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Background & Aims: Bacterial infections can trigger the development of organ failure(s) and acute-on-chronic liver failure (ACLF). Geographic variations in bacteriology and clinical practice could lead to worldwide differences in ACLF epidemiology, phenotypes and associated outcomes. Herein, we aimed to evaluate regional differences in bacterial infection-related ACLF in patients with cirrhosis admitted to hospital. Methods: This post hoc analysis included 1,175 patients with decompensated cirrhosis (with bacterial infection on admission or nosocomial infection) from 6 geographic regions worldwide. Clinical, laboratory and microbiological data were collected from the diagnosis of infection. Patients were followed-up for organ failure(s) and ACLF development according to the EASL-CLIF criteria from enrolment to discharge/death. Results: A total of 333 patients (28%) had ACLF at diagnosis of infection, while 230 patients developed ACLF after diagnosis of infection, resulting in an overall rate of bacterial infection related-ACLF of 48%, with rates differing amongst different geographic regions (38% in Southern Europe vs. 75% in the Indian subcontinent). Bacterial infection related-ACLF more frequently developed in younger patients (55 ± 13 vs. 58 ± 14 years), males (73% vs. 62%), patients with alcohol-related cirrhosis (59% vs. 45%) and those with a higher baseline MELD score (25 ± 11 vs. 16 ± 5) (all p

Introduction and aim Thromboxane (TX) A2 was identified as an important vasoconstrictor during Zymosan induced portal perfusion pressure (PP) increase. We aimed at investigating whether hepatic steatosis influences the extent of TXA2-induced portal hypertension. Materials and methods Sprague–Dawley rats were randomly divided into control and steatosis (induced by the special diet) groups. PP and TXB2 (stable degradation product of TXA2) in the perfusate were measured after in situ liver perfusion with Zymosan (150 μg/ml, 40–46 min) or U46619 (TXA2 analog, 0.1 μM/ml, 40–46 min). The number of Kupffer cell (KC) was measured by immunohistochemistry with CD163. Results Zymosan induced more TXB2 production and a higher PP increase in control group than in steatosis group despite more CD163 positive KCs in fatty livers. PP and TXB2 efflux revealed a strong correlation in control group and a moderate correlation in steatosis group. Contrary to the effect of Zymosan, U46619 induced a much higher PP increase in steatosis group than in control group. Conclusion Severe steatosis increased number of KCs, however, PP increase and TXB2 efflux caused by Zymosan infusion in fatty livers were lower than those in healthy livers. In contrast, TXA2 analog caused higher PP increase in fatty livers. Targeting the more sensitive response to TXA2 in fatty livers might be a potential therapy of severe steatosis.

The data presented in this article describe the fatty acid composition of chow, liver tissue and isolated liver mitochondria from mice fed for 6–24 weeks with a high caloric western diet (WD) in comparison to control diet (normal diet, ND). The fatty acid composition was measured via gas chromatography flame ionization detection (GC-FID). Moreover, WD-induced mitochondrial protein changes are presented in this work and were analyzed by mass spectrometry (LC–MS/MS). For further interpretation and discussion of the presented data please refer to the research article entitled “Mitochondrial adaptation in steatotic mice” (Einer et al., 2017) [1] .

Kupffer cells (KCs) have a major role in liver injury, and cysteinyl-leukotrienes (Cys-LTs) are known to be involved as well. The KC-mediated pathways for the production and secretion of Cys-LT in cholestatic liver injury have not yet been elucidated. Here, we hypothesized that KC activation by Toll-like receptor ligands results in Cys-LT-mediated microcirculatory alterations and liver injury in acute cholestasis. We hypothesized further that this situation is associated with changes in the secretion and production of Cys-LT. One week after bile duct ligation (BDL), livers showed typical histological signs of cholestatic liver injury. Associated microcirculatory disturbances caused increased basal and maximal portal pressure following KC activation. These differences were determined in BDL livers compared with sham-operated livers in vivo (KC activation by LPS 4 mg/kg b.w.) and in isolated perfused organs (KC activation by Zymosan A, 150 μg/ml). Treatment with the 5-lipoxygenase inhibitor MK-886 alone did not alter portal perfusion pressure, lactate dehydrogenase (LDH) efflux, or bile duct proliferation in BDL animals. Following KC activation, portal perfusion pressure increased. The degree of cell injury was attenuated by MK-886 (3 μM) treatment as estimated by LDH efflux. In normal rats, a large amount of Cys-LT efflux was found in the bile. Only a minor amount was found in the effluent perfusate. In BDL livers, the KC-mediated Cys-LT efflux into the sinusoidal system increased, although the absolute Cys-LT level was still grossly lower than the biliary excretion in sham-operated livers. In conclusion, our results indicate that treatment with Cys-LT inhibitors might be a relevant target for attenuating cholestatic liver damage.

Recent studies have shown that the risk of variceal bleeding in patients with liver cirrhosis increases with infections such as spontaneous bacterial peritonitis (SBP). In this study, we hypothesized that pretreatment with intraperitoneal LPS may escalate portal hypertension. In fibrotic livers (4 weeks after bile duct ligation, BDL), the activation of Kupffer cells (KCs) by zymosan (150 microg/ml) in the isolated non-recirculating liver perfusion system resulted in a transient increase in portal perfusion pressure. Pretreatment with intraperitoneal LPS (1 mg/kg body weight (b.w.) for 3 h) increased basal portal perfusion pressure, and prolonged the zymosan-induced increase from transient to a long-lasting increase that was sustained until the end of the experiments in BDL but not in sham-operated animals. Pretreatment with gadolinium chloride (10 mg/kg b.w.), MK-886 (0.6 mg/kg b.w.), Ly171883 (20 microM) or BM 13.177 (20 microM) reduced the maximal and long-lasting pressure increase in BDL animals by approximately 50-60%. The change in portal perfusion pressure was paralleled by a long-lasting production of cysteinyl leukotriene (Cys-LT) and thromboxane (TX) after LPS pretreatment. However, the response to vasoconstrictors was not altered by intraperitoneal LPS. Western blot analyses showed an increased Toll-like receptor (TLR)4 and MyD88 expression after LPS pretreatment. In vivo experiments confirmed that intraperitoneal LPS increased basal portal pressure, and extended the portal pressure increase produced by intraportal zymosan or by LPS infusion. In conclusion, upregulation of TLR4 and MyD88 expression in fibrotic livers confers hypersensitivity to LPS. This may lead to escalation of portal hypertension by production of TX and Cys-LT after endotoxin-induced KC activation. Therefore, LT inhibitors may represent a promising treatment option in addition to early administration of antibiotics in SBP.

Background/Aims Cirrhotic patients show an increased risk of variceal bleeding upon bacterial infections. Kupffer cells (KC) constitute the first macrophage population to become activated by bacterial β-glucans and endotoxins derived from the gut. We therefore investigated whether and how KC activation increases portal pressure. Methods KC in normal and fibrotic livers from bile duct ligated (BDL) rats were activated by the β-glucan component of zymosan in vivo and during isolated rat liver perfusion. Results Activation of KC in normal livers resulted in a severalfold increase of portal pressure in vivo as well as in isolated perfused liver preparations. This increase and the accompanying 40-fold stimulation of hepatic prostaglandin F 2α /D 2 and thromboxane A 2 (TxA 2 ) production in isolated perfused livers were attenuated by KC blockade. The TxA 2 synthase inhibitor furegrelate and the TxA 2 receptor antagonist BM 13.177 reduced the increase of portal perfusion pressure supporting TxA 2 as pivotal vasoconstrictor released by activated KC. Importantly, a more pronounced vasopressor response in fibrotic livers was related to a raise in KC density and a 10-fold increase of TxA 2 production after KC activation. Conclusions KC activated by β-glucans increase portal pressure through the release of TxA 2 . This vasopressor response is augmented in BDL induced fibrosis.

Background/Aims Failure to induce apoptosis triggered by members of the death receptor family has been described in hepatocellular carcinoma (HCC) and sensitization of malignant cells to pro-apoptotic molecules such as TRAIL has been proposed as an alternative cancer therapy. Limiting to this approach are the resistance of many tumor cells to TRAIL and safety concerns about the toxicity of TRAIL in normal hepatocytes. Methods We here explored the possibility that the protooncogene c-Src, known to be overexpressed in a variety of tumors, could be specifically responsible for the loss of response to receptor-mediated apoptosis. Results Cotreatment of several hepatoma cell lines with the Src inhibitor PP2 potently sensitized these cells to TRAIL and CD95, dramatically decreasing effective doses of TRAIL to as low as 1ng/ml. Remarkably, Src-inhibition did not synergize with TRAIL signaling in primary hepatocytes. Specific siRNAs showed that the effect was due to blockade of p60 c-Src and occurred through increased recruitment of caspase 8. Conclusions We provide evidence that p60 c-Src is an important and effective suppressor of receptor-mediated apoptosis in hepatoma cells but not in primary human hepatocytes. Inhibition of Src sensitizes tumor cells to apoptosis and decreases effective doses of TRAIL to therapeutic concentrations.

BACKGROUND: Crigler-Najjar syndrome type 1 (CNS1) is characterized by severe unconjugated hyperbilirubinemia from birth, caused by total failure of UDP-glucuronyltransferase activity. Only orthotopic liver transplantation (OLT) offers the prospect of cure. However, because the onset of neurologic deficits is unpredictable, timing of OLT remains difficult. METHODS: In our transplant center, 3 patients underwent early OLT for CNS1. Two of them (7 yr, 12 yr) showed mild to moderate neurologic deficits only few weeks before OLT, another patient (4 yr) had no signs of bilirubin encephalopathy. All patients required extensive phototherapy to control bilirubin levels. Thus, OLT was performed shortly after the onset of neurologic symptoms or as a prophylactic procedure, respectively. RESULTS: OLT was uneventful in all recipients. One of the symptomatic patients (7 yr) completely recovered from neurologic deficits at 36 months on OLT, whereas the other patient (12 yr) significantly improved symptoms at 27 months of OLT. These patients, including the 4-year-old boy, attend school at appropriate grades now. CONCLUSIONS: Irreversible brain damage (kernicterus) may occur in the course of CNS1 disease. Because no alternative treatment options are available at this time, OLT should be performed as a preventive procedure to counteract severe CNS-related complications.

Hyperlipoproteinemia is frequent following liver transplantation and may lead to atherosclerosis. Lipid-lowering agents may be useful, but could interfere with the function of the transplanted organ and with immunosuppression. We therefore evaluated in a prospective, randomized, open-labeled cross-over trial the effect of two frequently used 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (pravastatin 10 mg d(-1) and cerivastatin 0.1 mg d(-1)) in hyperlipoproteinemic patients after liver transplantation.Sixteen patients (6.3 +/- 2.0 years post-transplantation, cyclosporine n = 11, tacrolimus n = 5) with hyperlipoproteinemia (cholesterol 246 +/- 42, triglycerides 191 +/- 87, low-density lipoprotein (LDL)-cholesterol 161 +/- 35, high-density lipoprotein (HDL)-cholesterol 44 +/- 11 mg d(-1)) were included. Treatment periods of 6 weeks were separated by a 4-week washout period.Both medications were tolerated well, no effects on serum concentrations of liver enzymes or immunosuppressive agents were observed. Cerivastatin and pravastatin decreased (P0.001) cholesterol by 21 +/- 10% and 15 +/- 10%, LDL-cholesterol by 27 +/- 14% and 17 +/- 15%, respectively, while triglyceride and HDL-cholesterol concentrations did not change significantly. LDL/HDL-cholesterol markedly improved (P0.001) by 29 +/- 16% (cerivastatin) and 16 +/- 16% (pravastatin). Cerivastatin was more potent than pravastatin in patients receiving cyclosporine A, while there was no significant difference in patients receiving tacrolimus.Low-dose cerivastatin and pravastatin significantly improve lipid profiles following liver transplantation without affecting liver function or immunosuppression.

Background/Aims: The cardiovascular hormone Atrial Natriuretic Peptide (ANP) attenuates activation of the pro-inflammatory transcription factor NF-κB in macrophages. ANP was also shown to protect from ischemia-reperfusion injury of the rat liver. This study aimed to investigate the effects of this immunomodulatory hormone and its second messenger cGMP on the activation of the two redox-sensitive transcription factors AP-1 and NF-κB and the expression of corresponding pro-inflammatory target genes during ischemia and reperfusion of the liver. The identification of the mechanisms underlying the protection by ANP should reveal new aspects concerning the pathomechanisms of ischemia/reperfusion injury. Methods: Rat livers were perfused with and without ANP or 8-Br-cGMP preceding 24 h of cold storage in University of Wisconsin solution. During reperfusion NF-κB and AP-1 DNA binding activities were determined in freeze-clamped liver samples by electrophoretic mobility shift assay. Protein levels of p50, p65, and of IκB were determined by Western blot. mRNA coding for inducible nitric oxide synthase, cyclooxygenase-2, and TNF-α was determined by RT-PCR and Northern blot. Results: After 45 min of reperfusion DNA binding activities of NF-κB were increased, whereas in ANP pre-treated livers this effect was markedly reduced. AP-1, another important redox-sensitive transcription factor, was activated and in the course of reperfusion the subunit composition of AP-1 changed as assessed by supershift assays. ANP markedly reduced binding activities of both forms of AP-1. 8-Br-cGMP mimicked the effects of ANP on NF-κB and AP-1. Neither inducible nitric oxide synthase nor cyclooxygenase-2 mRNA could be detected. In contrast, a profound expression of transcripts coding for TNF-α was detected in the course of reperfusion and ANP markedly reduced TNF-α mRNA expression. Conclusion: ANP seems to mediate its protective effect during ischemia and reperfusion by reducing the activation of NF-κB and AP-1 via cGMP. The reduced binding activity of these redox-sensitive transcription factors was accompanied by a diminished mRNA expression of TNF-α, a cytokine known to be involved in cellular damage in ischemia reperfusion injury.

s / Digestive and Liver Disease 48S (2016) e1–e17 e17 showed that 70% of patients has insufficient intake of calories and 40-60% inadequate water and sodium intake, despite the nutritional indications provided by the hepatologist. The weekly macronutrient intake was insufficient in 70-95% of cases, with the exception of sugar, which was excessive in 87% of cases including diabetic patients. Conclusions: The intake of total calories anddifferentmacronutrients is inadequate in 80-90% of outpatients with cirrhosis, due to both scarce adherence to the indications given by the hepatologist and low knowledge of nutritional facts. Muscle wasting is very often present even in less advanced disease. This highlights the need of a personalized nutritional approach, including educational and motivational aspects. http://dx.doi.org/10.1016/j.dld.2015.12.047

Torasemide is a new loop diuretic with a longer half-life and longer action than furosemide in healthy subjects. In order to evaluate the pharmacodynamic effects, single oral doses of furosemide (80 mg) and torasemide (20 mg), which were equipotent in healthy subjects, were given to 14 patients with cirrhosis and ascites. Before the study patients underwent an equilibration period of 4 days without diuretics. The drugs were alternated following a randomized double-blind cross-over design after a wash-out period of at least 2 days. Urine was collected at defined intervals for 24 h after drug administration and blood samples were taken before, 6 h and 24 h after medication. Torasemide induced greater cumulative 24 h diuresis (2863 ± 343 vs. 2111 ± 184 ml, p p p p

The role of atrial natriuretic peptide (ANP) and potential defects of ANP in liver disease are reviewed. Patients with cirrhosis of the liver show no decrease of ANP plasma concentrations nor changes in the pattern of ANP immunoreactivity nor changes of splanchnic ANP clearance. The renal effects of exogenously administered as well as endogenously released ANP are blunted in cirrhosis, in particular in patients with ascites. This seems due to increased activity of sodium-retaining hormonal systems and changes of the renal ANP receptor status. Pharmacological inhibition of ANP-degradation or clearance may yield therapeutic potential.

The tumor suppressor p53 exerts important protective functions towards DNA-damaging agents. Its inactivation by allelic deletions or point mutations within the P53 gene as well as complex formation of wildtype p53 with cellular or viral proteins is a common and crucial event in carcinogenesis. Mutations increase the half-life of the p53 protein allowing the immunohistochemical detection and anti-p53 antibody formation. Distinct G to T point mutations in codon 249 leading to a substitution of the basic amino acid arginine by the neutral amino acid serine are responsible for the altered functionality of the mutant gene product and were originally identified in 8 of 16 Chinese and 5 of 10 African HCC patients. Both groups are frequently exposed to mycotoxin contaminations of their food. Today an average P53 gene mutation rate of 25% is assumed for high-aflatoxin B1-exposure regions. This is double the rate observed in low-aflatoxin B1-exposure countries. Although many HCC patients displaying P53 mutations also suffer from HBV infection, which itself can lead to rearrangements of P53 coding regions or induce the synthesis of viral proteins possibly interacting with p53, the specific G to T transversion within codon 249 of the P53 gene seems to directly reflect the extent of aflatoxin B1 exposure.

The existence of marked elevations of ascitic fluid cholesterol has been observed in patients with peritoneal carcinomatosis compared to patients with cirrhosis and has been found useful in differential diagnosis. This finding could be caused by an enhanced movement of plasma lipoproteins into the peritoneal cavity. To test this hypothesis we determined the fasting concentrations of total, high density lipoprotein (HDL)- and low density lipoprotein (LDL)-cholesterol, apolipoprotein-A1 (apo-A1) and apolipoprotein-B (apo-B) in serum and ascites of 17 patients with cirrhosis and 16 patients with peritoneal carcinomatosis. The movement of proteins from plasma to ascites was calculated from the ascites/serum concentration ratios of six different sized proteins with a molecular mass ranging from 54 kDa to 971 kDa. Mean values (mg/dl) for total cholesterol (92.6 vs. 21.0), HDL-cholesterol (15.6 vs. 1.8), LDL-cholesterol (63.4 vs. 16.1), apo-A1 (50.2 vs. 13.6) and apo-B (41.2 vs. 12.9) in ascites were significantly higher in peritoneal carcinomatosis than in cirrhosis. These differences could only partially be explained by the higher serum concentrations of these parameters in peritoneal carcinomatosis, but were mainly due to a lower selectivity for the movement of plasma proteins and lipoproteins into ascites (mean ascites/serum (A/S) ratio: 0.30-0.77) in peritoneal carcinomatosis as compared to cirrhosis (mean ascites/serum ratio: 0.11-0.21). In both groups about 85% of the total cholesterol in serum and ascites consisted of HDL- and LDL-cholesterol. These findings support the hypothesis that elevations in ascitic cholesterol in peritoneal carcinomatosis compared to cirrhosis are mainly caused by the increased movement of plasma HDL and LDL into the peritoneal cavity.

In 11 patients with decompensated cirrhosis and deteriorating renal function, the effect of the vasoconstrictor substance 8-ornithin vasopressin (ornipressin; POR 8; Sandoz, Basel, Switzerland) on renal function, hemodynamic parameters, and humoral mediators was studied. Ornipressin was infused at a dose of 6 IU/h over a period of 4 hours. During ornipressin infusion an improvement of renal function was achieved as indicated by significant increases in inulin clearance (+65%), paraaminohippuric acid clearance (+49%), urine volume (+45%), sodium excretion (+259%), and fractional elimination of sodium (+130%). The hyperdynamic circulation was reversed to a nearly normal circulatory state. The increase in systemic vascular resistance (+60%) coincided with a decrease of a previously elevated renal vascular resistance (-27%) and increase in renal blood flow (+44%). The renal fraction of the cardiac output increased from 2.3% to 4.7% (P less than 0.05). A decline of the elevated plasma levels of noradrenaline (2.08-1.13 ng/mL; P less than 0.01) and renin activity (27.6-14.2 ng.mL-1.h-1; P less than 0.01) was achieved. The plasma concentration of the atrial natriuretic factor increased in most of the patients, but slightly decreased in 3 patients. The decrease of renal vascular resistance and the increase of renal blood flow and of the renal fraction of cardiac output play a key role in the beneficial effect of ornipressin on renal failure. These changes develop by an increase in mean arterial pressure, the reduction of the sympathetic activity, and probably of an extenuation of the splanchnic vasodilation. A significant contribution of atrial natriuretic factor is less likely. The present findings implicate that treatment with ornipressin represents an alternative approach to the management of functional renal failure in advanced liver cirrhosis.

In order to assess the effect of food ingestion on splanchnic disposal of human alpha-atrial natriuretic peptide (ANF), hepatic-intestinal removal of ANF was determined before and after a test meal. Hepatic venous and arterial plasma samples were obtained from six subjects, most of whom had only disorders of minor degree. Hepatic blood flow (HBF) increased significantly after meal ingestion (1.10 +/- 0.17 [SEM] to 1.51 +/- 0.26 L/min, P less than .01). Baseline arterial ANF (10.9 +/- 3.1 pmol/L) did not change significantly. In contrast, hepatic venous ANF increased after meal intake (5.7 +/- 2.0 to 8.4 +/- 1.9 pmol/L, P less than .05), and accordingly the splanchnic fractional extraction decreased (0.53 +/- 0.09 to 0.35 +/- 0.08), although this was not statistically significant. Splanchnic clearance of ANF increased from 347 +/- 90 mL/min to a maximal value of 615 +/- 158 mL/min (P less than .05). Splanchnic removal of ANF was 3.0 +/- 0.5 pmol/min before and increased to a maximum value (7.1 +/- 2.2 pmol/min, P less than .05) 35 minutes after ingestion of the meal. Our results showed enhanced splanchnic removal of ANF after food intake. This is due to increased hepatic-intestinal clearance of the peptide consequent on increased splanchnic blood flow, rather than altered fractional extraction of ANF.

Liver Failure Group, Institute for Liver and Digestive Health, Royal Free Hospital, London, United Kingdom; Data Management Center, EASL-CLIF Consortium, Barcelona, Spain; Hopital Paul Brousse, Villejuif, France; Hospital Clinic, University of Barcelona, IDIBAPS, CIBEReHD, IRSIN, Spain; Policlinico St Orsola Malpighi, Bologna, Italy; Hopital Beaujon, Clichy, France; University of Padua, Padova, Italy; University of Turin, Torino, Italy; University Hospital Gasthuisberg, Leuven, Belgium; University Hospital Bonn, Bonn, Germany; J.W Goethe University Hospital, Frankfurt, Germany; Erasme Hospital Brussels, Brussels, Belgium; University of Munich, Klinikum der LMU, Munich, Germany; King’s College Hospital, London, United Kingdom; EASL-CLIF Consortium, Barcelona, Spain

1038 MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) ACTS AS AN ANTI-FIBROTIC CYTOKINE IN EXPERIMENTAL LIVER FIBROSIS VIA CD74 AND AMPK D. Heinrichs, M. Jung, C. Offermanns, M.-L. Berres, A. Nellen, P. Schmitz, L. Leng, R. Bucala, C. Trautwein, C. Weber, J. Bernhagen, H.E. Wasmuth. University Hospital Aachen, Aachen, Germany; Yale University School of Medicine, New Haven, CT, USA E-mail: dheinrichs@ukaachen.de