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Acetaminophen (APAP) hepatotoxicity induces endoplasmic reticulum (ER) stress which triggers the unfolded protein response (UPR) in hepatocytes. However, the mechanisms underlying ER stress remain poorly understood, thus reducing the options for exploring new pharmacological therapies for patients with hyperacute liver injury. Eight-to-twelve-week-old C57BL/6J Xbp1-floxed (Xbp1f/f) and hepatocyte-specific knockout Xbp1 mice (Xbp1∆hepa) were challenged with either high dose APAP [500 mg/kg] and sacrificed at early (1–2 h) and late (24 h) stages of hepatotoxicity. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Pharmacological inhibition of XBP1 using pre-treatment with STF-083010 [STF, 75 mg/kg] and autophagy induction with Rapamycin [RAPA, 8 mg/kg] or blockade with Chloroquine [CQ, 60 mg/kg] was also undertaken in vivo. Cytoplasmic expression of XBP1 coincided with severity of human and murine hyperacute liver injury. Transcriptional and translational activation of the UPR and sustained activation of JNK1/2 were major events in APAP hepatotoxicity, both in a human hepatocytic cell line and in a preclinical model. Xbp1∆hepa livers showed decreased UPR and JNK1/2 activation but enhanced autophagy in response to high dose APAP. Additionally, blockade of XBP1 splicing by STF, mitigated APAP-induced liver injury and without non-specific off-target effects (e.g., CYP2E1 activity). Furthermore, enhanced autophagy might be responsible for modulating CYP2E1 activity in Xbp1∆hepa animals. Genetic and pharmacological inhibition of Xbp1 specifically in hepatocytes ameliorated APAP-induced liver injury by enhancing autophagy and decreasing CYP2E1 expression. These findings provide the basis for the therapeutic restoration of ER stress and/or induction of autophagy in patients with hyperacute liver injury., Ministerio de Economía, Comercio y Empresa, German Research Foundation, Depto. de Inmunología, Oftalmología y ORL, Fac. de Medicina, TRUE, pub

Alcohol-related liver disease (ALD) ranges from never-decompensated ALD (ndALD) to the life-threatening decompensated phenotype, known as alcohol-related hepatitis (AH). A multidimensional study of the clinical, histological and molecular features of these subtypes is lacking. Two large cohorts of patients were recruited in an international, observational multicentre study: a retrospective cohort of patients with ndALD (n=110) and a prospective cohort of patients with AH (n=225). Clinical, analytical, immunohistochemistry and hepatic RNA microarray analysis of both disease phenotypes were performed. Age and mean alcohol intake were similar in both groups. AH patients had greater aspartate amino transferase/alanine amino transferase ratio and lower gamma-glutamyl transferase levels than in ndALD patients. Patients with AH demonstrated profound liver failure and increased mortality. One-year mortality was 10% in ndALD and 50% in AH. Histologically, steatosis grade, ballooning and pericellular fibrosis were similar in both groups, while advanced fibrosis, Mallory-Denk bodies, bilirubinostasis, severe neutrophil infiltration and ductular reaction were more frequent among AH patients. Transcriptome analysis revealed a profound gene dysregulation within both phenotypes when compare to controls. While ndALD was characterised by deregulated expression of genes involved in matrisome and immune response, the development of AH resulted in a marked deregulation of genes involved in hepatocyte reprogramming and bile acid metabolism. Despite comparable alcohol intake, AH patients presented with worse liver function compared with ndALD patients. Bilirubinostasis, severe fibrosis and ductular reaction were prominent features of AH. AH patients exhibited a more profound deregulation of gene expression compared with ndALD patients.

Background & aimsOnly a minority of excess alcohol drinkers develop cirrhosis. We developed and evaluated risk stratification scores to identify those at highest risk.MethodsThree cohorts (GenomALC-1: n = 1,690, GenomALC-2: n = 3,037, UK Biobank: relevant n = 6,898) with a history of heavy alcohol consumption (≥80 g/day (men), ≥50 g/day (women), for ≥10 years) were included. Cases were participants with alcohol-related cirrhosis. Controls had a history of similar alcohol consumption but no evidence of liver disease. Risk scores were computed from up to 8 genetic loci identified previously as associated with alcohol-related cirrhosis and 3 clinical risk factors. Score performance for the stratification of alcohol-related cirrhosis risk was assessed and compared across the alcohol-related liver disease spectrum, including hepatocellular carcinoma (HCC).ResultsA combination of 3 single nucleotide polymorphisms (SNPs) (PNPLA3:rs738409, SUGP1-TM6SF2:rs10401969, HSD17B13:rs6834314) and diabetes status best discriminated cirrhosis risk. The odds ratios (ORs) and (95% CIs) between the lowest (Q1) and highest (Q5) score quintiles of the 3-SNP score, based on independent allelic effect size estimates, were 5.99 (4.18-8.60) (GenomALC-1), 2.81 (2.03-3.89) (GenomALC-2), and 3.10 (2.32-4.14) (UK Biobank). Patients with diabetes and high risk scores had ORs of 14.7 (7.69-28.1) (GenomALC-1) and 17.1 (11.3-25.7) (UK Biobank) compared to those without diabetes and with low risk scores. Patients with cirrhosis and HCC had significantly higher mean risk scores than patients with cirrhosis alone (0.76 ± 0.06 vs. 0.61 ± 0.02, p = 0.007). Score performance was not significantly enhanced by information on additional genetic risk variants, body mass index or coffee consumption.ConclusionsA risk score based on 3 genetic risk variants and diabetes status enables the stratification of heavy drinkers based on their risk of cirrhosis, allowing for the provision of earlier

Alcohol-related liver disease (ALD) ranges from never-decompensated ALD (ndALD) to the life-threatening decompensated phenotype, known as alcohol-related hepatitis (AH). A multidimensional study of the clinical, histological and molecular features of these subtypes is lacking., SCOPUS: ar.j, info:eu-repo/semantics/published

Drug induced liver injury (DILI) is an important cause acute liver failure. Here the authors report that serum Mg2+ serum levels decrease in patients with DILI as well as in preclinical animal models treated with acetaminophen overdose, and that early intervention targeting the Mg2+ transporter Cyclin M4 may be beneficial for acetaminophen overdose in preclinical models. Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury.

Only a minority of excess alcohol drinkers develop cirrhosis. We developed and evaluated risk stratification scores to identify those at highest risk., SCOPUS: ar.j, info:eu-repo/semantics/published

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury.

Management of long-term immunosuppression following liver transplantation (LT) remains empirical. Surveillance liver biopsies in combination with transcriptional profiling could overcome this challenge by identifying recipients with active alloimmune-mediated liver damage despite normal liver tests, but has low applicability. Our aim was to investigate the utility of non-invasive tools in stratifying stable long-term LT recipients according to their immunological risk and need for immunosuppression. We conducted a cross-sectional multicentre study of 190 adult liver recipients assessed to determine their eligibility to participate in an immunosuppression withdrawal trial. Patients had stable liver allograft function and had been transplanted for non-autoimmune non-replicative viral liver disease >3 years before inclusion. We performed histological, immunogenetic and serological studies and measured the intrahepatic transcript levels of an 11-gene classifier highly specific for T cell-mediated rejection (TCMR). 36% patients harboured clinically-silent fibro-inflammatory liver lesions, which were scored as moderate-to-severe in 22%. The severity of liver allograft damage was positively associated with TCMR-related transcripts, class II donor-specific antibodies (DSA), ALT/AST and liver stiffness measurements (LSM), and negatively correlated with serum creatinine and tacrolimus trough levels. Liver biopsies were stratified according to their TCMR transcript levels using a cut-off derived from biopsies with clinically-significant TCMR. Two multivariable prediction models, integrating ALT/LSM or ALT/class II DSA, respectively, had a high discriminative capacity in classifying patients with or without alloimmune damage. The latter model had a good performance in an independent cohort of 156 liver biopsies obtained from paediatric liver recipients with similar inclusion/exclusion criteria. ALT, class II DSA and LSM are valuable tools to non-invasively identify stable LT re

Laboratory tests can play an important role in assessment of alcoholic patients, including for evaluation of liver damage and as markers of alcohol intake. Evidence on test performance should lead to better selection of appropriate tests and improved interpretation of results. We compared laboratory test results from 1578 patients between cases (with alcoholic cirrhosis; 753 men, 243 women) and controls (with equivalent lifetime alcohol intake but no liver disease; 439 men, 143 women). Comparisons were also made between 631 cases who had reportedly been abstinent from alcohol for over 60 days and 364 who had not. ROC curve analysis was used to estimate and compare tests ability to distinguish patients with and without cirrhosis, and abstinent and drinking cases. The best tests for presence of cirrhosis were INR and bilirubin, with areas under the ROC curve (AUCs) of 0.91 ± 0.01 and 0.88 ± 0.01, respectively. Confining analysis to patients with no current or previous ascites gave AUCs of 0.88 ± 0.01 for INR and 0.85 ± 0.01 for bilirubin. GGT and AST showed discrimination between abstinence and recent drinking in patients with cirrhosis, including those without ascites, when appropriate (and for GGT, sex-specific) limits were used. For AST, a cut-off limit of 85 units/L gave 90% specificity and 37% sensitivity. For GGT, cut-off limits of 288 units/L in men and 138 units/L in women gave 90% specificity for both and 40% sensitivity in men, 63% sensitivity in women. INR and bilirubin show the best separation between patients with alcoholic cirrhosis (with or without ascites) and control patients with similar lifetime alcohol exposure. Although AST and GGT are substantially increased by liver disease, they can give useful information on recent alcohol intake in patients with alcoholic cirrhosis when appropriate cut-off limits are used.

Background: Chronic liver injury can lead to the development of liver fibrosis and cirrhosis but only in a minority of patients. Currently, it is not clear which factors determine progression to fibrosis. We investigated whether DNA\methylation profile as determined by pyrosequencing can distinguish patients with mild from those with advanced/severe fibrosis in non-alcoholic liver disease (NAFLD) and alcoholic liver disease (ALD). To this end, paraffin-embedded liverbiopsies were collected from patients with biopsy-proven NAFLD or ALD, as well as paraffin-embedded normal liver resections, genomic DNA isolated, bisulfite converted and pyrosequencing assays used to quantify DNA methylation at specific CpGs within PPAR alpha, PPAR alpha, TGF beta 1, Collagen 1A1 and PDGF alpha genes. Furthermore, we assessed the impact of age, gender and anatomical location within the liver on patterns of DNA methylation inthe same panel of genes. Results: DNA methylation at specific CpGs within genes known to affect fibrogenesis distinguishes between patients with mild from those with severe fibrosisin both NAFLD and ALD, although same CpGs are not equally represented in both etiologies. In normal liver, age, gender or anatomical location had no significant impact on DNA methylation patterns in the liver. Conclusions: DNA methylation status at specific CpGs may be useful as part of a wider set of patient data for predicting progression to liver fibrosis., EASL Physician Scientist Sheila Sherlock Fellowship; Medical Research Council (MRC); National Institute on Alcohol Abuse and Alcoholism (NIAAA); NIHR Newcastle Biomedical Research Centre

Acetaminophen (APAP) is the active component of many medications used to treat pain and fever worldwide. Its overuse provokes liver injury and it is the second most common cause of liver failure. Mitochondrial dysfunction contributes to APAP-induced liver injury but the mechanism by which APAP causes hepatocyte toxicity is not completely understood. Therefore, we lack efficient therapeutic strategies to treat this pathology. Here we show that APAP interferes with the formation of mitochondrial respiratory supercomplexes via the mitochondrial negative regulator MCJ, and leads to decreased production of ATP and increased generation of ROS. In vivo treatment with an inhibitor of MCJ expression protects liver from acetaminophen-induced liver injury at a time when N-acetylcysteine, the standard therapy, has no efficacy. We also show elevated levels of MCJ in the liver of patients with acetaminophen overdose. We suggest that MCJ may represent a therapeutic target to prevent and rescue liver injury caused by acetaminophen.

Acetaminophen (APAP) is the active component of many medications used to treat pain and fever worldwide. Its overuse provokes liver injury and it is the second most common cause of liver failure. Mitochondrial dysfunction contributes to APAP-induced liver injury but the mechanism by which APAP causes hepatocyte toxicity is not completely understood. Therefore, we lack efficient therapeutic strategies to treat this pathology. Here we show that APAP interferes with the formation of mitochondrial respiratory supercomplexes via the mitochondrial negative regulator MCJ, and leads to decreased production of ATP and increased generation of ROS. In vivo treatment with an inhibitor of MCJ expression protects liver from acetaminophen-induced liver injury at a time when N-acetylcysteine, the standard therapy, has no efficacy. We also show elevated levels of MCJ in the liver of patients with acetaminophen overdose. We suggest that MCJ may represent a therapeutic target to prevent and rescue liver injury caused by acetaminophen.

BackgroundThe risk of alcohol-related liver cirrhosis increases with increasing alcohol consumption, but many people with very high intake escape from liver disease. We postulate that susceptibility to alcoholic cirrhosis has a complex genetic component and propose that this can be dissected through a large and sufficiently powered genomewide association study (GWAS).MethodsThe GenomALC Consortium comprises researchers from Australia, France, Germany, Switzerland, United Kingdom, and United States, with a joint aim of exploring the genetic and genomic basis of alcoholic cirrhosis. For this National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism funded study, we are recruiting high-risk drinkers who are either cases (with alcoholic cirrhosis) or controls (drinking comparable amounts over similar time, but free of significant liver disease). Extensive phenotypic data are obtained using semistructured interviews and patient records, and blood samples are collected.ResultsWe have successfully recruited 859 participants including 538 matched case-control samples as of September 2014, using study-specific inclusion-exclusion criteria and data collection protocols. Of these, 580 are cases (442 men and 138 women) and 279 are controls (205 men and 74 women). Duration of excessive drinking was slightly greater in cases than controls and was significantly less in women than men. Cases had significantly lower lifetime alcohol intake than controls. Both cases and controls had a high prevalence of reported parental alcohol problems, but cases were significantly more likely to report that a father with alcohol problems had died from liver disease (odds ratio 2.53, 95% confidence interval 1.31 to 4.87, p = 0.0055).ConclusionsRecruitment of participants for a GWAS of alcoholic cirrhosis has proved feasible across countries with multiple sites. Affected patients often consume less alcohol than unaffected ones, emphasizing the existence of individual

BackgroundThe risk of alcohol-related liver cirrhosis increases with increasing alcohol consumption, but many people with very high intake escape from liver disease. We postulate that susceptibility to alcoholic cirrhosis has a complex genetic component and propose that this can be dissected through a large and sufficiently powered genomewide association study (GWAS).MethodsThe GenomALC Consortium comprises researchers from Australia, France, Germany, Switzerland, United Kingdom, and United States, with a joint aim of exploring the genetic and genomic basis of alcoholic cirrhosis. For this National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism funded study, we are recruiting high-risk drinkers who are either cases (with alcoholic cirrhosis) or controls (drinking comparable amounts over similar time, but free of significant liver disease). Extensive phenotypic data are obtained using semistructured interviews and patient records, and blood samples are collected.ResultsWe have successfully recruited 859 participants including 538 matched case-control samples as of September 2014, using study-specific inclusion-exclusion criteria and data collection protocols. Of these, 580 are cases (442 men and 138 women) and 279 are controls (205 men and 74 women). Duration of excessive drinking was slightly greater in cases than controls and was significantly less in women than men. Cases had significantly lower lifetime alcohol intake than controls. Both cases and controls had a high prevalence of reported parental alcohol problems, but cases were significantly more likely to report that a father with alcohol problems had died from liver disease (odds ratio 2.53, 95% confidence interval 1.31 to 4.87, p = 0.0055).ConclusionsRecruitment of participants for a GWAS of alcoholic cirrhosis has proved feasible across countries with multiple sites. Affected patients often consume less alcohol than unaffected ones, emphasizing the existence of individual