Premortem anticoagulation timing and dose in donation after circulatory death: multicentre study of associations with graft function.

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.

Donation after circulatory determination of death in western Canada: a multicentre study of donor characteristics and critical care practices.

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.

RéSUMé: OBJECTIF: Le don d'organes après décès cardiocirculatoire (DDC) est pratiqué au Canada depuis 2006. De nombreux aspects touchant à la prise en charge des donneurs demeurent controversés. MéTHODE: Nous avons réalisé une étude de cohorte multicentrique auprès de donneurs potentiels de DDC dans l'Ouest canadien (2008­2017), ainsi qu'auprès des récipiendaires de leurs organes, afin de décrire les caractéristiques des donneurs et les pratiques de soins intensifs, ainsi que la relation entre ces éléments et la survie à un an des récipiendaires et des organes greffés. RéSULTATS: Au total, 257 patients provenant de quatre provinces ont subi une interruption des traitements de survie en vue d'un possible DDC. La proportion de patients décédés dans les deux heures suivant l'interruption des traitements de survie allait de 67 % à 88 % dans toutes les provinces à l'étude (P = 0,06) et pouvait être prédite par une profondeur du coma plus importante (P = 0,01), la perte de la réaction pupillaire à la lumière ou des réflexes cornéens (P = 0,02), et l'utilisation de vasopresseurs (P = 0,01). Des différences significatives ont été observées entre les différentes provinces dans les intervalles de temps entre le début de l'hypotension et le décès (9­11 min; P = 0,02) et entre le décès et la canulation vasculaire (7­10 min; P < 0,001). Il y avait divergence dans l'administration d'héparine avant le décès (82-96 %; P = 0,03), notamment en ce qui concerne le moment d'administration (avant vs après l'interruption des traitements de survie; P < 0,001) et la posologie (≥ 300 vs < 300 unités·kg−1; P < 0,001). Le don après décès cardiocirculatoire a permis de procurer des organes pour 321 greffes rénales, 81 greffes hépatiques et 50 greffes pulmonaires. La survie à un an du récipiendaire et du greffon ne différait pas d'une province à l'autre (allant de 85 à 90 %, P = 0,45). Les prédicteurs de décès ou de la défaillance du greffon incluaient l'âge plus avancé du récipiendaire (rapport de cotes [RC] par année, 1,04; intervalle de confiance [IC] 95 %, 1,01 à 1,07) et un donneur de sexe masculin (RC, 3,35; IC 95 %, 1,39 à 8,09), mais pas les intervalles de temps entre l'interruption des traitements de survie et la canulation, ni les pratiques liées à l'utilisation d'héparine. CONCLUSION: Il existe une importante variabilité dans les pratiques de soins intensifs pour le DDC dans l'Ouest canadien, mais cette variabilité n'a pas résulté en différences significatives en matière de survie des récipiendaires ou des greffons. Des recherches supplémentaires sont nécessaires afin d'aiguiller la prise en charge optimale des donneurs potentiels de DDC.

Intraoperative continuous renal replacement therapy during liver transplantation: a pilot randomized-controlled trial (INCEPTION).

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.

RéSUMé: OBJECTIF: Notre but était d'évaluer la faisabilité d'un traitement substitutif peropératoire continu de l'insuffisance rénale pendant une greffe hépatique, notamment en matière de recrutement, d'adhésion au protocole, et de suivi. MéTHODE: Dans cette étude randomisée contrôlée non aveugle pilote réalisée auprès d'adultes recevant une greffe hépatique avec un score MELD (Model for End-Stage Liver Disease) ≥ 25 et une insuffisance rénale aiguë préopératoire (RIFLE - RISQUÉ ou plus élevé) et/ou un taux de filtration glomérulaire estimé < 60 mL·min−1·1,73 m−2, les patients ont été randomisés à recevoir un traitement substitutif peropératoire continu de l'insuffisance rénale (le traitement) ou les soins habituels (la norme). Les critères d'évaluation principaux étaient la faisabilité et les événements indésirables. L'analyse principale était l'analyse du projet thérapeutique (intention-to-treat; n = 32) et l'analyse secondaire était l'analyse selon le protocole (n = 28). RéSULTATS: L'étude a été précocement interrompue en raison du recrutement lent de patients et du manque de fonds. Soixante patients ont été recrutés et 32 (53 %) ont été randomisés (n = 15 traitement; n = 17 norme). Le score MELD moyen (écart type) était de 36 (8), 81 % (n = 26) des patients souffraient de cirrhose; 69 % (n = 22) ont reçu un traitement substitutif de l'insuffisance rénale préopératoire; 66 % (n = 21) ont reçu une greffe hépatique à partir de l'unité de soins intensifs. Quatre patients (n = 2 traitement, n = 2 norme) n'ont pas reçu de greffe hépatique après la randomisation. Sept patients (41 %) alloués au groupe norme sont passés dans le groupe traitement en période peropératoire. Trois patients ont été perdus au suivi au cours de la première année. Aucun événement indésirable n'est survenu en association au traitement substitutif peropératoire continu de l'insuffisance rénale. Aucune différence en matière de survie à un an n'a été observée (traitement, 71 % [n = 10/14] vs norme, 93 % [n = 14/15]; risque relatif, 0,77; intervalle de confiance 95 %, 0,54 à 1,1). Dans l'analyse selon le protocole (n = 28 ont reçu un traitement après la randomisation - n = 20 traitement, n = 8 norme), la survie à un an était de 92 % et les complications périopératoires étaient semblables dans les deux groupes. Un seul patient recevait de la dialyse un an après la greffe hépatique. CONCLUSION: Dans cette étude randomisée pilote, le traitement substitutif peropératoire continu de l'insuffisance rénale s'est avéré faisable et sécuritaire, et aucune différence en matière de complications n'a été observée. Les taux de transfert d'un groupe à l'autre étaient élevés. Malgré une sévérité préopératoire élevée de la maladie, la survie à un an était excellente. Ces données peuvent être utiles pour concevoir une étude multicentrique plus importante. ENREGISTREMENT DE L'éTUDE: www.clinicalTrials.gov (NCT01575015); enregistrée le 12 avril 2012.

AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver.

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.

Metabolism of a Phenylarsenical in Human Hepatic Cells and Identification of a New Arsenic Metabolite.

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 IC50 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 IC50 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.

Oxidative Stress Attenuates Lipid Synthesis and Increases Mitochondrial Fatty Acid Oxidation in Hepatoma Cells Infected with Hepatitis C Virus.

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 ß-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 ß-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 ß-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 ß-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.

Hepatitis C Virus-Induced Degradation of Cell Death-Inducing DFFA-Like Effector B Leads to Hepatic Lipid Dysregulation.

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.

Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

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).

STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

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.

Clinical Impact of Portal Vein Thrombosis Prior to Liver Transplantation: A Retrospective Cohort Study.

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.

Total tumor volume and alpha-fetoprotein for selection of transplant candidates with hepatocellular carcinoma: A prospective validation.

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.

Comorbidities have a limited impact on post-transplant survival in carefully selected cirrhotic patients: a population-based cohort study.

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.

Characterization of lipid and lipoprotein metabolism in primary human hepatocytes.

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.

Productive hepatitis C virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation.

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.

Three-dimensional tumor volume and serum alpha-fetoprotein are predictors of hepatocellular carcinoma recurrence after liver transplantation: refined selection criteria.

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.

Chimeric rodents with humanized liver: bridging the preclinical/clinical trial gap in ADME/toxicity studies.

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.

Arylacetamide deacetylase: a novel host factor with important roles in the lipolysis of cellular triacylglycerol stores, VLDL assembly and HCV production.

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.

A model for dropout assessment of candidates with or without hepatocellular carcinoma on a common liver transplant waiting list.

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.

Additive effect of sirolimus and anti-death receptor 5 agonistic antibody against hepatocellular carcinoma.

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.