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Acetaminophen (APAP) intoxication is the foremost cause of drug-induced liver failure in developed countries. The only pharmacologic treatment option, N-acetylcysteine (NAC), is not effective for patients who are admitted too late and/or who have excessive liver damage, emphasizing the need for alternative treatment options. APAP intoxication results in hepatocyte death and release of danger signals, which further contribute to liver injury, in part by hepatic monocyte/macrophage infiltration and activation. Metallothionein (MT) 1 and 2 have important danger signaling functions and might represent novel therapeutic targets in APAP overdose. Therefore, we evaluated hepatic MT expression and the effect of anti-MT antibodies on the transcriptional profile of the hepatic macrophage population and liver injury following APAP overdose in mice. Hepatic MT expression was significantly induced in APAP-intoxicated mice and abundantly present in human livers. APAP intoxication in mice resulted in increased serum transaminase levels, extended necrotic regions on liver histology and induced expression of proinflammatory markers, which was significantly less pronounced in mice treated with anti-MT antibodies. Anti-MT antibody therapy attenuated proinflammatory macrophage polarization, as demonstrated by RNA sequencing analyses of isolated liver macrophages and in LPS-stimulated bone marrow-derived macrophages. Importantly, NAC and anti-MT antibodies were equally effective whereas administration of anti-MT antibody in combination with NAC exceeded the efficiency of both monotherapies in APAP-induced liver injury (AILI). We conclude that the neutralization of secreted MTs using a monoclonal antibody is a novel therapeutic strategy as mono- or add-on therapy for AILI. In addition, we provide evidence suggesting that MTs in the extracellular environment are involved in macrophage polarization.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. HCC typically develops on a background of chronic inflammation and fibrosis with tumor associated macrophages (TAMs) playing an important role in chronic inflammation-induced HCC and progression. However, the liver harbors unique macrophages, resident liver Kupffer cells (KCs) and monocyte-derived macrophages (Mo-Mφ), and their contribution to HCC and to the population of TAMs is incompletely known. Here, we characterized the tumor microenvironment and the proportion and transcriptional profile of hepatic macrophages (Mφ) in two commonly used HCC mouse models. A gradually increased expression of inflammatory, immune regulatory, fibrotic and cell proliferation pathways and markers was observed during diethylnitrosamine (DEN)- and non-alcoholic steatohepatitis (NASH)-induced HCC development. The transcriptional phenotypes of isolated hepatic Mφ subsets were clearly distinct and shifted during HCC development, with mixed pro-inflammatory and tumor-promoting expression profiles. There were marked differences between the models as well, with Mφ in NASH-HCC exhibiting a more immunomodulatory phenotype, in conjunction with an upregulation of lipid metabolism genes. Our data show that at least some infiltrated macrophages display expression of pro-tumoral markers, and that Kupffer cells are part of the population of TAMs and enhance tumor progression. These insights are useful to further unravel sequential pathogenic events during hepatocarcinogenesis and direct future development of new treatment strategies for HCC.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. For advanced HCC, there is still an unmet need for more effective therapeutic strategies. HCC is typically associated with hypoxia and the hypoxia-inducible factor (HIF) regulatory pathway plays an important role in HCC development and progression. Therefore, we investigated the therapeutic potential of isoform-specific HIF-1α and HIF-2α antisense oligonucleotides (ASOs), along with their effect on the inflammatory and fibrotic component of the tumor microenvironment (TME), in an experimental HCC mouse model. Based on its efficacy and safety, a dosage regimen of 20 mg/kg intraperitoneal injection of HIFα ASO twice per week was selected for further investigation in a preventive and therapeutic setting in a N,N-diethylnitrous amide (DEN)-induced HCC mouse model. DEN administration resulted in 100% tumor formation and HIFα ASO administration led to effective and selective hepatic downregulation of its target genes. HIFα ASO treatment had no effect on tumor numbers, but even enhanced the increased hepatic expression of HCC tumor markers, α-fetoprotein and glypican-3, compared to scrambled control ASO treatment in HCC mice. Especially HIF-1α ASO treatment resulted in an enhanced increase of monocytes and monocyte-derived macrophages in the liver and an enhanced hepatic upregulation of inflammatory markers. Both HIFα ASOs aggravated liver fibrosis in HCC mice compared to scrambled ASO treatment. The observed effects of our dosing regimen for HIF-1α and HIF-2α ASO treatment in the DEN-induced HCC mouse model discourage the use of HIFα isoforms as targets for the treatment of HCC.

Introduction : Auto-immune hepatitis (AIH) is a rare condition which primarily affects young women. Several diagnostic scoring systems exist based on clinical, biochemical, immunologic and histologic characteristics of AIH. Additionally, prognostic parameters can be identified. The purpose of this literary review is to compare the clinical value, strengths and limitations of these diagnostic and prognostic scoring systems. Methods : A literature search was performed in two databases and selected based on diagnostic and prognostic criteria. Only studies concerning AIH in adults were included. Results : The backbone of scoring systems remains the revised AIH criteria published in 1999 and the simplified from 2008. The revised system shows a higher sensitivity, lower specificity and lower diagnostic accuracy compared to the simplified. Limitations to these scoring systems include limited diagnostic accuracy in acute or fulminant liver failure, insufficient inclusion of atypical auto-antibodies and lacking diagnostic power in presence of overlap syndromes. Concerning these overlap syndromes, the Paris criteria show a higher diagnostic accuracy compared to the scoring systems for AIH. Presently, no clinical prognostic scoring systems are available. However, a first system based on response to treatment accurately predicts long-term survival in AIH. Conclusion : Diagnostic scoring systems are useful in diagnosing AIH and have complementary value. However, they are no sub- stitute for the gold standard of appropriate clinical assessment and are mostly useful in defining cohorts for research purposes. An evolution towards a more dynamic scoring system, using prognostic parameters and the progression of typical features, seems more valuable than the current diagnostic systems.

Non-alcoholic fatty liver disease (NAFLD) is a crucial health problem with a prevalence that is increasing concurrently with the obesity epidemic on a global scale. Steatosis, nonalcoholic steatohepatitis (NASH), hepatocellular carcinoma (HCC), cirrhosis, and advanced fibrosis constitute the disease spectrum covered by NAFLD. NASH-related cirrhosis and HCC is currently the second most common indication for liver transplantation. Although lifestyle modifications, especially weight loss, effectively reduces the liver injury in NASH, adherence in the clinical setting is low. Potential treatments for NASH are still under investigation in phase 2–3 studies. Bariatric surgery can improve metabolic components and cause great weight loss. Therefore, bariatric surgery may reverse the pathological liver changes in NAFLD and NASH patients. However, complications such as liver failure after bariatric surgery can occur. This chapter will give an overview of the benefits and pitfalls of bariatric surgery in patients with NAFLD, liver transplant candidates and post-liver transplant patients.

Purpose Obese subjects with nonalcoholic fatty liver disease (NAFLD) are more prone to develop additional metabolic disturbances such as systemic insulin resistance (IR) and type 2 diabetes. NAFLD is defined by hepatic steatosis, lobular inflammation, ballooning and stage of fibrosis, but it is unclear if and which components could contribute to IR. Objective To assess which histological components of NAFLD associate with IR in subjects with obesity, and if so, to what extent. Methods This cross-sectional study included 78 obese subjects (mean age 46 ± 11 years; BMI 42.2 ± 4.7 kg/m2). Glucose levels were analysed by hexokinase method and insulin levels with electrochemiluminescence. Homeostasis model assessment-estimated insulin resistance (HOMA-IR) was calculated. Liver biopsies were evaluated for histological components of NAFLD. Results A positive association between overall NAFLD Activity Score and HOMA-IR was found (r s = 0.259, P = 0.022). As per individual components, lobular inflammation and fibrosis stage were positively associated with HOMA-IR, glucose and insulin levels (P P Conclusion In this group of obese subjects, a major contributing histological component of NAFLD to the relation between NAFLD severity and IR seems to be the grade of hepatic lobular inflammation. Although no causal relationship was assessed, preventing or mitigating this inflammatory response in obesity might be of importance in controlling obesity-related metabolic disturbances.

The prevalence of obesity and metabolic diseases such as type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) has risen dramatically over the past decades. At present, bariatric surgery is the most effective treatment for this global health problem, through effects on food intake, gut hormone secretion, metabolic signaling pathways, and adipose tissue dysfunction. The liver occupies a central role in carbohydrate, protein, and lipid metabolism. Notably, a reduction in hepatic fat content and an improvement in hepatic insulin resistance are among the earliest beneficial effects of bariatric surgery, which has therefore emerged as an attractive treatment option for NAFLD. However, as the scope and popularity of weight loss surgery have expanded, new questions have arisen regarding its safety in patients with liver cirrhosis, the outcome of liver transplantation in patients with a history of bariatric surgery, and over incidental reports of liver failure following surgery. Studies in humans and rodents have also linked bariatric surgery to an increased risk of developing alcohol use disorder, a major risk factor for liver disease. This review integrates data from clinical and translational research to delineate both the beneficial impact of bariatric surgery on the liver and the potential risks involved.

Background & Aims Nonalcoholic fatty liver disease (NAFLD) has become the most common pediatric liver disease. The intrauterine and early life environment can have an important impact on long-term metabolic health. We investigated the impact of maternal prepregnancy obesity, (pre)gestational diabetes, breastfeeding, and birth anthropometrics/preterm birth on the development of NAFLD in children and adolescents. Methods A comprehensive search was performed in MEDLINE, PubMed Central, EMBASE, and grey literature databases through August 2020. The primary outcome was the prevalence of pediatric NAFLD, whereas the histologic severity of steatohepatitis and/or fibrosis were secondary outcomes. Study selection, data extraction, and quality assessment were performed by 2 independent reviewers. Results Our systematic review included 33 articles. Study heterogeneity regarding patient populations, diagnostic tools, and overall quality was considerable. Eight studies determined the impact of maternal prepregnancy overweight/obesity and identified this as a possible modifiable risk factor for pediatric NAFLD. Conversely, 8 studies investigated (pre)gestational diabetes, yet the evidence on its impact is conflicting. Breastfeeding was associated with a reduced risk for NAFLD, steatohepatitis, and fibrosis, especially in studies that evaluated longer periods of breastfeeding. Being born preterm or small for gestational age has an unclear impact on the development of NAFLD, although an early catch-up growth might drive NAFLD. Conclusions In a systematic review, we found that maternal prepregnancy overweight and obesity were associated with an increased risk of pediatric NAFLD. Breastfeeding might be protective against the development of NAFLD when the duration of breastfeeding is sufficiently long (≥6 months).

Background The poor prognosis of advanced HCC and limited efficacy of current systemic treatments emphasize the need for new or combined targeted therapies. The development of HCC is a multistage process in which liver injury appears in a complex microenvironment associated with oxidative stress. NOX enzymes are the main source of ROS during hepatocarcinogenesis and NOX1 in particular has shown correlation with poor prognosis of HCC patients. This study evaluates the effect of pharmacological NOX1 inhibition on the development and progression of HCC and its effect on the tumor microenvironment. Methods The in vitro cytotoxic effects of the NOX1 inhibitor GKT771 (Genkyotex) on human Huh7 and Hep3B and murine Hepa1-6 HCC cell lines, the human THP1 monocyte cell line and mouse macrophages were evaluated via MTT, LDH activity and CaspGlo® assays. In order to induce in vivo HCC, male SV129 wild-type mice received weekly IP injections of diethylnitrosamine (DEN) (35 mg/kg) for 20–25 weeks. Mice were treated with vehicle or GKT771 (30 mg/kg) via oral gavage, daily or twice daily, in preventive and therapeutic studies. The liver damage was evaluated for inflammation, angiogenesis, fibrosis and HCC development via histology, RT-qPCR, multiplex analyses and ROS levels. Results A concentration-dependent reduction in cellular activity of the human HCC cell lines without cytotoxicity was observed. GKT771 treatment reduced LPS-induced pro-inflammatory bone-marrow derived macrophage polarization. DEN injections resulted in 100 % tumor formation and the induction of HCC markers which could be reduced by twice daily dosing of GKT771 at early onset of advanced HCC. DEN-induced HCC resulted in an upregulation of pro-inflammatory, angiogenic and fibrotic markers which was less pronounced in GKT771 treated mice in all treatment regimens. In line, liver fibrosis was induced in HCC mice and this to a lesser extend upon GKT771 treatment. Conclusions NOX1 inhibition showed to be safe and well tolerated and was able to attenuate the induction of a pro-inflammatory, angiogenic and pro-fibrotic microenvironment suggesting that this might be a promising adjuvant therapeutic strategy in the treatment of advanced HCC.

Background Impact of portal vein thrombosis (PVT) on the clinical course in liver transplant candidates remains unclear. This study aims to identify prevalence and risk factors for PVT, assess outcome after liver transplantation (LT) in patients with PVT and study the effect of anticoagulation. Methods This single-center retrospective cohort study was performed from January 2006 until June 2016. Patients were stratified according to presence of PVT. Risk factors and outcome were assessed using logistic regression and survival analysis. Results Among 390 adults who underwent orthotopic LT, PVT occurred in 40 (10.3%). In, respectively, 10 (25%), 7 (17.5%), and 23 (57.5%) patients, PVT was identified at time of evaluation for transplantation, on the waiting list and during transplantation. A beneficial trend was present favoring the use of anticoagulation for PVT resolution (n = 3/7 vs 0/9; p = .062). Patient and graft survival were similar between the groups after a median follow-up of 5 years. However, 1-year patient survival was significantly lower (p = .031) in patients with PVT. Conclusion Portal vein thrombosis occurred in 10% of patients awaiting LT was undiagnosed in 50% until moment of LT and had a deleterious effect on 1-year survival. Anticoagulation showed a beneficial trend on recanalization of PVT and survival rate.

Background and aims Obesity, diabetes and associated non-alcoholic steatohepatitis (NASH) are rising risk factors for hepatocellular carcinoma (HCC). Macrophages are important immune cells involved in inflammation and tumour development. Macrophage inositol-requiring enzyme 1 alpha (IRE1α), an ER-stress protein, has been shown to be involved in macrophage cytokine production, and myeloid-specific IRE1α knock-out (myeloid IRE1α-KO) mice showed reduced weight gain during high-fat diet feeding. However, the effect of myeloid IRE1α on NASH and subsequent HCC development has not been examined. Here, we characterized the transcriptional profile of the hepatic macrophage population in a diabetes-NASH-HCC mouse model, and investigated the effect of myeloid-specific IRE1α deletion on the phenotype of hepatic macrophage subsets and experimental NASH-HCC development. Methods Mice with non-functional myeloid IRE1α were created by crossing Ire1a floxed mice with Lysm-Cre mice. Two-day old myeloid IRE1α-KO and wild type (WT) mice were subcutaneously injected with streptozotocin (STZ), and male mice were fed a high-fat, -sucrose, -cholesterol diet (Western diet, WD) from the age of 4 weeks until 21 weeks. Control myeloid IRE1α-KO and WT mice received a PBS injection and were fed a matched control diet. These mice were evaluated for obesity, diabetes, NASH and HCC. The hepatic macrophage population was evaluated by flow cytometry and RNA sequencing on FACS-isolated macrophage subsets. Results STZ-injection and WD feeding resulted in an impaired glucose tolerance, advanced NASH with fibrosis, and HCC development. Myeloid IRE1α-KO STZ mice showed lower fasting glucose levels at the start of WD feeding, and an improved glucose tolerance and attenuated HCC development after 17 weeks of WD feeding despite a similar degree of liver steatosis and inflammation compared to WT mice. Transcriptomic analysis of WT liver Kupffer cells, macrophages and monocytes revealed phenotypical changes in those cell subsets during NASH-HCC development. Isolated liver Kupffer cells and macrophages from mice with a myeloid IRE1α deletion showed downregulated pathways involved in immune system activation and metabolic pathways (only in Kupffer cells), whereas pathways involved in cell division and metabolism were upregulated in monocytes. These transcriptional differences were attenuated during NASH-HCC development. Conclusion Our results show that myeloid-specific IRE1α deletion results in an altered transcriptional profile of hepatic macrophages and dampens diabetes-induced NASH-HCC development, possibly by attenuated diabetes induction.

Men with obesity often present with low testosterone (T) and sex hormone-binding globulin (SHBG) levels. Several mechanisms for this have been proposed, but as SHBG is secreted by hepatocytes and sex steroids undergo hepatic metabolization, this study investigates whether severity and histological components of nonalcoholic fatty liver disease (NAFLD) are associated with sex steroid levels in obese men. This cross-sectional study included 80 obese men (age: 46 ± 11 years; body mass index: 42.2 ± 5.5 kg m−2). Serum levels of total T and estradiol (E2) were measured using liquid chromatography coupled with tandem mass spectroscopy (LC/MS-MS) and SHBG and gonadotropins by immunoassay. Liver biopsies were evaluated using Steatosis, Activity, and Fibrosis scoring. Participants with steatohepatitis had similar median (1st quartile–3rd quartile) total T levels (7.6 [5.0–11.0] nmol l−1 vs 8.2 [7.2–10.9] nmol l−1; P = 0.147), lower calculated free T (cFT) levels (148.9 [122.9–188.8] pmol l−1 vs 199.5 [157.3–237.6] pmol l−1; P = 0.006), and higher free E2/T ratios (10.0 [6.4–13.9] x10-3 vs 7.1 [5.7–10.7] x10-3; P = 0.026) compared to men with only nonalcoholic fatty liver. Among the histological components of NAFLD, only steatosis was independently associated with total T (rs = −0.331, P = 0.003) and cFT levels (rs = −0.255, P = 0.025). Obese men with steatohepatitis have even lower cFT levels compared to those without, an association mainly driven by grade of steatosis. Whether this reflects a subgroup of men with a more severe obesity-related phenotype or results from direct relations between hepatic steatosis and sex steroid metabolism needs further investigation.

Recent studies have indicated that patients with prior bariatric surgery (BS) are at risk for developing alcohol use disorder. We explored patient demographics and impact on liver disease severity in 11 patients with prior BS listed for transplantation due to alcoholic liver disease, and compared these with 177 patients without BS. BS patients were younger with a female predominance and presented with more severe decompensation. The timeframe between diagnosis, listing, and transplantation was significantly shorter in patients with prior BS. The incidence of post-transplant complications and 3-year survival rate was comparable. In conclusion, alcoholic liver disease with rapid decompensation may develop after BS, warranting rapid identification with referral to a transplant center. Attention should be paid to pre-surgery screening for alcohol overuse risk factors.

To evaluate the outcome of early transjugular portosystemic shunt (TIPS) treatment in patients with a trial-compatible high-risk variceal bleeding and secondly to disclose other predictors of early mortality.A cohort study was conducted on patients referred for a TIPS procedure with or without combined variceal embolization to control acute esophageal variceal bleeding. A total of 32 patients with Child-Pugh C score less than 14 or Child-Pugh B plus active bleeding at endoscopy, admitted for early-TIPS treatment (72 h), were included.We noted one (3.7%) failure to control bleeding and no rebleeding during 1-year follow-up. Ten (31.3%) patients died within 6 weeks after TIPS placement. Early mortality was associated with model for end-stage liver disease (MELD) score (P=0.025), MELD score of at least 19 (P=0.008) and hemodynamic instability at time of admission (P=0.001). If hemodynamic instability is associated with a high MELD score, the 6-week mortality peaks at 77.8% (P=0.000).This study confirms the excellent survival results of early-TIPS treatment for acute variceal bleeding in a selected patient group with a low MELD score. Poor survival in hemodynamically unstable patients with high MELD scores (≥19) contests the guidelines that patients with Child-Pugh class C cirrhosis or Child-Pugh class B with active bleeding on endoscopy should deliberately receive preemptive TIPS treatment after endoscopic haemostasis.

Autoimmune hepatitis (AIH) is a rare cause of chronic liver disease. The backbone of treatment is immunosuppressive medication, typically prednisolone as induction therapy and azathioprine as a maintenance therapy. Side effects of the long-term use of systemic corticosteroids are well known and have led to the use of alternative induction regimens. An attractive alternative is budesonide, a nonhalogenated glucocorticosteroid characterized by a high first-pass effect in the liver (90%), resulting in a high topical anti-inflammatory activity and a low systemic activity. It should be stressed that budesonide is contraindicated in patients with established cirrhosis with portal hypertension and portocaval shunting. In this case report, we present the first case of adrenal insufficiency following treatment with budesonide for AIH.

We tried to identify structural and functional liver aberrances in a palliated Fontan population and sought to determine useful screening modalities, in order to propose a screening protocol to detect patients at risk. Twenty nine patients, median age 23.7 years (interquartile range (IQR) 20.5-27.2) and median Fontan interval 19.7 years (IQR 4.5-21.4), were prospectively studied with echocardiography, blood analysis (including serum fibrosis scores Forns, APRI and FIB4), liver imaging (ultrasound (US), Doppler), and shear wave elastography to determine liver stiffness (LS). Laboratory tests predominantly showed abnormal values for gamma-glutamyltransferase. Forns index indicated moderate fibrosis in 29% of patients and correlated with Fontan interval (p = 0.034). US liver morphology was deviant in 46% of patients, with surface nodularity in 21% and nodular hyperplasia in 29%. Doppler assessment of flow velocities was within normal ranges for most patients. LS (mean 10.4 ± 3.7 kPa) was elevated in 96% of our population and higher LS values were significantly related to longer Fontan interval (p = 0.018). Adolescent and adult Fontan patients show moderate signs of liver dysfunction. Usefulness of serum parameters and fibrosis scores in post-Fontan screening remains ambiguous. The high percentage of morphologic liver changes in palliated patients supports the use of US in periodic follow-up. LS likely overestimates fibrosis due to liver congestion, arguing for the need of validation through sequential measurements. Screening should minimally encompass US assessment in combination with selective liver fibrosis scores. The role of LS measurement in Fontan follow-up and liver screening needs to be further elucidated.

Hepatic encephalopathy (HE) is a neurological complication of hepatic dysfunction and portosystemic shunting. It is highly prevalent in patients with cirrhosis and is associated with poor outcomes. New insights into the role of peripheral origins in HE have led to the development of innovative treatment strategies like faecal microbiota transplantation. However, this broadening of view has not been applied fully to perturbations in the central nervous system. The old paradigm that HE is the clinical manifestation of ammonia-induced astrocyte dysfunction and its secondary neuronal consequences requires updating. In this review, we will use the holistic concept of the neurogliovascular unit to describe central nervous system disturbances in HE, an approach that has proven instrumental in other neurological disorders. We will describe HE as a global dysfunction of the neurogliovascular unit, where blood flow and nutrient supply to the brain, as well as the function of the blood-brain barrier, are impaired. This leads to an accumulation of neurotoxic substances, chief among them ammonia and inflammatory mediators, causing dysfunction of astrocytes and microglia. Finally, glymphatic dysfunction impairs the clearance of these neurotoxins, further aggravating their effect on the brain. Taking a broader view of central nervous system alterations in liver disease could serve as the basis for further research into the specific brain pathophysiology of HE, as well as the development of therapeutic strategies specifically aimed at counteracting the often irreversible central nervous system damage seen in these patients. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL).

Purpose: Cirrhosis is a major risk factor for the development of hepatocellular carcinoma but remains underdiagnosed in the compensated stage. Fibrosis progression and cirrhosis are associated with changes in blood serum glycomic profiles. Previously, the serum glycomics-based GlycoCirrhoTest was shown to identify 50% to 70% of compensated cirrhosis cases in chronic liver disease cohorts, at >90% specificity. This study assessed GlycoCirrhoTest for the risk of hepatocellular carcinoma development in compensated cirrhosis. Experimental Design: Serum glycomics were analyzed in sera of 133 patients, with compensated cirrhosis collected between 1995 and 2005 in a surveillance protocol for hepatocellular carcinoma using an optimized glycomic technology on a DNA sequencer. Results: Baseline GlycoCirrhoTest values were significantly increased in patients who developed hepatocellular carcinoma after a median follow-up of 6.4 years as compared with patients who did not. For patients with a baseline GlycoCirrhoTest exceeding 0.2, the HR for hepatocellular carcinoma development over the entire study (Cox regression) was 5.1 [95% confidence interval (CI), 2.2–11.7; P < 0.001], and the HR for hepatocellular carcinoma development within 7 years was 12.1 (95% CI, 2.8–51.6; P = 0.01) based on the cut-off value optimized in the same cohort. An absolute increase in GlycoCirrhoTest of 0.2 was associated with an HR of 10.29 (95% CI, 3.37–31.43; P < 0.001) for developing hepatocellular carcinoma. In comparison, the HR for the development of hepatocellular carcinoma within 7 years for AFP levels above the optimal cutoff in this study (5.75 ng/mL) was 4.65 (95% CI, 1.59–13.61). Conclusions: This prognostic study suggests that GlycoCirrhoTest is a serum biomarker that identifies compensated cirrhotic patients at risk for developing hepatocellular carcinoma. Screening strategies could be guided by a positive test on GlycoCirrhoTest. Clin Cancer Res; 23(11); 2750–8. ©2016 AACR.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide and is strongly associated with obesity, dyslipidemia and insulin resistance. NAFLD often presents as simple steatosis (NAFL) but can progress to non-alcoholic steatohepatitis (NASH) and fibrosis. Current non-invasive biomarkers are not tailored to identify significant (⩾F2) fibrosis, although recent guidelines recommend a stringent follow-up of this patient population. We and others have reported on the role of pathological angiogenesis in the pathogenesis of NAFLD, highlighting pro-angiogenic factors as potential diagnostic markers.To investigate the applicability of angiogenic and endothelial dysfunction markers as non-invasive diagnostic tools for NASH or NASH-associated fibrosis in obese patients.In a prospective cross-sectional study, male patients undergoing bariatric surgery (n=61) and control patients (n=35) were recruited. Serum protein levels and visceral adipose tissue gene expression of endothelial dysfunction and angiogenic markers were analyzed by multiplex bead-based assay and quantitative RT-PCR, respectively. For validation, we recruited a second cohort of patients undergoing bariatric surgery (n=40) and a cohort of NAFLD patients from our outpatient clinic (n=30).We identified serum vascular cell adhesion molecule-1 (VCAM-1) as an independent predictor for ⩾F2 fibrosis (median 14.0 vs 8.7 ng mlVCAM-1 levels are able to accurately predict significant (⩾F2) fibrosis in NAFLD patients.

Background & Aims Peroxisome proliferator-activated receptors (PPARs) are essential regulators of whole-body metabolism, but also modulate inflammation in immune cells, notably macrophages. We compared the effects of selective PPAR agonists to those of the pan-PPAR agonist lanifibranor in non-alcoholic fatty liver disease (NAFLD), and studied isoform-specific effects on hepatic macrophage biology. Methods Lanifibranor or selective PPARα (fenofibrate), PPARγ (pioglitazone) and PPARδ (GW501516) agonists were therapeutically administered in choline-deficient, amino acid-defined high-fat diet (CDAA-HFD)- and Western diet (WD)-fed mouse models of NAFLD. Acute liver injury was induced by carbon tetrachloride (CCl4). The role of PPARs on macrophage functionality was studied in isolated hepatic macrophages, bone marrow-derived macrophages stimulated with palmitic acid, and circulating monocytes from patients with NAFLD. Results Lanifibranor improved all histological features of steatohepatitis in CDAA-HFD-fed mice, including liver fibrosis, thereby combining and exceeding specific effects of the single PPAR agonists. Its potent anti-steatotic efficacy was confirmed in a 3D liver biochip model with primary cells. Infiltrating hepatic monocyte-derived macrophages were reduced following PPAR agonist administration, especially with lanifibranor, even after short-term treatment, paralleling improved steatosis and hepatitis. Lanifibranor similarly decreased steatosis, liver injury and monocyte infiltration in the WD model. In the acute CCl4 model, neither single nor pan-PPAR agonists directly affected monocyte recruitment. Hepatic macrophages isolated from WD-fed mice displayed a metabolically activated phenotype. Lanifibranor attenuated the accompanying inflammatory activation in both murine palmitic acid-stimulated bone marrow-derived macrophages, as well as patient-derived circulating monocytes, in a PPARδ-dependent fashion. Conclusion Pan-PPAR agonists combine the beneficial effects of selective PPAR agonists and may counteract inflammation and disease progression more potently. PPARδ agonism and lanifibranor directly modulate macrophage activation, but not infiltration, thereby synergizing with beneficial metabolic effects of PPARα/γ agonists. Lay summary Peroxisome proliferated-activated receptors (PPARs) are essential regulators of metabolism and inflammation. We demonstrated that the pan-PPAR agonist lanifibranor ameliorated all aspects of non-alcoholic fatty liver disease in independent experimental mouse models. Non-alcoholic fatty liver disease and fatty acids induce a specific polarization status in macrophages, which was altered by lanifibranor to increase expression of lipid handling genes, thereby decreasing inflammation. PPAR isoforms have differential therapeutic effects on fat-laden hepatocytes, activated hepatic stellate cells and inflammatory macrophages, supporting the clinical development of pan-PPAR agonists.

Hepatocyte death during acetaminophen (APAP) intoxication elicits a reactive inflammatory response, with hepatic recruitment of neutrophils and monocytes, which further aggravates liver injury. Neutrophil elastase (NE), secreted by activated neutrophils, carries degradative and cytotoxic functions and maintains a proinflammatory state. We investigated NE as a therapeutic target in acetaminophen-induced liver injury (AILI). C57BL/6 mice were administered a toxic dose of APAP, 2 h prior to receiving the NE inhibitor sivelestat, N-acetylcysteine (NAC), or a combination therapy, and were euthanized after 24 and 48 h. Upon APAP overdose, neutrophils and monocytes infiltrate the injured liver, accompanied by increased levels of NE. Combination therapy of NAC and sivelestat significantly limits liver damage, as evidenced by lower serum transaminase levels and less hepatic necrosis compared to mice that received APAP only, and this to a greater extent than NAC monotherapy. Lower hepatic expression of proinflammatory markers was observed in the combination treatment group, and flow cytometry revealed significantly less monocyte influx in livers from mice treated with the combination therapy, compared to untreated mice and mice treated with NAC only. The potential of NE to induce leukocyte migration was confirmed in vitro. Importantly, sivelestat did not impair hepatic repair. In conclusion, combination of NE inhibition with sivelestat and NAC dampens the inflammatory response and reduces liver damage following APAP overdose. This strategy exceeds the standard of care and might represent a novel therapeutic option for AILI.

Angiogenesis contributes to the development of nonalcoholic steatohepatitis (NASH) and promotes inflammation, fibrosis, and progression to hepatocellular carcinoma (HCC). Angiopoietin-2 (Ang-2) is a key regulator of angiogenesis. We aimed to investigate the role of Ang-2 and its potential as a therapeutic target in NASH using human samples, in vivo mouse models, and in vitro assays. Serum Ang-2 levels were determined in 104 obese patients undergoing bariatric surgery and concomitant liver biopsy. The effect of the Ang-2/Tie2 receptor inhibiting peptibody L1-10 was evaluated in the methionine-choline deficient (MCD) and streptozotocin-western diet nonalcoholic fatty liver disease mouse models, and in vitro on endothelial cells and bone marrow-derived macrophages. The hepatic vasculature was visualized with mu CT scans and scanning electron microscopy of vascular casts. Serum Ang-2 levels were increased in patients with histological NASH compared with patients with simple steatosis and correlated with hepatic CD34 immunoreactivity as a marker of hepatic angiogenesis. Serum and hepatic Ang-2 levels were similarly increased in mice with steatohepatitis. Both preventive and therapeutic L1-10 treatment reduced hepatocyte ballooning and fibrosis in MCD diet-fed mice and was associated with reduced hepatic angiogenesis and normalization of the vascular micro-architecture. Liver-isolated endothelial cells and monocytes from MCD-fed L1-10-treated mice showed reduced expression of leukocyte adhesion and inflammatory markers, respectively, compared with cells from untreated MCD diet-fed mice. In the streptozotocin-western diet model, therapeutic Ang-2 inhibition was able to reverse NASH and attenuate HCC progression. In vitro, L1-10 treatment mitigated increased cytokine production in lipopolysaccharide-stimulated endothelial cells but not in macrophages. Conclusion: Our findings provide evidence for Ang-2 inhibition as a therapeutic strategy to target pathological angiogenesis in NASH.

© 2019 Elsevier Ltd. All rights reserved., Background: Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods: In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH, non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2-F3, or F1 with at least one accompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpoints for the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2-F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings: Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1-F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2-F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1-F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation: Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes.

Objective This study aimed to evaluate whether circulating levels and/or visceral adipose tissue (VAT) expression of recently described adipokines associate with histopathological severity of nonalcoholic fatty liver disease (NAFLD), independent of obesity and insulin resistance. Methods Serum levels of adiponectin, omentin, chemerin, monocyte chemoattractant protein-1, and secreted frizzled-related protein 4 were measured using enzyme-linked immunosorbent assay in 81 patients with obesity and NAFLD and 18 lean control subjects. Expression in VAT was measured using real-time PCR and histopathological grading was scored using the NAFLD activity score (NAS). Results When NAFLD patients were subdivided into groups with simple steatosis, borderline nonalcoholic steatohepatitis (NASH), and NASH, adiponectin serum levels and omentin expression were lower in NASH versus simple steatosis patients. Serum adiponectin was generally lower with higher histopathological grading. Chemerin VAT expression was negatively associated with NAS (r = −0.331, P = 0.022) and steatosis score (r = −0.335, P = 0.020), independent of age, BMI, and HOMA-IR. In addition, adjusting for chemerin VAT expression in a multivariate model explained part of the association between NAS and HOMA-IR. Conclusions These findings suggest that lower VAT expression of chemerin in patients with obesity may be involved in the pathophysiology of hepatic steatosis, potentially by modulating the link between insulin resistance and NAFLD.

Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators.

Overnutrition and a sedentary lifestyle have resulted in the expansion of human obesity and associated metabolic complications. Nonalcoholic fatty liver disease has become the most common chronic liver disease in Western developed countries and can range from simple hepatic steatosis to a combination of steatosis, inflammation, and ballooning degeneration (nonalcoholic steatohepatitis). Obesity and its related liver disease are both risk factors for hepatocellular carcinoma, the incidence of which is expected to increase rapidly. The pathogenesis of nonalcoholic fatty liver disease and its progression to nonalcoholic steatohepatitis and hepatocellular carcinoma involve a deregulated lipid metabolism and a disruption of immune homeostasis and tissue integrity and are associated with a state of chronic inflammation. Macrophages are immune cells essential for maintenance of organ function and homeostasis but can also contribute to tissue damage and maintain a proinflammatory response. Their function depends on their origin, and tissue and can be converted based on local environmental cues. Resident liver macrophages, Kupffer cells, which function as sentinels, provide a first defense and are assisted by infiltrating monocytes in cases of hepatic insult. Until now, the contribution of tissue-residing and infiltrating macrophages to the onset and progression of nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and hepatocellular carcinoma has been only partially unraveled. This review summarizes the current knowledge on the contribution of macrophage subsets to obesity-driven fatty liver disease and its complications and sheds light on still unexplored areas.

BACKGROUND & AIMS: Primary nonfunction (PNF) is a rare complication after liver transplantation that requires urgent retransplantation. PNF is associated with livers from extended criteria donors. Clinical and biochemical factors have not been identified that reliably associate with graft function after liver transplantation. Serum patterns of N-glycans associate with changes in the liver. We analyzed perfusate from grafted liver to identify protein glycosylation patterns associated with PNF. METHODS: We performed a prospective study of consecutive patients who underwent liver transplantation (66 patients, from 1 center, in the derivation set, and 56 patients, from 2 centers, in the validation set) in Belgium, from October 1, 2011, through April 30, 2017. All donor grafts were transported using cold static storage, and perfusate samples were collected from the livers by flushing of hepatic veins before transplantation. Protein-linked N-glycans were isolated from perfusate samples and analyzed with a multicapillary electrophoresis-based ABI3130 sequencer. We compared glycan patterns between patients with vs without PNF of transplanted livers. PNF was defined as the need for urgent retransplantation when a graft had no evidence of function, after exclusion of other causes, such as hepatic artery thrombosis or acute cellular rejection. RESULTS: The relative abundance of a single glycan, agalacto core-alpha-1,6-fucosylated biantennary glycan (NGA2F) was significantly increased in perfusate of livers given to 4 patients who developed PNF after liver transplantation compared with livers given to patients who did not develop PNF. Level of NGA2F identified patients with PNF with 100% accuracy. This glycomarker was the only factor associated with PNF in multivariate analysis in the derivation and the validation sets (P < .0001). CONCLUSIONS: In an analysis of patients who underwent liver transplantation, we associated graft perfusate level of glycan NGA2F present on perfusate proteins with development of PNF with 100% accuracy, and validated this finding in a separate cohort of patients. This biomarker might be used to assess grafts before transplantation, especially when high-risk organs are under consideration. ispartof: Gastroenterology vol:154 issue:5 pages:1361-1368 ispartof: location:United States status: published

Hepatopulmonary syndrome (HPS) is a severe complication of cirrhosis with increased risk of mortality. Pulmonary microvascular alterations are key features of HPS, but underlying mechanisms are incompletely understood, and studies on HPS are limited to rats. Placental growth factor (PlGF), a pro-angiogenic molecule that is selectively involved in pathological angiogenesis, may play an important role in HPS development, however, its role has never been investigated. In this study, we validated an HPS model by common bile duct ligation (CBDL) in mice, investigated the kinetic changes in pulmonary angiogenesis and inflammation during HPS development, and provide evidence for a novel therapeutic strategy by targeting pathological angiogenesis. Mice with CBDL developed hypoxemia and intrapulmonary shunting on a background of liver fibrosis. Pulmonary alterations included increased levels of pro-angiogenic and inflammatory markers, which was confirmed in serum of human HPS patients. Increased PlGF production in HPS mice originated from alveolar type II cells and lung macrophages, demonstrated by immunofluorescent stainings. Dysfunctional vessel formation in CBDL mice was visualized by microscopy on vascular corrosion casts. Both prophylactic and therapeutic anti-PlGF (αPlGF) antibody treatment impeded HPS development, as demonstrated by significantly less intrapulmonary shunting and improved gas exchange. αPlGF treatment decreased endothelial cell dysfunction in vivo and in vitro, and was accompanied by reduced pulmonary inflammation. Importantly, αPlGF therapy did not affect liver alterations, supporting αPlGF's capability to directly target the pulmonary compartment. Conclusion: CBDL in mice induces HPS, which is mediated by PlGF production. αPlGF treatment improves experimental HPS by counteracting pulmonary angiogenesis and might be an attractive therapeutic strategy for human HPS. This article is protected by copyright. All rights reserved.

Background Hepatitis E virus (HEV) infection is increasingly recognized as a cause of hepatitis in developed countries. A high HEV IgG seroprevalence in humans and pigs is reported as well as sporadic clinical cases of autochtonous HEV but there are currently no data available on the clinical burden of HEV in Belgium. Objectives The objective of the current study was to evaluate the actual clinical burden of HEV infections in our tertiary care center in Flanders, Belgium. Study design In the setting of Ghent University Hospital, patients were assessed for the presence of HEV IgG and IgM as well as HEV RNA if no other cause was found for one of the following clinical presentations: a) elevation of liver enzymes in post-liver transplant; b) suspicion of acute or toxic hepatitis; c) unexplainable elevation of liver enzymes; d) cirrhosis with acute-on-chronic exacerbation. Results In a period of 39 months (January 2011–April 2014) 71 patients were enrolled. HEV IgG was found positive in 13 (18,3%) patients; HEV IgM in 6 patients (8,5%) and HEV RNA in 4 (5,6%) patients. All HEV IgM/RNA positive patients were male, aged 41–63, and classified in the clinical groups a), b) or d). HEV IgG seroprevalence was slightly higher but not significantly different from the seroprevalence in the general population in this region in Belgium previously reported to be 14% (p-value 0.41) by our group. Conclusions HEV should be considered as a cause of liver pathology especially in middle-aged men with elevation of liver enzymes.

Background: In patients with hepatocellular carcinoma (HCC), the outcome after liver transplantation (LT) is excellent if tumor characteristics are within the Milan criteria (MC). Expanded Asan criteria (AC) have not yet been validated in Western countries. Methods: A total of 76 patients with HCC underwent LT. Patients were divided and compared according to Milan, UCSF, and Asan criteria. Differences between pre- and post-operative assessment were evaluated. Overall survival (OS) and disease-free survival (DFS) were compared between groups. Predictors of recurrence were investigated. Results: Asan criteria provided 26% and 15% more criteria-fitting patients than MC and UCSF pre-operatively while 49% and 35% at pathological evaluation. Discrepancy between pre- and post-operative evaluation was 32% for MC, 33% for UCSF, and 18% for AC (p=0.06). After a median follow-up of 70.5months, patients exceeding MC but fulfilling Asan had comparable 5-yr OS and DFS to patients fulfilling MC (p=0.17; p=0.29). Patients exceeding UCSF but fulfilling AC had comparable 5-yr OS and DFS to patients fulfilling UCSF (p=0.26; p=0.32). Number of nodules, macro-vascular invasion, capsular invasion, and exceeding AC predicted recurrence at multivariate analysis (p=0.01, 0.03, 0.01, 0.02, respectively). Conclusions: The extension to AC allows increasing the number of patients eligible for LT without affecting OS and DFS.

Kupffer cells (KCs) and monocyte-derived macrophages are implicated in non-alcoholic steatohepatitis (NASH) pathogenesis but their functions remain unclear due to the lack of specific markers to distinguish between the different cell types. Additionally, it is unclear if multiple subsets of KCs are present during NASH. Here, we characterized the liver macrophage subsets during methionine/choline deficient (MCD) diet-induced NASH and recovery. We observed a significant reduced contribution of Ly6CloClec4F+Tim4+KCs to the hepatic macrophage pool in MCD fed mice, which normalized during recovery. Ly6CloClec4F-Tim4- monocyte-derived macrophages increased during MCD feeding and returned to baseline during recovery. Ly6CloClec4F+Tim4- monocyte-derived KCs developed during initial recovery but did not self-renew as their numbers were reduced after full recovery. Initial recovery from MCD diet feeding was further characterized by increased proportions of Ki-67+ proliferating KCs. In conclusion, the hepatic macrophage pool undergoes substantial albeit transient changes during NASH and recovery, with the KC pool being maintained by proliferation and differentiation of short-lived monocyte-derived KCs.

The role of endoplasmic reticulum stress and the unfolded protein response (UPR) in cholestatic liver disease and fibrosis is not fully unraveled. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, has been shown to reduce endoplasmic reticulum (ER) stress and counteract apoptosis in different pathologies. We aimed to investigate the therapeutic potential of TUDCA in experimental secondary biliary liver fibrosis in mice, induced by common bile duct ligation. The kinetics of the hepatic UPR and apoptosis during the development of biliary fibrosis was studied by measuring markers at six different timepoints post-surgery by qPCR and Western blot. Next, we investigated the therapeutic potential of TUDCA, 10 mg/kg/day in drinking water, on liver damage (AST/ALT levels) and fibrosis (Sirius red-staining), in both a preventive and therapeutic setting. Common bile duct ligation resulted in the increased protein expression of CCAAT/enhancer-binding protein homologous protein (CHOP) at all timepoints, along with upregulation of pro-apoptotic caspase 3 and 12, tumor necrosis factor receptor superfamily, member 1A (TNFRsf1a) and Fas-Associated protein with Death Domain (FADD) expression. Treatment with TUDCA led to a significant reduction of liver fibrosis, accompanied by a slight reduction of liver damage, decreased hepatic protein expression of CHOP and reduced gene and protein expression of pro-apoptotic markers. These data indicate that TUDCA exerts a beneficial effect on liver fibrosis in a model of cholestatic liver disease, and suggest that this effect might, at least in part, be attributed to decreased hepatic UPR signaling and apoptotic cell death.

Background & Aims Hepatopulmonary syndrome is a complication of chronic liver disease resulting in increased morbidity and mortality. It is caused by intrapulmonary vascular dilations and arteriovenous connections with devastating influence on gas exchange. The pathogenesis is not completely understood but evidence mounts for angiogenesis. Aims of this study were [1] to identify angiogenic factors in serum of patients with hepatopulmonary syndrome and [2] to study the possibility to predict its presence by these factors. Methods Multiplex assays were used to measure the concentration of angiogenic factors in patients with (n = 30) and without hepatopulmonary syndrome (n = 30). Diagnosis was based on the presence of gas exchange abnormality and intrapulmonary vasodilations according to published guidelines. Results Patients with and without hepatopulmonary syndrome had similar MELD scores (median: 11.2 vs. 11.6; P = 0.7), Child–Pugh score (P = 0.7) and PaCO2 values (median: 35 vs. 37; P = 0.06). PaO2 and P(A-a) O2 gradient were significantly different (respectively median of 80 vs. 86, P = 0.02; and 24 vs. 16, P = 0.004). Based on area under the curve (AUC) data and P-values, the best predictors were vascular cell adhesion molecule 1 (VCAM1) (AUC = 0.932; P

When cells are subjected to stress by changes in their extracellular environment, unfolded proteins accumulate in the endoplasmic reticulum (ER), causing ER stress. This initiates the unfolded protein response (UPR), a signal transduction cascade aiming at restoring cellular homeostasis. The UPR and angiogenesis are involved in the pathogenesis of many diseases such as cancer, pulmonary diseases and chronic liver diseases (CLDs) including alcoholic liver disease, non-alcoholic steatohepatitis and hepatitis B. This review summarizes the upcoming knowledge of the interaction between the UPR and angiogenesis in physiological angiogenesis and in different CLDs and other diseases.

Fanconi anemia (FA) is an inherited bone marrow failure syndrome due to defective DNA inter-strand cross-link repair. Hematopoietic stem cell transplantation (HSCT) is curative for pancytopenia, but may not prevent the development of non-hematological malignancies. We describe a 26-year-old male patient with FA and Marfan syndrome who in 1994 underwent successful HSCT with bone marrow stem cells from his human leukocyte antigen (HLA)-identical sister. In 2006, three lesions in the liver were detected and resected. The three lesions all showed activation of the β-catenin pathway and were histologically characterized by a highly differentiated steatotic hepatocellular carcinoma (HCC) with remnants of the underlying adenoma from which it arose, a hepatocellular adenoma with foci of well-differentiated HCC, and a cholestatic adenoma. Risk factors for the emergence of HCC included FA itself, the use of androgens for a period of 3 years preceding HSCT and toxicity of the conditioning regimen. Because of the danger of developing additional HCC, liver transplantation was proposed, taking into consideration that immunosuppression would increase the risk of other malignancies. By using part of the liver of the sister, who already acted as bone marrow donor 13 years earlier, immunosuppression could be avoided. Liver transplantation was performed in 2007 without complication. Five years after liver transplantation the patient is doing well. This case is twofold special being the first case reporting FA co-occurring with Marfan syndrome and being the first reported case of FA treated for HCC by liver transplantation from a HLA-identical sibling donor without the use of immunosuppression.

Portopulmonary hypertension (POPH), a complication of chronic liver disease, may be a contraindication to liver transplantation (LT) because of the elevated risk of peritransplant and posttransplant morbidity and mortality. Because POPH is frequently asymptomatic, screening with echocardiography is recommended. The only reliable technique, however, for diagnosing POPH is right heart catheterization (RHC). The aims of this study were to evaluate the current estimated systolic pulmonary artery pressure (sPAP) cutoff value of 30 mm Hg and to determine a better cutoff value. One hundred fifty-two patients underwent pretransplant echocardiography between January 2005 and December 2010. These echocardiographic results were compared with pulmonary artery pressures measured during the pretransplant workup or at the beginning of the transplantation procedure (both by catheterization). With a cutoff value of 30 mm Hg, 74 of the 152 patients met the criteria for POPH on echocardiography, although the diagnosis was confirmed in only 7 patients during catheterization; this resulted in a specificity of 54%. It would have been more accurate to use a cutoff value of 38 mm Hg, which had a maximal specificity of 82% and, at the same time, guaranteed a sensitivity and negative predictive value of 100%. With the incorporation of the presence or absence of right ventricular dilatation, the specificity even increased to 93% for this new cutoff value. In conclusion, the prevalence of POPH was 4.6% among LT candidates in this study. We can recommend that LT candidates with an sPAP > 38 mm Hg should be referred for RHC. With the cutoff value increased from 30 to 38 mm Hg, the number of patients undergoing invasive RHC during their evaluation could be safely reduced.

Portopulmonary hypertension (POPH) is a part of group 1 pulmonary hypertension (pulmonary hypertension associated with portal hypertension). Liver transplantation (LTx) may be curative, but is usually restricted to patients with mild-to-moderate POPH. The presence of severe POPH may be a contraindication to transplantation because of the elevated risk of peritransplantation and post-transplantation morbidity and mortality. This report describes a series of seven patients with onset of moderate (two patients) or severe (five patients) POPH before LTx, of whom six were treated with oral vasodilator therapy for POPH. Although previous studies recommend aggressive parenteral prostacyclin therapy (epoprostenol), we describe the opportunity to treat cases of severe POPH with an oral phosphodiesterase type 5 inhibitor (sildenafil) and/or an endothelin receptor antagonist (bosentan/ambrisentan) as a bridge to successful LTx in selected patients.

As different hepatitis C virus (HCV) genotypes respond differently to initiated therapy, correct HCV genotyping is essential. A potential risk for misclassification of the intergenotypic HCV circulating recombinant form (CRF) 2k/1b strains exists, depending on the genotyping method used. The aim was to investigate the differences in HCV genotyping methods with regard to CRF 2k/1b and to gain insight in the prevalence of the CRF 2k/1b. Genotyping results by Versant HCV Genotype Assay were compared with nonstructural protein 5B (NS5B) sequencing. In total, from November 2001 until March 2015, 3296 serum samples were analyzed by Versant HCV Genotype Assay. As misclassified CRF is harbored among HCV genotype 2, we further focused our search on 142 (4.3%) samples positive for HCV genotype 2. On 116 (81.7%) retrieved samples, the NS5B sequencing was performed. Twelve out of the 116 retrieved samples (10.3%) were classified as CRF 2k/1b by sequencing of the NS5B region. Ten of these 12 samples were originally misclassified as genotype 2a or 2c, while 2 of them were misclassified as genotype 2. Our results show that the current prevalence of CRF 2k/1b is underestimated. The importance of correct HCV genotyping is emphasized, considering the tailored choice of treatment regimen and overall prognosis.

Background & Aims Acetaminophen overdose in mice is characterized by hepatocyte endoplasmic reticulum stress, which activates the unfolded protein response, and centrilobular hepatocyte death. We aimed at investigating the therapeutic potential of tauroursodeoxycholic acid, a hydrophilic bile acid known to have anti-apoptotic and endoplasmic reticulum stress reducing capacities, in experimental acute liver injury induced by acetaminophen overdose. Methods Mice were injected with 300 mg/kg acetaminophen, two hours prior to receiving tauroursodeoxycholic acid, N-acetylcysteine or a combination therapy, and were euthanized 24 hours later. Liver damage was assessed by serum transaminases, liver histology, TUNEL staining, expression profiling of inflammatory, oxidative stress, unfolded protein response, apoptotic and pyroptotic markers. Results Acetaminophen overdose resulted in a significant increase in serum transaminases, hepatocyte cell death, unfolded protein response activation, oxidative stress, NLRP3 inflammasome activation, caspase 1 and pro-inflammatory cytokine expressions. Standard of care, N-acetylcysteine and, to a lesser extent, tauroursodeoxycholic treatment were associated with significantly lower transaminase levels, hepatocyte death, unfolded protein response activation, oxidative stress markers, caspase 1 expression and NLRP3 levels. Importantly, the combination of N-acetylcysteine and tauroursodeoxycholic acid improved serum transaminase levels, reduced histopathological liver damage, UPR-activated CHOP, oxidative stress, caspase 1 expression, NLRP3 levels, IL-1β levels and the expression of pro-inflammatory cytokines and this to a greater extend than N-acetylcysteine alone. Conclusions These findings indicate that a combination strategy of N-acetylcysteine and tauroursodeoxycholic acid surpasses the standard of care in acetaminophen-induced liver injury in mice and might represent an attractive therapeutic opportunity for acetaminophen-intoxicated patients. This article is protected by copyright. All rights reserved.

The pandemic rise in obesity has resulted in an increased incidence of metabolic complications. Non-alcoholic fatty liver disease is the hepatic manifestation of the metabolic syndrome and has become the most common chronic liver disease in large parts of the world. The adipose tissue expansion and hepatic fat accumulation characteristics of these disorders compromise local oxygen homeostasis. The resultant tissue hypoxia induces adaptive responses to restore oxygenation and tissue metabolism and cell survival. Hypoxia-inducible factors (HIFs) function as master regulators of this hypoxia adaptive response, and are in turn hydroxylated by prolyl hydroxylases (PHDs). PHDs are the main cellular oxygen sensors and regulate HIF proteasomal degradation in an oxygen-dependent manner. HIFs and PHDs are implicated in numerous physiological and pathological conditions. Extensive research using genetic models has revealed that hypoxia signaling is also a key mechanism in adipose tissue dysfunction, leading to adipose tissue fibrosis, inflammation and insulin resistance. Moreover, hypoxia affects liver lipid metabolism and deranges hepatic lipid accumulation. This review summarizes the molecular mechanisms through which the hypoxia adaptive response affects adipocyte and hepatic metabolism, and the therapeutic possibilities of modulating HIFs and PHDs in obesity and fatty liver disease.

The gold standard for the diagnosis of acute cellular rejection (ACR) is a liver biopsy. The quest for an alternative non-invasive biomarkers has been long and is ongoing. However, an efficient and useful biomarker has not been developed yet. In this manuscript, we review all possible candidate biomarkers that have been studied in recent years, starting with cytokines and ending with an overview of different newly discovered "omics". Promising paths are being explored but a valid non-invasive biomarker has not been discovered yet.

In Wilson's disease, liver transplantation can constitute the only option for patients presenting with fulminant hepatic failure or decompensated liver disease unresponsive to drug therapy. We report the case of a 29-year-old woman receiving a liver transplant for end-stage Wilson's disease who developed neurological complications after transplantation. After an accurate evaluation of possible differential causes of neurological complications developing as the result of liver transplantation, moyamoya disease was diagnosed. Moyamoya disease is a rare cerebrovascular disease of unknown etiology. However, data exist supporting a possible role for some immunosuppressive regimens in determining the peculiar vascular alterations observed in moyamoya disease. To the best of our knowledge, the association with post-transplantation state for Wilson's disease has not been previously described.