1.04; 95% confidence interval [CI],1.01 to 1.07) and male donor sex (OR, 3.35; 95% CI, 1.39 to 8.09), but not time intervals between WLST and cannulation or practices related to heparin use., Conclusion: There is significant variability in critical care DCD practices in western Canada, but this has not resulted in significant differences in recipient or graft survival. Further research is required to guide optimal management of potential DCD donors.

Purpose: To evaluate the feasibility of intraoperative continuous renal replacement therapy (IoCRRT) during liver transplantation (LT), in terms of recruitment, protocol adherence, and ascertainment of follow-up., Methods: In this pilot randomized open-label controlled trial in adults receiving LT with a Model for End-Stage Liver Disease (MELD) score ≥ 25 and preoperative acute kidney injury (RIFLE - RISK or higher) and/or estimated glomerular filtration rate < 60 mL·min -1 ·1.73 m -2 , patients were randomized to receive IoCRRT or standard of care (SOC). Primary endpoints were feasibility and adverse events. Primary analysis was intention-to-treat (n = 32) and secondary analysis was per-protocol (n = 28)., Results: The trial was stopped early because of slow patient accrual and inadequate funding. Sixty patients were enrolled and 32 (53%) were randomized (n = 15 IoCRRT; n = 17 SOC). Mean (standard deviation) MELD was 36 (8), 81% (n = 26) had cirrhosis; 69% (n = 22) received preoperative RRT; 66% (n = 21) received LT from the intensive care unit. Four patients (n = 2 IoCRRT, n = 2 SOC) did not receive LT post-randomization. Seven patients (41%) allocated to SOC crossed over intraoperatively to IoCRRT. Three patients were lost to follow-up at one year. No adverse events occurred related to IoCRRT. There were no differences in survival at one year (IoCRRT, 71% [n = 10/14] vs SOC, 93% [n = 14/15]; risk ratio, 0.77; 95% confidence interval, 0.54 to 1.1). In the per-protocol analysis (n = 28 received IoCRRT after randomization - n = 20 IoCRRT, n = 8 SOC), one-year survival was 92% and perioperative complications were similar between groups. Only one patient was receiving dialysis one year after LT., Conclusion: In this pilot randomized trial, IoCRRT was feasible and safe with no difference in complications. Crossover rates were high. Despite high preoperative severity of illness, one-year survival was excellent. These data can inform the design of a larger multicentre trial., Trial Registration: www.clinicalTrials.gov (NCT01575015); registered 12 April, 2012.

Introduction: A primary acinar cell carcinoma (ACC) of the liver was incidentally diagnosed in a clinically asymptomatic 80-year-old man. This study aimed to delineate critical diagnostic characteristics of an ACC originating uniquely from the liver to improve its future identification., Presentation of Case: Enhanced MRI revealed a heterogenous, cystic 7.7 × 11.1 × 10.4 cm tumour occupying hepatic segments II and III. The mass demonstrated mild diffuse enhancement in hepatic arterial phase with minimal portal venous washout in a liver without cirrhotic features. A central stellate T2-hyperintense necrotic scar and outer capsule were apparent. No primary lesion or metastasis outside the liver was discernable. Post-left hepatic lobectomy, the tumour immunophenotype was atypical for presumptive diagnoses of hepatocellular carcinoma (HCC) or cholangiocarcinoma. Extensive morphologic workup on electron microscopy definitively diagnosed primary hepatic ACC by establishing presence of secretory zymogen-like granules, intracytoplasmic microvilli and acinar cell differentiation. Cytopathology revealed cellular lumen expressing PAS-positive diastase-resistant granular cytoplasmic contents., Discussion: This case showcased the novel utility of electron microscopy that was crucial in yielding the definitive diagnosis. The previous literature on hepatic ACC was compiled here in context of the present case. The mechanism of hepatic acinar cell localization was also discussed., Conclusion: Primary hepatic ACC may easily be confused for other lesions due to nonspecific imaging patterns. Specifically, the presence of a central scar without risk factors for HCC can favour a diagnosis of benign entities such as focal nodular hyperplasia (FNH). Electron microscopy presents an important tool to identify primary hepatic ACC and may improve future patient outcomes., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The complete life cycle of the hepatitis C virus (HCV) can be recapitulated in vivo using immunodeficient mice that have had their livers extensively repopulated with human hepatocytes. These human liver chimeric mouse models have enabled the study of many aspects of the HCV life cycle, including antiviral interventions that have helped to shape the curative landscape that is available today. The first human liver chimeric mouse model capable of supporting the HCV life cycle was generated in SCID-uPA mice. Although other human liver chimeric mouse models have since been developed, the SCID-uPA mouse model remains one of the most robust in vivo systems available for HCV studies. This chapter reviews development, validation and application of the SCID-uPA mouse model, and discusses their potential application for studying other liver-centric diseases and pathogens and for the design and testing of vaccine candidates for the eradication of HCV.

Deciding who should receive a liver transplant (LT) depends on both urgency and utility. Most survival scores are validated through discriminative tests, which compare predicted outcomes between patients. Assessing post-transplant survival utility is not discriminate, but should be "calibrated" to be effective. There are currently no such calibrated models. We developed and validated a novel calibrated model to predict individual survival after LT for Primary Sclerosing Cholangitis (PSC). We applied a software tool, PSSP, to adult patients in the Scientific Registry of Transplant Recipients (n = 2769) who received a LT for PSC between 2002 and 2013; this produced a model for predicting individual survival distributions for novel patients. We also developed an appropriate evaluation measure, D-calibration, to validate this model. The learned PSSP model showed an excellent D-calibration (p = 1.0), and passed the single-time calibration test (Hosmer-Lemeshow p-value of over 0.05) at 0.25, 1, 5 and 10 years. In contrast, the model based on traditional Cox regression showed worse calibration on long-term survival and failed at 10 years (Hosmer-Lemeshow p value = 0.027). The calculator and visualizer are available at: http://pssp.srv.ualberta.ca/calculator/liver&#95;transplant&#95;2002. In conclusion we present a new tool that accurately estimates individual post liver transplantation survival.

Environmental contamination and human consumption of chickens could result in potential exposure to Roxarsone (3-nitro-4-hydroxyphenylarsonic acid), an organic arsenical that has been used as a chicken feed additive in many countries. However, little is known about the metabolism of Roxarsone in humans. The objective of this research was to investigate the metabolism of Roxarsone in human liver cells and to identify new arsenic metabolites of toxicological significance. Human primary hepatocytes and hepatocellular carcinoma HepG2 cells were treated with 20 or 100 μM Roxarsone. Arsenic species were characterized using a strategy of complementary chromatography and mass spectrometry. The results showed that Roxarsone was metabolized to more than 10 arsenic species in human hepatic cells. A new metabolite was identified as a thiolated Roxarsone. The 24 h IC 50 values of thiolated Roxarsone for A549 lung cancer cells and T24 bladder cancer cells were 380 ± 80 and 42 ± 10 μM, respectively, more toxic than Roxarsone, whose 24 h IC 50 values for A549 and T24 were 9300 ± 1600 and 6800 ± 740 μM, respectively. The identification and toxicological studies of the new arsenic metabolite are useful for understanding the fate of arsenic species and assessing the potential impact of human exposure to Roxarsone.

O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.The role of O-glycosylation in the malaria life cycle is largely unknown. Here, the authors identify a Plasmodium protein O-fucosyltransferase and show that it is important for normal trafficking of a subset of surface proteins, particularly CSP and TRAP, and efficient infection of mosquito and vertebrate hosts.

The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA-like protein, merozoite apical erythrocyte-binding ligand (MAEBL). MAEBL-deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages., (© 2017 John Wiley & Sons Ltd.)

Malaria sporozoites are deposited into the skin by mosquitoes and infect hepatocytes. The molecular basis of how Plasmodium falciparum sporozoites migrate through host cells is poorly understood, and direct evidence of its importance in vivo is lacking. Here, we generated traversal-deficient sporozoites by genetic disruption of sporozoite microneme protein essential for cell traversal (PfSPECT) or perforin-like protein 1 (PfPLP1). Loss of either gene did not affect P. falciparum growth in erythrocytes, in contrast with a previous report that PfPLP1 is essential for merozoite egress. However, although traversal-deficient sporozoites could invade hepatocytes in vitro, they could not establish normal liver infection in humanized mice. This is in contrast with NF54 sporozoites, which infected the humanized mice and developed into exoerythrocytic forms. This study demonstrates that SPECT and perforin-like protein 1 (PLP1) are critical for transcellular migration by P. falciparum sporozoites and demonstrates the importance of cell traversal for liver infection by this human pathogen., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: To identify the impact of portal vein thrombosis (PVT) and associated medical and surgical factors on outcomes post liver transplant (LT)., Material and Methods: Two analyses were performed. Analysis One: cohort study of 505 consecutive patients who underwent LT (Alberta) between 01/2002-12/2012. PVT was identified in 61 (14%) patients. Analysis Two: cohort study of 144 consecutive PVT patients from two sites (Alberta and London) during the same period. Cox multivariable survival analysis was used to identify independent associations with post-LT mortality., Results: In Analysis One (Alberta), PVT was not associated with post-LT mortality (log rank p = 0.99). On adjusted analysis, complete/occlusive PVT was associated with increased mortality (Hazard Ratio (HR) 8.4, p &lt; 0.001). In Analysis Two (Alberta and London), complete/occlusive PVT was associated with increased mortality only on unadjusted analysis (HR 3.7, p = 0.02). On adjusted analysis, Hepatitis C (HR 2.1, p = 0.03) and post-LT portal vein re-occlusion (HR 3.2, p = 0.01) were independently associated with increased mortality., Conclusion: Well-selected LT patients who had PVT prior to LT had similar post-LT outcomes to non-PVT LT recipients. Subgroups of PVT patients who did worse post-LT (complete/occlusive thrombosis pre-LT, Hepatitis C or post-LT portal vein re-occlusion) warrant closer evaluation in listing and management post-LT.

Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation., Conclusion: The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. (Hepatology 2016;64:1922-1933)., Competing Interests: Statement: The authors have declared that no conflict of interest exists., (© 2016 by the American Association for the Study of Liver Diseases.)

Background: We have previously described the existence of two phenotypically distinct cell subsets in ALK-positive anaplastic large cell lymphoma (ALK + ALCL) based on their differential responsiveness to a Sox2 reporter (SRR2), with reporter-responsive (RR) cells being more tumorigenic and chemoresistant than reporter-unresponsive (RU) cells. However, the regulator(s) of RU/RR dichotomy are not identified. In this study, we aim to delineate the key regulator(s) of RU/RR dichotomy., Methods: JASPER motif match analysis was used to identify the putative factors binding to SRR2 sequence. SRR2 probe pull-down assay and quantitate real-time PCR were performed to analyze the regulation of Sox2 transcriptional activity by MYC. Methylcellulose colony formation assay, chemoresistance to doxorubicin and mouse xenograft study were performed to investigate the biological functions of MYC. PCR array and western blotting were executed to study related signaling pathways that regulate MYC expression. Immunofluorescence and immunohistochemistry assay were initiated to evaluate the expression of MYC and its correlation with its regulator by chi-square test analysis in human primary tumor cells., Results: We identified MYC as a potential regulator of RU/RR dichotomy. In support of its role, MYC was highly expressed in RR cells compared to RU cells, and inhibition of MYC substantially decreased the Sox2/SRR2 binding, Sox2 transcriptional activity, chemoresistance, and methylcellulose colony formation. In contrast, enforced expression of MYC in RU cells conferred the RR phenotype. The Wnt/β-catenin pathway, a positive regulator of MYC, was highly active in RR but not RU cells. While inhibition of this pathway in RR cells substantially decreased MYC expression and SRR2 reporter activity, experimental activation of this pathway led to the opposite effects in RU cells. Collectively, our results support a model in which a positive feedback loop involving Wnt/β-catenin/MYC and Sox2 contributes to the RR phenotype. In a mouse xenograft model, RU cells stably transfected with MYC showed upregulation of the Wnt/β-catenin/MYC/Sox2 axis and increased tumorigenecity. Correlating with these findings, there was a significant correlation between the expression of active β-catenin and MYC in ALK + ALCL primary tumor cells., Conclusions: A positive feedback loop involving the Wnt/β-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subset in ALK + ALCL.

Unlabelled: Individuals chronically infected with hepatitis C virus (HCV) commonly exhibit hepatic intracellular lipid accumulation, termed steatosis. HCV infection perturbs host lipid metabolism through both cellular and virus-induced mechanisms, with the viral core protein playing an important role in steatosis development. We have recently identified a liver protein, the cell death-inducing DFFA-like effector B (CIDEB), as an HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. In this study, we investigated the biological significance and molecular mechanism of this downregulation. HCV infection in a mouse model downregulated CIDEB in the liver tissue, and knockout of the CIDEB gene in a hepatoma cell line results in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very-low-density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis is further supported by the requirement of the HCV core and its LD localization for CIDEB downregulation, which utilize a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB., Importance: Our data demonstrate that HCV infection of human hepatocytesin vitroandin vivoresults in CIDEB downregulation via a proteolytic cleavage event. Reduction of CIDEB protein levels by HCV or gene editing, in turn, leads to multiple aspects of lipid dysregulation, including LD stabilization. Consequently, CIDEB downregulation may contribute to HCV-induced hepatic steatosis., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Cytopathic effects are currently believed to contribute to hepatitis C virus (HCV)-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species, which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase. The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate reactive oxygen species-mediated activation of AMP-activated kinase in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated, and β-oxidation is enhanced in these cells. A striking increase in nuclear peroxisome proliferator-activated receptor α, which plays a dominant role in the expression of β-oxidation genes after ligand-induced activation, was also observed, and we provide evidence that peroxisome proliferator-activated receptor α is constitutively activated in these cells. The combination of attenuated lipid synthesis and enhanced β-oxidation is not conducive to lipid accumulation, yet cellular lipids still accumulated during this stage of infection. Notably, the serum in the culture media was the only available source for polyunsaturated fatty acids, which were elevated (2-fold) in the infected cells, implicating altered lipid import/export pathways in these cells. This study also provided the first in vivo evidence for enhanced β-oxidation during HCV infection because HCV-infected SCID/Alb-uPA mice accumulated higher plasma ketones while fasting than did control mice. Overall, this study highlights the reprogramming of hepatocellular lipid metabolism and bioenergetics during HCV infection, which are predicted to impact both the HCV life cycle and pathogenesis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Carbohydrate response element binding protein (ChREBP) regulates insulin-independent de novo lipogenesis. Recently, a novel ChREBPβ isoform was identified. The purpose of the current study was to define the effect of dietary carbohydrates (CHO) and obesity on the transcriptional activity of ChREBP isoforms and their respective target genes. Mice were subjected to fasting-refeeding of high-CHO diets. In all three CHO-refeeding groups, mice failed to induce ChREBPα, yet ChREBPβ increased 10- to 20-fold. High-fat fed mice increased hepatic ChREBPβ mRNA expression compared to chow-fed along with increased protein expression. To better assess the independent effect of fructose on ChREBPα/β activity, HepG2 cells were treated with fructose ± a fructose-1,6-bisphosphatase inhibitor to suppress gluconeogenesis. Fructose treatment in the absence of gluconeogenesis resulted in increased ChREBP activity. To confirm the existence of ChREBPβ in human tissue, primary hepatocytes were incubated with high-glucose and the expression of ChREBPα and -β was determined. As with the animal models, glucose induced ChREBPβ expression while ChREBPα was decreased. Taken together, ChREBPβ is more responsive to changes in dietary CHO availability than the -α isoform. Diet-induced obesity increases basal expression of ChREBPβ, which may increase the risk of developing hepatic steatosis, and fructose-induced activation is independent of gluconeogenesis.

Background. Since 2002, the Model of End-Stage Liver Disease (MELD) has been used for allocation of liver transplants (LT) in the USA. In Canada, livers were allocated by the CanWAIT algorithm. The aim of this study was to compare the abilities of MELD, Child-Pugh (CP), and CanWAIT status to predict 3-month and 1-year mortality before LT in Canadian patients and to describe the use of MELD in Canada. Methods. Validation of MELD was performed in 320 patients listed for LT in Alberta (1998-2002). In October 2014, a survey of MELD use by Canadian LT centers was conducted. Results. Within 1 year of listing, 47 patients were removed from the waiting list (29 deaths, 18 too ill for LT). Using logistic regression, the MELD and CP were better than the CanWAIT at predicting 3-month (AUROC: 0.79, 0.78, and 0.59; p = 0.0002) and 1-year waitlist mortality (AUROC: 0.70, 0.70, and 0.55; p = 0.0023). Beginning in 2004, MELD began to be adopted by Canadian LT programs but its use was not standardized. Conclusions. Compared with the CanWAIT system, the MELD score was significantly better at predicting LT waitlist mortality. MELD-sodium (MELD-Na) has now been adopted for LT allocation in Canada.

The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis., (© 2015 by The American Society of Hematology.)

Approximately 3% of the world׳s population suffers from chronic infections with hepatitis C virus (HCV). Although current treatment regimes are capable of effectively eradicating HCV infection from these patients, the cost of these combinations of direct-acting antivirals are prohibitive. Approximately 80% of untreated chronic HCV carriers will be at high risk for developing severe liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. A vaccine is urgently needed to lessen this global burden. Besides humans, HCV infection can be experimentally transmitted to chimpanzees, and this is the best model for studies of HCV infection and related innate and adaptive immune responses. Although the chimpanzee model yielded valuable insight, limited availability, high cost and ethical considerations limit their utility. The only small animal models of robust HCV infection are highly immunodeficient mice with human chimeric livers. However, these mice cannot be used to study adaptive immune responses and therefore a more relevant animal model is needed to assist in vaccine development. Novel strains of immunodeficient mice have been developed that allow for the engraftment of human hepatopoietic stem cells, as well as functional human lymphoid cells and tissues, effectively creating human immune systems in otherwise immunodeficient mice. These humanized mice are rapidly emerging as pre-clinical bridges for numerous pathogens that, like HCV, only cause infectious disease in humans. This review highlights the potential these new models have for changing the current landscape for HCV research and vaccine development., (Copyright © 2015. Published by Elsevier B.V.)

Background: Improving estimation of long-term survival of patients with end-stage liver disease after orthotopic liver transplantation (OLT) would optimize decisions on eligibility for transplant. We aimed to externally validate previously derived Charlson Comorbity Index for OLT (CCI-OLT); subsequently, we developed a new model to predict 5-year mortality after transplant., Material and Methods: This single center retrospective cohort study included 524 consecutive adult cirrhotic patients who underwent OLT in 2002-2012. External validation of CCI-OLT used Kaplan-Meier method. Derivation of the new predictive model used Cox proportional hazards regression., Results: One-, 3-, and 5-year cumulative survival after OLT was 89%, 80%, and 73%, respectively. CCI-OLT was not associated with 5-year mortality after transplant (P = 0.34). We derived and internally validated a new predictive model of 5-year mortality after OLT based on six pre-transplant characteristics of patients: age, body mass index, hepatitis C, hepatic encephalopathy, intensive care unit stay at transplant, and live donor (C-index = 0.64). We further developed a nomogram to estimate individual probability of 1-, 3-, and 5-year survival after OLT., Conclusions: In our cohort, CCI-OLT was not associated with survival following transplant. The new predictive model discriminative capacity was only modest, suggesting that pre-transplant characteristics are of limited value in predicting post-transplant outcomes in thoroughly selected patients.

Aims/hypothesis: There are potential advantages to the low-temperature (-196 °C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years., Methods: Human islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes., Results: The cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation., Conclusions/interpretation: We have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.

Unlabelled: The selection of liver transplantation (LT) candidates with hepatocellular carcinoma (HCC) is currently validated based on Milan criteria. The use of extended criteria has remained a matter of debate, mainly because of the absence of prospective validation. The present prospective study recruited patients according to the previously proposed total tumor volume (TTV; ≤115 cm(3) )/alpha-fetoprotein (AFP; ≤400 ng/mL) score. Patients with AFP >400 ng/mL were excluded, and, as such, the Milan group was modified to include only patients with AFP <400 ng/mL; these patients were compared to patients beyond Milan, but within TTV/AFP. From January 2007 to March 2013, 233 patients with HCC were listed for LT. Of them, 195 patients were within Milan and 38 beyond Milan, but within TTV/AFP. The average follow-up from listing was 33.9 ± 24.9 months. Risk of dropout was higher for patients beyond Milan, but within TTV/AFP (16 of 38; 42.1%), than for those within Milan (49 of 195 [25.1%]; P = 0.033). In parallel, intent-to-treat survival from listing was lower in patients beyond Milan (53.8% vs. 71.6% at 4 years; P < 0.001). After a median waiting time of 8 months, 166 patients were transplanted, 134 within Milan criteria, and 32 beyond Milan but within TTV/AFP. They demonstrated acceptable and similar recurrence rates (4.5% vs. 9.4%; P = 0.138) and post-transplant survivals (78.7% vs. 74.6% at 4 years; P = 0.932)., Conclusion: Based on the present prospective study, HCC LT candidate selection could be expanded to the TTV (≤115 cm(3) )/AFP (≤400 ng/mL) criteria in centers with at least 8-month waiting time. An increased risk of dropout on the waiting list can be expected, but with equivalent and satisfactory post-transplant survival., (© 2015 by the American Association for the Study of Liver Diseases.)

Arsenic is a proven human carcinogen and is associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high-affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)(2)]. In rats, the related canalicular transporter Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)(3) and MMA(GS)(2). The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic efflux was detected only across the basolateral membrane for 9 out of 14 SCHH preparations, 5 had both basolateral and canalicular efflux. Basolateral transport of arsenic was temperature- and GSH-dependent and inhibited by the MRP inhibitor MK-571. Canalicular efflux was completely lost after GSH depletion suggesting MRP2-dependence. Treatment of SCHH with As(III) (0.1-1 µM) dose-dependently increased MRP2 and MRP4 levels, but not MRP1, MRP6, or aquaglyceroporin 9. Treatment of SCHH with oltipraz (Nrf2 activator) increased MRP4 levels and basolateral efflux of arsenic. In contrast, oltipraz increased MRP2 levels without increasing biliary excretion. These results suggest arsenic basolateral transport prevails over biliary excretion and is mediated at least in part by MRPs, most likely including MRP4., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Cirrhotic patients with Model for End-stage Liver Disease (MELD) score ≥ 40 have high risk for death without liver transplant (LT)., Objective: To evaluate these patients' outcomes after LT., Methods: The present study analyzed a retrospective cohort of 519 cirrhotic adult patients who underwent LT at a single Canadian centre between 2002 and 2012. Primary exposure was severity of liver disease measured by MELD score at LT (≥ 40 versus < 40). Primary outcome was duration of first intensive care unit (ICU) stay after LT. Secondary outcomes were duration of first hospital stay after LT, rate of ICU readmission, re-LT and survival rates., Results: On the day of LT, 5% (28 of 519) of patients had a MELD score ≥ 40. These patients had longer first ICU stays after LT (14 versus two days; P < 0.001). MELD score ≥ 40 at LT was independently associated with first ICU stay after LT ≥ 10 days (OR 3.21). These patients had longer first hospital stays after LT (45 versus 18 days; P < 0.001); however, there was no significant difference in the rate of ICU readmission (18% versus 22%; P = 0.58) or re-LT rate (4% versus 4%; P = 1.00). Cumulative survival at one month, three months, one year, three years and five years was 98%, 96%, 90%, 79% and 72%, respectively. There was no significant difference in cumulative survival stratified according to MELD score ≥ 40 versus < 40 at LT (P = 0.59)., Conclusions: Cirrhotic patients with MELD score ≥ 40 at LT utilize greater postoperative health resources; however, they derive similar long-term survival benefit from LT.

We have recently described a novel phenotypic dichotomy within estrogen receptor-positive breast cancer cells; the cell subset responsive to a Sox2 regulatory region (SRR2) reporter (RR cells) are significantly more tumorigenic than the reporter unresponsive (RU) cells. Here, we report that a similar phenomenon also exists in triple negative breast cancer (TNBC), with RR cells more tumorigenic than RU cells. First, examination of all 3 TNBC cell lines stably infected with the SRR2 reporter revealed the presence of a cell subset exhibiting reporter activity. Second, RU and RR cells purified by flow cytometry showed that RR cells expressed higher levels of CD44, generated more spheres in a limiting dilution mammosphere formation assay, and formed larger and more complex structures in Matrigel. Third, within the CD44(High)/CD24- tumor-initiating cell population derived from MDA-MB-231, RR cells were significantly more tumorigenic than RU cells in an in vivo SCID/Beige xenograft mouse model. Examination of 4 TNBC tumors from patients also revealed the presence of a RR cell subset, ranging from 1.1-3.8%. To conclude, we described a novel phenotypic heterogeneity within TNBC, and the SRR2 reporter responsiveness is a useful marker for identifying a highly tumorigenic cell subset within the CD44(High)/CD24-tumor-initiating cell population.

For selected patients with type 1 diabetes, β-cell replacement is the treatment of choice, either by islet transplantation (ITX) or whole pancreas transplantation (PTX). When either modality fails, current practice is to consider retransplantation, or return to exogenous insulin. We investigate outcomes with PTX after failed ITX (PAI), and ITX after failed PTX (IAP). All patients receiving PAI or IAP at a single institution were identified. Donor and recipient variables were documented, including transplant outcomes analyzed for insulin requirement and metabolic control. Five subjects were listed for PAI, and 2 received transplants. Of the 4 listed for IAP, 3 have received transplants. The mean waitlist time was 4.5 ± 4.1 y for PAI and 0.35 ±0 .4 y for IAP (p = 0.08). Metabolic control was excellent after PAI, with 2/2 insulin-independent. After IAP, 1/2 achieved insulin independence and good metabolic control after 2 islet infusions. The third could not receive 2(nd) infusion and presented c-peptide levels < 0.1 nmol/L. Both strategies are feasible. The outcomes after PAI in our center must be offset by much longer waitlist time due to the sensitization status of these patients. Data from multicentre experience will allow more robust comparative outcomes to be made, the current observations being restricted to a limited patient set.

Purpose: Intensive care unit (ICU) readmission negatively impacts patients' outcomes. We aimed to characterize and determine risk factors for ICU readmission within the initial hospital stay after liver transplant (LT)., Materials and Methods: The reference cohort included 369 LT recipients from a Canadian center between 2005 and 2012. One control was randomly selected per each case of ICU readmission within the initial hospital stay after LT. Survival analysis used the Kaplan-Meier method. Associations were studied by conditional logistic regression., Results: Fifty-two (14%) LT recipients were readmitted to the ICU within the initial hospital stay after LT; they had longer first hospital stay (P < .001) and lower 1-month to 2-year cumulative survival (P < .001). Respiratory failure was the major cause of ICU readmission (61%). Respiratory rate at discharge from the first ICU stay after LT was an independent risk factor for ICU readmission (odds ratio = 1.24). The cutoff point more than 20 breaths per minute prognosticated ICU readmission with a specificity of 90% and a positive predictive value of 80%., Conclusions: Intensive care unit readmission within the initial hospital stay after LT negatively impacts LT recipients' outcomes. Monitoring respiratory rate at discharge from the first ICU stay after LT is important to prevent readmission., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Total tumor volume (TTV), as a better predictor of hepatocellular carcinoma (HCC) recurrence after liver transplant, has been explored by our center. Some tumors are not typically spherical but rather ellipsoid or spheroid, and calculating their TTV based on one dimension only may overestimate their volume and exclude them from candidacy for transplantation. Our aim was to study the actual tumor volume (ATV) calculated using the ellipsoid formula and assess its impact on recurrence. HCC patients transplanted between 1990 and 2010 at University of Alberta Hospital were analyzed. Tumor volumes were calculated using both formulas: [(4/3) πr(3)] (r = max. radius) and [(4/3) πabc] (a, b, c = the 3 radiuses). A total of 115 patients were included with a mean follow-up of 4.99 ± 4.23 yr. Five-yr recurrence-free survival was 79.8%. Univariate analysis for predictors of recurrence included: maximum tumor diameter, ATV, TTV, and alpha-fetoprotein (AFP) ≥ 400 ng/mL. Multivariate analysis showed that ATV and AFP ≥ 400 ng/mL were the only predictors of recurrence. Combining both variables provides better predication of recurrence with accuracy that exceeds 80%. Three-dimensional calculation of tumor volume is of critical importance for the group of patients with ellipsoid tumors where volumes are overestimated with the spherical formula and could lead to inappropriate exclusion from transplant., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

1. Immunocompromised mice with humanized livers were developed in the mid-1990s to allow the study of human hepatotropic viruses, which normally replicate only in higher primates. The production of the uPA/SCID mouse was the vanguard of these models and remains the most widely worked upon model for an ever increasing range of applications. 2. Since toxicology is conducted in laboratory animal species with the implicit intent of predicting the outcome of accidental, or intentional, human exposure, the potential for using an in vivo model with a humanised metabolism opens up the possibility of better predicting the human response following exposure to drugs and industrial chemicals. Chimeric humanised mice provide the tool for bridging between the non-clinical laboratory safety and metabolism studies, carried out in rodent and non-rodent species, and the first in man clinical trials. 3. Chimeric mice carrying a human liver have now been validated against a wide range of different drugs and chemical classes, and have been shown to clearly differentiate metabolically from the recipient mouse, and to show metabolic pathways more similar to those expected from human liver. 4. This review critically appraises the available animal models carrying human livers and where future developments would improve the existing systems.

This prospective inception cohort study determines kindergarten-entry neurocognitive abilities and explores their predictors following liver transplantation at age <3 yr. Of 52 children transplanted (1999-2008), 33 (89.2%) of 37 eligible survivors had psychological assessment at age 54.7 (8.4) months: 21 with biliary atresia, seven chronic cholestasis, and five acute liver failure. Neurocognitive scores (mean [s.d.], 100 [15]) as tested by a pediatric-experienced psychologist did not differ in relation to age group at transplant (≤12 months and >12 months): FSIQ, 93.9 (17.1); verbal (VIQ), 95.3 (16.5); performance (PIQ), 94.3 (18.1); and VMI, 90.5 (15.9), with >70% having scores ≥85, average or above. Adverse predictors from the pretransplant, transplant, and post-transplant (30 days) periods using univariate linear regressions for FSIQ were post-transplant use of inotropes, p = 0.029; longer transplant warm ischemia time, p = 0.035; and post-transplant highest serum creatinine, (p = 0.04). For PIQ, they were pretransplant encephalopathy, p = 0.027; post-transplant highest serum creatinine, p = 0.034; and post-transplant inotrope use, p = 0.037. For VMI, they were number of post-transplant infections, p = 0.019; post-transplant highest serum creatinine, p = 0.025; and lower family socioeconomic index, p = 0.039. Changes in care addressing modifiable predictors, including reducing acute post-transplant illness, pretransplant encephalopathy, transplant warm ischemia times, and preserving renal function, may improve neurocognitive outcomes., (© 2013 John Wiley & Sons A/S.)

Background & Aims: Despite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10-20% of cases after liver transplantation, and the use of potent adjuvant anticancer drugs would be welcome. The aim of this study was to evaluate the efficiency of a combined therapy of rapamycin (sirolimus) and anti-death receptor (DR)5 monoclonal antibody (mAb) on HCC., Methods: We first assessed the side effects of anti-DR5 mAb administration in vivo by giving various doses of anti-DR5 mAb. Cell proliferation assays were then performed using mouse Hepa1-6 cells or human Huh7 cells to quantify the relative cell viability under various concentrations of sirolimus, anti-DR5 mAb or a combination. Next, one million Hepa1-6 cells were transplanted into C.B17-SCID-beige mice subcutaneously, and four groups were created: (1) untreated, (2) anti-DR5 mAb alone, (3) sirolimus alone and (4) anti-DR5 mAb + sirolimus., Results: Anti-DR5 mAb (200 and 300 μg/day) induced liver dysfunction with partial necrosis of the liver, but 100 μg/day was well tolerated with transaminitis, but normal bilirubin and only minor histological liver damage. In vitro, anti-DR5 mAb lysed Hepa1-6 and Huh7 cells in a dose-dependent manner, and combinations of sirolimus and anti-DR5 mAb demonstrated an additive effect. In vivo studies demonstrated that tumour sizes were significantly smaller in the combined therapy group than in the monotherapy groups., Conclusions: Combining sirolimus and low-dose anti-DR5 mAb has a significant effect against HCC. This strategy represents a potential novel approach for the management of HCC., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Aims: Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production., Methods: We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches., Results: Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle., Conclusions: This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Aim: Cytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. To date, there is no established method to evaluate the effects of human CMV (HCMV) treatments in vivo except for human clinical trials. In the current study, we describe the development of a mouse model that supports the in vivo propagation of HCMV., Methods: One million viable human hepatocytes, purified from human livers, were injected into the spleens of severe combined immunodeficient/albumin linked-urokinase type plasminogen activator transgenic mice. A clinical strain of HCMV was inoculated in mice with confirmed human hepatocyte engraftment or in non-chimeric controls. Infection was monitored through HCMV titers in the plasma. Mice were administrated ganciclovir (50 mg/kg per day, i.p.) beginning at 2 days post-HCMV inoculation, or human liver natural killer (NK) cells (20 × 10(6)  cells/mouse, i.v.) 1 day prior to HCMV inoculation., Results: Chimeric mice that received HCMV showed high plasma titers of HCMV DNA on days 1 and 6 that became undetectable by day 11 post-inoculation. In contrast, non-transplanted mice had only residual plasma inoculum detection at day 1 and no detectable viremia thereafter. The levels of HCMV DNA were reduced by ganciclovir treatment or by human liver NK cell adoptive transfer, while HCMV-infected chimeric mice that were not treated sustained viremia during the follow up., Conclusion: Human liver chimeric mice provide an in vivo model for the study of acute HCMV infection of hepatocytes., (© 2012 The Japan Society of Hepatology.)

Background: Although factors associated with an increased risk of recurrence after liver transplantation for hepatocellular carcinoma (HCC) have been extensively studied, the history of patients with a post-transplant recurrence is poorly known., Methods: Patients experiencing a post-transplant HCC recurrence from 1996 to 2011 in two transplant programs were included. Demographic, transplant, and post-recurrence variables were assessed., Results: Thirty patients experienced an HCC recurrence-22 men and 8 women with a mean age of 55 ± 6 years. Sixteen (53 %) were outside the Milan criteria at the time of transplantation. Most recurrences (60 %) appeared within the first 18 months after transplantation, ranging between 1.7 and 109 months (median 14.2 months). Mean post-recurrence survival was 33 ± 31 months. On univariate analysis, total tumor volume (TTV; p = 0.047), microvascular invasion (p = 0.011), and time from transplant to recurrence (p = 0.001) predicted post-recurrence survival. On multivariate analysis, both time from transplant to recurrence (p = 0.001) and history of rejection (p = 0.043), but not the location of the recurrence or the type of recurrence treatment, predicted post-recurrence survival., Conclusion: This study suggests that patients with early post-transplant HCC recurrence have worse outcomes. Those with a history of graft rejection have better survivals, possibly due to more active anti-cancer immunity.

Introduction: Critically ill cirrhosis patients awaiting liver transplantation (LT) often receive prioritization for organ allocation. Identification of patients most likely to benefit is essential. The purpose of this study was to examine whether the Sequential Organ Failure Assessment (SOFA) score can predict 90-day mortality in critically ill recipients of LT and whether it can predict receipt of LT among critically ill cirrhosis listed awaiting LT., Methods: We performed a multicenter retrospective cohort study consisting of two datasets: (a) all critically-ill cirrhosis patients requiring intensive care unit (ICU) admission before LT at five transplant centers in Canada from 2000 through 2009 (one site, 1990 through 2009), and (b) critically ill cirrhosis patients receiving LT from ICU (n = 115) and those listed but not receiving LT before death (n = 106) from two centers where complete data were available., Results: In the first dataset, 198 critically ill cirrhosis patients receiving LT (mean (SD) age 53 (10) years, 66% male, median (IQR) model for end-stage liver disease (MELD) 34 (26-39)) were included. Mean (SD) SOFA scores at ICU admission, at 48 hours, and at LT were 12.5 (4), 13.0 (5), and 14.0 (4). Survival at 90 days was 84% (n = 166). In multivariable analysis, only older age was independently associated with reduced 90-day survival (odds ratio (OR), 1.07; 95% CI, 1.01 to 1.14; P = 0.013). SOFA score did not predict 90-day mortality at any time. In the second dataset, 47.9% (n = 106) of cirrhosis patients listed for LT died in the ICU waiting for LT. In multivariable analysis, higher SOFA at 48 hours after admission was independently associated with lower probability of receiving LT (OR, 0.89; 95% CI, 0.82 to 0.97; P = 0.006). When including serum lactate and SOFA at 48 hours in the final model, elevated lactate (at 48 hours) was also significantly associated with lower likelihood of receiving LT (0.32; 0.17 to 0.61; P = 0.001)., Conclusions: SOFA appears poor at predicting 90-day survival in critically ill cirrhosis patients after LT, but higher SOFA score and elevated lactate 48 hours after ICU admission are associated with a lower probability receiving LT. Older critically ill cirrhosis patients (older than 60) receiving LT have worse 90-day survival and should be considered for LT with caution.

Primary rodent hepatocytes and hepatoma cell lines are commonly used as model systems to elucidate and study potential drug targets for metabolic diseases such as obesity and atherosclerosis. However, if therapies are to be developed, it is essential that our knowledge gained from these systems is translatable to that of human. Here, we have characterized lipid and lipoprotein metabolism in primary human hepatocytes for comparison to rodent primary hepatocytes and human hepatoma cell lines. Primary human hepatocytes were maintained in collagen coated dishes in confluent monolayers for up to 3 days. We found primary human hepatocytes were viable, underwent lipid synthesis, and were able to secret lipoproteins up to 3 days in culture. Furthermore, the lipoprotein profile secreted by primary human hepatocytes was comparable to that found in human plasma; this contrasts with primary rodent hepatocytes and human hepatoma cells. We also investigated the pharmacological effects of nicotinic acid (niacin, NA), a potent dyslipidemic drug, on hepatic lipid synthesis and lipoprotein secretion. We found NA increased the expression of ATP-binding cassette transporter A1 in primary human hepatocytes, which may potentially explain how NA increases plasma high-density lipoproteins in humans. In conclusion, primary human hepatocytes are a more relevant model to study lipid synthesis and lipoprotein secretion than hepatoma cells or rodent primary hepatocyte models. Further research needs to be done to maintain liver specific functions of primary human hepatocytes in prolonged cultures for these cells to be a viable model., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Unlabelled: In many countries, the allocation of liver grafts is based on the Model of End-stage Liver Disease (MELD) score and the use of exception points for patients with hepatocellular carcinoma (HCC). With this strategy, HCC patients have easier access to transplantation than non-HCC ones. In addition, this system does not allow for a dynamic assessment, which would be required to picture the current use of local tumor treatment. This study was based on the Scientific Registry of Transplant Recipients and included 5,498 adult candidates of a liver transplantation for HCC and 43,528 for non-HCC diagnoses. A proportional hazard competitive risk model was used. The risk of dropout of HCC patients was independently predicted by MELD score, HCC size, HCC number, and alpha-fetoprotein. When combined in a model with age and diagnosis, these factors allowed for the extrapolation of the risk of dropout. Because this model and MELD did not share compatible scales, a correlation between both models was computed according to the predicted risk of dropout, and drop-out equivalent MELD (deMELD) points were calculated., Conclusion: The proposed model, with the allocation of deMELD, has the potential to allow for a dynamic and combined comparison of opportunities to receive a graft for HCC and non-HCC patients on a common waiting list., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Cyclosporine A (CSA) has potent effects against hepatitis C virus (HCV) in vitro, but its clinical efficacy after liver transplantation (LT) is uncertain. We evaluated the impact of CSA and tacrolimus (TAC) with or without concomitant interferon (IFN) therapy on serum HCV titers in a chimeric mouse model of HCV infection. Six groups of HCV-infected mice received only the vehicle, IFN, CSA, CSA and IFN, TAC, or TAC and IFN for 4 weeks. The quantitative HCV polymerase chain reaction levels were determined after 1, 2, and 4 weeks of drug administration. There were no significant differences in the HCV titers after 4 weeks of treatment between the non-IFN-treated groups (log HCV titers: 3.5 ± 0.3 for the vehicle group, 4.4 ± 0.6 for the CSA group, and 4.3 ± 0.4 for the TAC group, P = 0.3). Although IFN had a consistent effect of reducing HCV titers across the groups, there was no significant impact of CSA on HCV levels when it was used alone or in combination with IFN at any time point. The 4-week HCV titers were as follows: 3.2 ± 0.3 for the IFN group, 4.7 ± 0.4 for the CSA/IFN group, and 4.0 ± 0.5 for the TAC/IFN group (P = 0.07). The CSA/IFN and TAC/IFN groups did not differ significantly (P = 0.6). Six of the 7 animals in the IFN group (85.7%) had an HCV titer decline ≥ 1 log, whereas in the test groups (CSA/IFN and TAC/IFN), 6 of 9 animals (66.7%) achieved a 1-log decline in the HCV titer (P = 1). Using this animal model, we could find no evidence supporting the routine use of CSA after LT in HCV-infected patients., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Primary human hepatocytes isolated from patient biopsies represent the most physiologically relevant cell culture model for hepatitis C virus (HCV) infection, but these primary cells are not readily accessible, display individual variability, and are largely refractory to genetic manipulation. Hepatocyte-like cells differentiated from pluripotent stem cells provide an attractive alternative as they not only overcome these shortcomings but can also provide an unlimited source of noncancer cells for both research and cell therapy. Despite its promise, the permissiveness to HCV infection of differentiated human hepatocyte-like cells (DHHs) has not been explored. Here we report a novel infection model based on DHHs derived from human embryonic (hESCs) and induced pluripotent stem cells (iPSCs). DHHs generated in chemically defined media under feeder-free conditions were subjected to infection by both HCV derived in cell culture (HCVcc) and patient-derived virus (HCVser). Pluripotent stem cells and definitive endoderm were not permissive for HCV infection whereas hepatic progenitor cells were persistently infected and secreted infectious particles into culture medium. Permissiveness to infection was correlated with induction of the liver-specific microRNA-122 and modulation of cellular factors that affect HCV replication. RNA interference directed toward essential cellular cofactors in stem cells resulted in HCV-resistant hepatocyte-like cells after differentiation. The ability to infect cultured cells directly with HCV patient serum, to study defined stages of viral permissiveness, and to produce genetically modified cells with desired phenotypes all have broad significance for host-pathogen interactions and cell therapy.

The development of calcineurin inhibitors (CNIs) led to marked improvements in patient and graft survival after liver transplantation (LTx). We have been left, however, with a dependence on immunosuppressive agents with nephrotoxicity, neurotoxicity, adverse impacts on cardiac risk profile, and risk for malignancy. These challenges need to be met against a dominance of hepatitis C virus (HCV) and hepatocellular carcinoma (HCC) as indications for liver transplant. Unmet needs for immunosuppression (IS) in LTx include: (1) Effective drugs that avoid CNIs toxicities. (2) Agents without adverse impact on HCV recurrence. (3) Compounds that minimize risk of HCC recurrence. New immunosuppressives will need to address the above needs while supporting patient and graft survival equivalent to those achievable with CNIs, ideally without important new toxicities. Two new classes of agents are currently in advanced clinical development: belatacept, and the mammalian target of rapamycin inhibitors (m-TORi). This manuscript will review evidence for a role for m-TORi in LTx in a range of clinical scenarios including patients with CNI nephrotoxicity or neurotoxicity, patients at risk of (or with) HCV recurrence, and patients at risk of HCC recurrence., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Percutaneous transhepatic portal access avoids surgery but is rarely associated with bleeding or portal venous thrombosis (PVT). We herein report our large, single-center experience of percutaneous islet implantation and evaluate risk factors of PVT and graft function. Prospective data were collected on 268 intraportal islet transplants (122 subjects). A portal venous Doppler ultrasound was obtained on Days 1 and 7 posttransplant. Therapeutic heparinization, complete ablation of the portal catheter tract with Avitene paste and limiting packed cell volume (PCV) to <5 mL completely prevented any portal thrombosis in the most recent 101 islet transplant procedures over the past 5 years. In the previous cumulative experience, partial thrombosis did not affect islet function. Standard liver volume correlated negatively (r =-0.257, p < 0.001) and PCV correlated positively with portal pressure rise (r = 0.463, p < 0.001). Overall, partial portal thrombosis occurred after 10 procedures (overall incidence 3.7%, most recent 101 patient incidence 0%). There were no cases of complete thrombosis and no patient developed sequelae of portal hypertension. In conclusion, portal thrombosis is a preventable complication in clinical islet transplantation, provided therapeutic anticoagulation is maintained and PCV is limited to <5 mL., (©Copyright 2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background & Aims: Liver transplantation is a recognized treatment for selected patients with hepatocellular carcinoma (HCC), but transplant criteria still need to be refined, especially in the case of more advanced or downstaged tumors., Methods: The present study investigated alpha-fetoprotein (AFP) as a predictor of outcome in 6817 patients listed with a diagnosis of HCC in the Scientific Registry of Transplant Recipients., Results: Local pre-transplant HCC treatment was used in 41% of patients on the waiting list. Patients with AFP levels>400 ng/ml at the time of listing who were downstaged to AFP ≤400 ng/ml had better intent-to-treat survival than patients failing to reduce AFP to ≤400 (81% vs. 48% at 3 years, p ≤0.001) and comparable survival to patients with stable AFP ≤400 ng/ml (74%, p = 0.14). Patients with AFP levels decreased ≤400 ng/ml and patients with levels persistently ≤400 ng/ml also had similar drop-out rates from the list (10% in both groups) and post-transplant survival rates (89% vs. 78% at 3 years, p = 0.11). Such an AFP downstaging was associated with good survivals whatever the level of the original AFP (even if originally>1000 ng/ml). Only the last pre-transplant AFP independently predicted survival (p ≤0.001), unlike AFP at listing or AFP changes., Conclusions: Overall, downstaging HCC patients with high AFP is feasible and leads to similar intent-to-treat and post-transplant survivals to those of patients with AFP persistently low. Only last AFP appears relevant for patient selection before transplantation and should be used in combination with morphological variables., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)