Hepatocyte-derived biomarkers predict liver-related events at 2 years in Child-Pugh class A alcohol-related cirrhosis.

BACKGROUND & AIMS: In patients with compensated alcohol-related cirrhosis, reliable prognostic biomarkers are lacking. Keratin-18 and hepatocyte-derived large extracellular vesicle (lEV) concentrations reflect disease activity, but their ability to predict liver-related events is unknown. METHODS: We measured plasma keratin-18 and hepatocyte lEV concentrations in 500 patients with Child-Pugh class A alcohol-related cirrhosis. The ability of these hepatocyte-derived biomarkers, alone or combined with model for end-stage liver disease (MELD) and FibroTest scores, to predict liver-related events at 2 years was analyzed, taking into account the alcohol consumption at inclusion and during follow-up. RESULTS: Keratin-18 and hepatocyte lEV concentrations increased with alcohol consumption. In patients without active alcohol consumption at enrollment (n = 419), keratin-18 concentration predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both keratin-18 concentrations >285 U/L and FibroTest >0.74 had a 24% cumulative incidence of liver-related events at 2 years, vs. 5% to 14% in other groups of patients. Similar results were obtained when combining keratin-18 concentrations >285 U/L with MELD >10. In patients with active alcohol consumption at enrollment (n = 81), hepatocyte lEVs predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both hepatocyte lEV concentrations >50 U/L and FibroTest >0.74 had a 62% cumulative incidence of liver-related events at 2 years, vs. 8% to 13% in other groups of patients. Combining hepatocyte lEV concentrations >50 U/L with MELD >10 had a lower discriminative ability. Similar results were obtained when using decompensation of cirrhosis, defined according to Baveno VII criteria, as an endpoint. CONCLUSION: In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers with FibroTest or MELD scores identifies patients at high risk of liver-related events, and could be used for risk stratification and patient selection in clinical trials. IMPACT AND IMPLICATIONS: In patients with compensated alcohol-related cirrhosis, reliable predictors of outcome are lacking. In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers (keratin-18 and hepatocyte-large extracellular vesicles) with FibroTest or MELD scores identifies those at high risk of liver-related events at 2 years. The identified patients at high risk of liver-related events are the target-of-choice population for intensive surveillance (e.g., referral to tertiary care centers; intensive control of risk factors) and inclusion in clinical trials.

Endothelial autophagy is not required for liver regeneration after partial hepatectomy in mice with fatty liver.

BACKGROUND & AIMS: Patients with non-alcoholic fatty liver disease (NAFLD) have impaired liver regeneration. Liver endothelial cells play a key role in liver regeneration. In non-alcoholic steatohepatitis (NASH), liver endothelial cells display a defect in autophagy, contributing to NASH progression. We aimed to determine the role of endothelial autophagy in liver regeneration following liver resection in NAFLD. METHODS: First, we assessed autophagy in primary endothelial cells from wild type mice fed a high fat diet and subjected to partial hepatectomy. Then, we assessed liver regeneration after partial hepatectomy in mice deficient (Atg5lox/lox ;VE-cadherin-Cre+ ) or not (Atg5lox/lox ) in endothelial autophagy and fed a high fat diet. The role of endothelial autophagy in liver regeneration was also assessed in ApoE-/- hypercholesterolemic mice and in mice with NASH induced by methionine- and choline-deficient diet. RESULTS: First, autophagy (LC3II/protein) was strongly increased in liver endothelial cells following hepatectomy. Then, we observed at 40 and 48 h and at 7 days after partial hepatectomy, that Atg5lox/lox ;VE-cadherin-Cre+ mice fed a high fat diet had similar liver weight, plasma AST, ALT and albumin concentration, and liver protein expression of proliferation (PCNA), cell-cycle (Cyclin D1, BrdU incorporation, phospho-Histone H3) and apoptosis markers (cleaved Caspase-3) as Atg5lox/lox mice fed a high fat diet. Same results were obtained in ApoE-/- and methionine- and choline-deficient diet fed mice, 40 h after hepatectomy. CONCLUSION: These results demonstrate that the defect in endothelial autophagy occurring in NASH does not account for the impaired liver regeneration occurring in this setting.

Comparison of intra- and inter-host genetic diversity in rabies virus during experimental cross-species transmission.

The development of high-throughput genome sequencing enables accurate measurements of levels of sub-consensus intra-host virus genetic diversity and analysis of the role played by natural selection during cross-species transmission. We analysed the natural and experimental evolution of rabies virus (RABV), an important example of a virus that is able to make multiple host jumps. In particular, we (i) analyzed RABV evolution during experimental host switching with the goal of identifying possible genetic markers of host adaptation, (ii) compared the mutational changes observed during passage with those observed in natura, and (iii) determined whether the colonization of new hosts or tissues requires adaptive evolution in the virus. To address these aims, animal infection models (dog and fox) and primary cell culture models (embryo brain cells of dog and fox) were developed and viral variation was studied in detail through deep genome sequencing. Our analysis revealed a strong unidirectional host evolutionary effect, as dog-adapted rabies virus was able to replicate in fox and fox cells relatively easily, while dogs or neuronal dog cells were not easily susceptible to fox adapted-RABV. This suggests that dog RABV may be able to adapt to some hosts more easily than other host variants, or that when RABV switched from dogs to red foxes it lost its ability to adapt easily to other species. Although no difference in patterns of mutation variation between different host organs was observed, mutations were common following both in vitro and in vivo passage. However, only a small number of these mutations also appeared in natura, suggesting that adaptation during successful cross-species virus transmission is a complex, multifactorial evolutionary process.

A defect in endothelial autophagy occurs in patients with non-alcoholic steatohepatitis and promotes inflammation and fibrosis.

BACKGROUND & AIMS: Previous studies demonstrated that autophagy is protective in hepatocytes and macrophages, but detrimental in hepatic stellate cells in chronic liver diseases. The role of autophagy in liver sinusoidal endothelial cells (LSECs) in non-alcoholic steatohepatitis (NASH) is unknown. Our aim was to analyze the potential implication of autophagy in LSECs in NASH and liver fibrosis. METHODS: We analyzed autophagy in LSECs from patients using transmission electron microscopy. We determined the consequences of a deficiency in autophagy: (a) on LSEC phenotype, using primary LSECs and an LSEC line; (b) on early stages of NASH and on advanced stages of liver fibrosis, using transgenic mice deficient in autophagy specifically in endothelial cells and fed a high-fat diet or chronically treated with carbon tetrachloride, respectively. RESULTS: Patients with NASH had half as many LSECs containing autophagic vacuoles as patients without liver histological abnormalities, or with simple steatosis. LSECs from mice deficient in endothelial autophagy displayed an upregulation of genes implicated in inflammatory pathways. In the LSEC line, deficiency in autophagy enhanced inflammation (Ccl2, Ccl5, Il6 and VCAM-1 expression), features of endothelial-to-mesenchymal transition (α-Sma, Tgfb1, Col1a2 expression) and apoptosis (cleaved caspase-3). In mice fed a high-fat diet, deficiency in endothelial autophagy induced liver expression of inflammatory markers (Ccl2, Ccl5, Cd68, Vcam-1), liver cell apoptosis (cleaved caspase-3) and perisinusoidal fibrosis. Mice deficient in endothelial autophagy treated with carbon tetrachloride also developed more perisinusoidal fibrosis. CONCLUSIONS: A defect in autophagy in LSECs occurs in patients with NASH. Deficiency in endothelial autophagy promotes the development of liver inflammation, features of endothelial-to-mesenchymal transition, apoptosis and liver fibrosis in the early stages of NASH, but also favors more advanced stages of liver fibrosis. LAY SUMMARY: Autophagy is a physiological process controlling endothelial homeostasis in vascular beds outside the liver. This study demonstrates that autophagy is defective in the liver endothelial cells of patients with non-alcoholic steatohepatitis. This defect promotes liver inflammation and fibrosis at early stages of non-alcoholic steatohepatitis, but also at advanced stages of chronic liver disease.

Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis.

Microvesicles (MVs) are extracellular vesicles released by cells following activation or apoptosis. Some MV subpopulations augment with cirrhosis severity and contribute to portal hypertension. This study aimed at determining if plasma MV levels can estimate the presence of hepatic venous pressure gradient (HVPG) ≥10 mm Hg and predict mortality in patients with advanced chronic liver disease. All patients with severe fibrosis or cirrhosis undergoing liver catheterization between 2013 and 2015 at two centers were prospectively included. We measured circulating levels of annexin V+ , platelet, leukocyte, endothelial, and hepatocyte MVs. The test cohort included 139 patients. Hepatocyte MV levels were 4.0-fold and 2.2-fold higher in patients with Child-Pugh C than in those with Child-Pugh A or B liver disease, respectively. Levels of other MV subpopulations were not influenced by liver disease severity. Hepatocyte MV levels correlated with HVPG but could not identify patients with HVPG ≥10 mm Hg. Hepatocyte MV level >65 U/L predicted 6-month mortality independently of Child-Pugh score and of Model for End-Stage Liver Disease (MELD). Patients with hepatocyte MV levels >65 U/L and MELD >15 had a higher 6-month mortality than other patients (23% versus 3%; P = 0.001). These findings were confirmed in a validation cohort including 103 patients. CONCLUSION: Circulating MV levels cannot identify patients with HVPG ≥10 mm Hg; by contrast, hepatocyte MV levels strongly improve prediction of 6-month mortality in patients with advanced chronic liver disease; therapies associated with decreased levels of circulating hepatocyte MV might be attractive strategies in patients with severe cirrhosis. (Hepatology 2018).

Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus in Multiple Carnivore Hosts.

The natural evolution of rabies virus (RABV) provides a potent example of multiple host shifts and an important opportunity to determine the mechanisms that underpin viral emergence. Using 321 genome sequences spanning an unprecedented diversity of RABV, we compared evolutionary rates and selection pressures in viruses sampled from multiple primary host shifts that occurred on various continents. Two major phylogenetic groups, bat-related RABV and dog-related RABV, experiencing markedly different evolutionary dynamics were identified. While no correlation between time and genetic divergence was found in bat-related RABV, the evolution of dog-related RABV followed a generally clock-like structure, although with a relatively low evolutionary rate. Subsequent molecular clock dating indicated that dog-related RABV likely underwent a rapid global spread following the intensification of intercontinental trade starting in the 15th century. Strikingly, although dog RABV has jumped to various wildlife species from the order Carnivora, we found no clear evidence that these host-jumping events involved adaptive evolution, with RABV instead characterized by strong purifying selection, suggesting that ecological processes also play an important role in shaping patterns of emergence. However, specific amino acid changes were associated with the parallel emergence of RABV in ferret-badgers in Asia, and some host shifts were associated with increases in evolutionary rate, particularly in the ferret-badger and mongoose, implying that changes in host species can have important impacts on evolutionary dynamics.

The immune response of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain: A transcriptomic attempt at identifying molecular actors.

The marine mussel Mytilus edulis, tolerant to a wide range of environmental changes, combines a key role as a sentinel species for environmental monitoring programs and a significant economic importance. Mortality events caused by infective agents and parasites have not been described in mussels, which suggests an efficient immune system. This study aims at identifying the molecular mechanisms involved in the early immune responses M. edulis' hemocytes challenged with Vibrio splendidus LGP32 strain during 2, 4 and 6 h. A total of 149,296 assembled sequences has been annotated and compared to KEGG reference pathways. Several immune related sequences were identified such as Toll-Like receptors (TLRs), transcription factors, cytokines, protease inhibitors, stress proteins and sequences encoding for proteins involved in cell adhesion, phagocytosis, oxidative stress, apoptosis and autophagy. Differential gene expression clustered 10 different groups of transcripts according to kinetics of transcript occurrence. Sequences were assigned to biological process gene ontology categories. Sequences encoding for galectins, fibrinogen-related proteins, TLRs, MyD88, some antimicrobial peptides, lysosomal hydrolases, heat shock proteins and protease inhibitors, as well as proteins of oxidative stress and apoptosis were identified as differently regulated during the exposure to V. splendidus LGP32. The levels of candidate transcripts were quantified in M. edulis' hemocytes exposed to V. splendidus LGP32 and 7SHRW by using branched DNA technology. Transcripts encoding for inhibitor kappa B, inhibitor of apoptosis proteins, tumor protein D54, serine/threonine-proteine kinase SIK2 were identified as up-regulated in hemocytes exposed to both strains.

Impact of microcystin-producing cyanobacteria on reproductive success of Lymnaea stagnalis (Gastropoda, Pulmonata) and predicted consequences at the population level.

Our previous studies showed that microcystin (MC)-accumulation in the gastropod Lymnaea stagnalis and effects on life-history traits (survival, growth, and fecundity) varied according to age, exposure pathway (MC-producing cyanobacteria or dissolved MC), and presence or not of additional non-toxic food. This study investigated effects of exposure to MC-producing cyanobacteria or to dissolved MC of parent and of parent and egg masses of L. stagnalis on hatching success, duration of embryonic development and neonate survival. Secondly, the potential impact of MC-producing cyanobacterial proliferations (blooms) on L. stagnalis population growth, depending on bloom frequencies and recovery duration of life traits after exposure, was evaluated using a modelling approach. Experimental results showed that embryonic development was shortened in case of parent exposure to toxic cyanobacteria. Parent and eggs exposure to dissolved MC extended embryonic development and reduced hatching percentage, suggesting a permeability of egg masses to MC. Whatever exposure, neonate survival was reduced. Neonates exposed to cyanobacteria accumulated MCs 24 h after hatching, suggesting very early cyanobacteria ingestion. Modelling results showed that L. stagnalis population growth was influenced by the recovery time of life-history traits after exposure. When setting the latest at 6 weeks according to previous experiments, a frequency of one to four blooms per year strongly affected population dynamics and induced up to a 80-weeks delay compared to control in time required for populations to grow from 1 to 1000 individuals. Results are discussed in terms of impact of intoxication pathways on parents, eggs and neonates, and on population dynamics of L. stagnalis.

Endothelial dysfunction markers predict short-term mortality in patients with severe alcoholic hepatitis.

OBJECTIVES: Alcoholic hepatitis (AH) is a severe condition characterized by a marked inflammatory response and high short-term mortality. Endothelial dysfunction (ED) is an early event in vascular and inflammatory disorders. The aim of this study is to evaluate ED in AH patients. METHODS: Prognostic value of ED biomarkers was evaluated in patients with severe AH (n = 67), compensated alcoholic cirrhosis (n = 15), heavy drinkers without liver disease (n = 15) and controls (n = 9), and in a validation cohort of 50 patients with AH. Gene expression of ED markers was analyzed in liver tissue. RESULTS: Plasma levels of ED markers such as vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin and von Willebrand factor (vWF) increased along alcohol-related liver disease (ALD) progression. Intergroup analysis showed a significant increase of these markers in AH patients. In addition, VCAM-1 showed a positive correlation with Maddrey, MELD and ABIC scores and inflammation parameters (i.e. C-reactive protein and LPS levels). Importantly, levels of VCAM-1 were higher in patients with increased mortality and were independently associated with short-term survival (90-day) when adjusted by ABIC score. These results were confirmed in an independent cohort of AH patients. In addition, severe AH patients showed altered hepatic expression of ED markers. CONCLUSIONS: In this study we show that advanced ALD and particularly severe AH is associated with an increase of ED biomarkers, which correlate with patient outcomes. These results suggest that ED may be a pathogenic event in AH and highlight endothelial factors as potential biomarkers in AH.

Interaction between the autophagy protein Beclin 1 and Na+,K+-ATPase during starvation, exercise, and ischemia.

Autosis is a distinct form of cell death that requires both autophagy genes and the Na+,K+-ATPase pump. However, the relationship between the autophagy machinery and Na+,K+-ATPase is unknown. We explored the hypothesis that Na+,K+-ATPase interacts with the autophagy protein Beclin 1 during stress and autosis-inducing conditions. Starvation increased the Beclin 1/Na+,K+-ATPase interaction in cultured cells, and this was blocked by cardiac glycosides, inhibitors of Na+,K+-ATPase. Increases in Beclin 1/Na+,K+-ATPase interaction were also observed in tissues from starved mice, livers of patients with anorexia nervosa, brains of neonatal rats subjected to cerebral hypoxia-ischemia (HI), and kidneys of mice subjected to renal ischemia/reperfusion injury (IRI). Cardiac glycosides blocked the increased Beclin 1/Na+,K+-ATPase interaction during cerebral HI injury and renal IRI. In the mouse renal IRI model, cardiac glycosides reduced numbers of autotic cells in the kidney and improved clinical outcome. Moreover, blockade of endogenous cardiac glycosides increased Beclin 1/Na+,K+-ATPase interaction and autotic cell death in mouse hearts during exercise. Thus, Beclin 1/Na+,K+-ATPase interaction is increased in stress conditions, and cardiac glycosides decrease this interaction and autosis in both pathophysiological and physiological settings. This crosstalk between cellular machinery that generates and consumes energy during stress may represent a fundamental homeostatic mechanism.

Characterization of Blood Immune Cells in Patients With Decompensated Cirrhosis Including ACLF.

Background and Aims: Patients with cirrhosis and acute-on-chronic liver failure (ACLF) have immunosuppression, indicated by an increase in circulating immune-deficient monocytes. The aim of this study was to investigate simultaneously the major blood-immune cell subsets in these patients. Material and Methods: Blood taken from 67 patients with decompensated cirrhosis (including 35 critically ill with ACLF in the intensive care unit), and 12 healthy subjects, was assigned to either measurements of clinical blood counts and microarray (genomewide) analysis of RNA expression in whole-blood; microarray (genomewide) analysis of RNA expression in blood neutrophils; or assessment of neutrophil antimicrobial functions. Results: Several features were found in patients with ACLF and not in those without ACLF. Indeed, clinical blood count measurements showed that patients with ACLF were characterized by leukocytosis, neutrophilia, and lymphopenia. Using the CIBERSORT method to deconvolute the whole-blood RNA-expression data, revealed that the hallmark of ACLF was the association of neutrophilia with increased proportions of macrophages M0-like monocytes and decreased proportions of memory lymphocytes (of B-cell, CD4 T-cell lineages), CD8 T cells and natural killer cells. Microarray analysis of neutrophil RNA expression revealed that neutrophils from patients with ACLF had a unique phenotype including induction of glycolysis and granule genes, and downregulation of cell-migration and cell-cycle genes. Moreover, neutrophils from these patients had defective production of the antimicrobial superoxide anion. Conclusions: Genomic analysis revealed that, among patients with decompensated cirrhosis, those with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils (that had a unique phenotype) and macrophages M0-like monocytes, and depletion of several lymphocyte subsets (including memory lymphocytes). All these lymphocyte alterations, along with defective neutrophil superoxide anion production, may contribute to immunosuppression in ACLF, suggesting targets for future therapies.

Erythrocyte-derived microvesicles induce arterial spasms in JAK2V617F myeloproliferative neoplasm.

Arterial cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms (MPNs). However, their mechanisms are poorly understood. The high prevalence of myocardial infarction without significant coronary stenosis or atherosclerosis in patients with MPNs suggests that vascular function is altered. The consequences of JAK2V617F mutation on vascular reactivity are unknown. We observe here increased responses to vasoconstrictors in arteries from Jak2V617F mice resulting from a disturbed endothelial NO pathway and increased endothelial oxidative stress. This response was reproduced in WT mice by circulating microvesicles isolated from patients carrying JAK2V617F and by erythrocyte-derived microvesicles from transgenic mice. Microvesicles of other cellular origins had no effect. This effect was observed ex vivo on isolated aortas, but also in vivo on femoral arteries. Proteomic analysis of microvesicles derived from JAK2V617F erythrocytes identified increased expression of myeloperoxidase as the likely mechanism accounting for their effect. Myeloperoxidase inhibition in microvesicles derived from JAK2V617F erythrocytes suppressed their effect on oxidative stress. Antioxidants such as simvastatin and N-acetyl cysteine improved arterial dysfunction in Jak2V617F mice. In conclusion, JAK2V617F MPNs are characterized by exacerbated vasoconstrictor responses resulting from increased endothelial oxidative stress caused by circulating erythrocyte-derived microvesicles. Simvastatin appears to be a promising therapeutic strategy in this setting.

Functional and molecular responses in Mytilus edulis hemocytes exposed to bacteria, Vibrio splendidus.

This study aims at examining the morphological, functional and molecular responses of Mytilus edulis hemocytes exposed to different strains of Gram-negative bacteria Vibrio splendidus (a virulent strain V. splendidus LGP32, V. splendidus LGP32 Δvsm without metalloprotease and an environmental type strain V. splendidus 7SHRW) at a 1:3 ratio for 2, 4, and 6 h. Our data showed that hemocytes could have a discriminative capacity towards microorganisms. Both V. splendidus LGP32 strains had an effect on hemocyte adhesion, phagocytosis abilities and oxidative burst, whereas the environmental strain 7SHRW induced weak and delayed hemocyte responses. At a molecular level, differential levels of candidate transcripts were measured in M. edulis hemocytes exposed to V. splendidus LGP32-GFP and 7SHRW. Mainly, a down-regulation of defensin was recorded in hemocytes exposed to V. splendidus LGP32. A significant up-regulation of lysozyme and proteasome 26S was observed at 2 h followed by a down-regulation at 4 and 6 h of exposure to the LGP32 strain. Similarly, SOD and GPx genes were up-regulated 2 h post-exposure to LGP32 strain and their expressions decreased after 4 and 6 h post-exposure. Further analysis is however needed in a near future to relate the transcript level variations with the physiological process.

Sequence analysis of a normalized cDNA library of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain.

In the past decades, reports on bivalves' pathogens and associated mortalities have steadily increased. To face pathogenic micro-organisms, bivalves rely on innate defenses established in hemocytes which are essentially based on phagocytosis and cytotoxic reactions. As a step towards a better understanding of the molecular mechanisms involved in the mussel Mytilus edulis innate immune system, we constructed and sequenced a normalized cDNA library specific to M. edulis hemocytes unchallenged (control) and challenged with Vibrio splendidus LGP32 strain for 2, 4 and 6 h. A total of 1,024,708 nucleotide reads have been generated using 454 pyrosequencing. These reads have been assembled and annotated into 19,622 sequences which we believe cover most of the M. edulis hemocytes transcriptome. These sequences were successfully assigned to biological process, cellular component, and molecular function Gene Ontology (GO) categories. Several transcripts related to immunity and stress such as some fibrinogen related proteins and Toll-like receptors, the complement C1qDC, some antioxidant enzymes and antimicrobial peptides have already been identified. In addition, Toll-like receptors signaling pathways and the lysosome and apoptosis mechanisms were compared to KEGG reference pathways. As an attempt for large scale RNA sequencing, this study focuses on identifying and annotating transcripts from M. edulis hemocytes regulated during an in vitro experimental challenge with V. splendidus. The bioinformatic analysis provided a reference transcriptome, which could be used in studies aiming to quantify the level of transcripts using high-throughput analysis such as RNA-Seq.