Cell networks in the mouse liver during partial hepatectomy.

In solid tissues homeostasis and regeneration after injury involve a complex interplay between many different cell types. The mammalian liver harbors numerous epithelial and non-epithelial cells and little is known about the global signaling networks that govern their interactions. To better understand the hepatic cell network, we isolated and purified 10 different cell populations from normal and regenerative mouse livers. Their transcriptomes were analyzed by bulk RNA-seq and a computational platform was used to analyze the cell-cell and ligand-receptor interactions among the 10 populations. Over 50,000 potential cell-cell interactions were found in both the ground state and after partial hepatectomy. Importantly, about half of these differed between the two states, indicating massive changes in the cell network during regeneration. Our study provides the first comprehensive database of potential cell-cell interactions in mammalian liver cell homeostasis and regeneration. With the help of this prediction model, we identified and validated two previously unknown signaling interactions involved in accelerating and delaying liver regeneration. Overall, we provide a novel platform for investigating autocrine/paracrine pathways in tissue regeneration, which can be adapted to other complex multicellular systems.

Senolytic treatment preserves biliary regenerative capacity lost through cellular senescence during cold storage.

Liver transplantation is the only curative option for patients with end-stage liver disease. Despite improvements in surgical techniques, nonanastomotic strictures (characterized by the progressive loss of biliary tract architecture) continue to occur after liver transplantation, negatively affecting liver function and frequently leading to graft loss and retransplantation. To study the biological effects of organ preservation before liver transplantation, we generated murine models that recapitulate liver procurement and static cold storage. In these models, we explored the response of cholangiocytes and hepatocytes to cold storage, focusing on responses that affect liver regeneration, including DNA damage, apoptosis, and cellular senescence. We show that biliary senescence was induced during organ retrieval and exacerbated during static cold storage, resulting in impaired biliary regeneration. We identified decoy receptor 2 (DCR2)-dependent responses in cholangiocytes and hepatocytes, which differentially affected the outcome of those populations during cold storage. Moreover, CRISPR-mediated DCR2 knockdown in vitro increased cholangiocyte proliferation and decreased cellular senescence but had the opposite effect in hepatocytes. Using the p21KO model to inhibit senescence onset, we showed that biliary tract architecture was better preserved during cold storage. Similar results were achieved by administering senolytic ABT737 to mice before procurement. Last, we perfused senolytics into discarded human donor livers and showed that biliary architecture and regenerative capacities were better preserved. Our results indicate that cholangiocytes are susceptible to senescence and identify the use of senolytics and the combination of senotherapies and machine-perfusion preservation to prevent this phenotype and reduce the incidence of biliary injury after transplantation.

Human biliary epithelial cells from discarded donor livers rescue bile duct structure and function in a mouse model of biliary disease.

Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease.

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.

Cellular Senescence in Liver Disease and Regeneration.

Cellular senescence is an irreversible cell cycle arrest implemented by the cell as a result of stressful insults. Characterized by phenotypic alterations, including secretome changes and genomic instability, senescence is capable of exerting both detrimental and beneficial processes. Accumulating evidence has shown that cellular senescence plays a relevant role in the occurrence and development of liver disease, as a mechanism to contain damage and promote regeneration, but also characterizing the onset and correlating with the extent of damage. The evidence of senescent mechanisms acting on the cell populations of the liver will be described including the role of markers to detect cellular senescence. Overall, this review intends to summarize the role of senescence in liver homeostasis, injury, disease, and regeneration.

Ductular Reaction Cells Display an Inflammatory Profile and Recruit Neutrophils in Alcoholic Hepatitis.

Chronic liver diseases are characterized by the expansion of ductular reaction (DR) cells and the expression of liver progenitor cell (LPC) markers. In alcoholic hepatitis (AH), the degree of DR expansion correlates with disease progression and short-term survival. However, little is known about the biological properties of DR cells, their impact on the pathogenesis of human liver disease, and their contribution to tissue repair. In this study, we have evaluated the transcriptomic profile of DR cells by laser capture microdissection in patients with AH and assessed its association with disease progression. The transcriptome analysis of cytokeratin 7-positive (KRT7+ ) DR cells uncovered intrinsic gene pathways expressed in DR and genes associated with alcoholic liver disease progression. Importantly, DR presented a proinflammatory profile with expression of neutrophil recruiting C-X-C motif chemokine ligand (CXC) and C-C motif chemokine ligand chemokines. Moreover, LPC markers correlated with liver expression and circulating levels of inflammatory mediators such as CXCL5. Histologically, DR was associated with neutrophil infiltration at the periportal area. In order to model the DR and to assess its functional role, we generated LPC organoids derived from patients with cirrhosis. Liver organoids mimicked the transcriptomic and proinflammatory profile of DR cells. Conditioned medium from organoids induced neutrophil migration and enhanced cytokine expression in neutrophils. Likewise, neutrophils promoted the proinflammatory profile and the expression of chemokines of liver organoids. Conclusion: Transcriptomic and functional analysis of KRT7+ cells indicate that DR has a proinflammatory profile and promote neutrophil recruitment. These results indicate that DR may be involved in the liver inflammatory response in AH, and suggest that therapeutic strategies targeting DR cells may be useful to mitigate the inflammatory cell recruitment in AH.

Generation of Hepatic Stellate Cells from Human Pluripotent Stem Cells Enables In Vitro Modeling of Liver Fibrosis.

The development of complex in vitro hepatic systems and artificial liver devices has been hampered by the lack of reliable sources for relevant cell types, such as hepatic stellate cells (HSCs). Here we report efficient differentiation of human pluripotent stem cells into HSC-like cells (iPSC-HSCs). iPSC-HSCs closely resemble primary human HSCs at the transcriptional, cellular, and functional levels and possess a gene expression profile intermediate between that of quiescent and activated HSCs. Functional analyses revealed that iPSC-HSCs accumulate retinyl esters in lipid droplets and are activated in response to mediators of wound healing, similar to their in vivo counterparts. When maintained as 3D spheroids with HepaRG hepatocytes, iPSC-HSCs exhibit a quiescent phenotype but mount a fibrogenic response and secrete pro-collagen in response to known stimuli and hepatocyte toxicity. Thus, this protocol provides a robust in vitro system for studying HSC development, modeling liver fibrosis, and drug toxicity screening.

A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl4-treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-ß1 (TGF-ß1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P < 0.05). The decrease of EndMT in cirrhotic livers correlated with a significant decrease in liver fibrosis (P < 0.05) and an improvement in the vascular disorganization rate (P < 0.05). We demonstrated the acquisition of the EndMT phenotype by a subpopulation of endothelial cells from cirrhotic livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization.NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-ß1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization.

Pentraxin-3 modulates lipopolysaccharide-induced inflammatory response and attenuates liver injury.

Acute-on-chronic liver injury is characterized by an important inflammatory response frequently associated with endotoxemia. In this context, acute-phase proteins such as Pentraxin-3 (PTX3) are released; however, little is known about their role in chronic liver disease. The aim of this study was to elucidate the role of PTX3 in liver injury. The role of PTX3 was evaluated in cultured human cells, liver tissue slices, and mice with acute-on-chronic liver injury. PTX3 expression was assessed in tissue and serum samples from 54 patients with alcoholic hepatitis. PTX3 expression was up-regulated in animal models of liver injury and strongly induced by lipopolysaccharide (LPS). Liver cell fractionation showed that macrophages and activated hepatic stellate cells were the main cell types expressing PTX3 in liver injury. Ex vivo and in vivo studies showed that PTX3 treatment attenuated LPS-induced liver injury, inflammation, and cell recruitment. Mechanistically, PTX3 mediated the hepatic stellate cell wound-healing response. Moreover, PTX3 modulated LPS-induced inflammation in human primary liver macrophages and peripheral monocytes by enhancing a TIR domain-containing adapter-inducing interferon-dependent response and favoring a macrophage interleukin-10-like phenotype. Additionally, hepatic and plasma PTX3 levels were increased in patients with alcoholic hepatitis, a prototypic acute-on-chronic condition; and its expression correlated with disease severity scores, endotoxemia, infections, and short-term mortality, thus suggesting that expression of PTX3 found in patients could be a counterregulatory response to injury. CONCLUSION: Experimental and human evidence suggests that, in addition to being a potential biomarker for alcoholic hepatitis, PTX3 participates in the wound-healing response and attenuates LPS-induced liver injury and inflammation; therefore, administration of PTX3 could be a promising therapeutic strategy in acute-on-chronic conditions, particularly those associated with endotoxemia. (Hepatology 2017;66:953-968).

LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis.

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development.

Integrative microRNA profiling in alcoholic hepatitis reveals a role for microRNA-182 in liver injury and inflammation.

OBJECTIVE: MicroRNAs (miRNAs) are well-known regulators of disease pathogenesis and have great potential as biomarkers and therapeutic targets. We aimed at profiling miRNAs in alcoholic hepatitis (AH) and identifying miRNAs potentially involved in liver injury. DESIGN: MiRNA profiling was performed in liver samples from patients with AH, alcohol liver disease, non-alcoholic steatohepatitis, HCV disease and normal liver tissue. Expression of miRNAs was assessed in liver and serum from patients with AH and animal models. Mimic and decoy miR-182 were used in vitro and in vivo to evaluate miR-182's biological functions. RESULTS: MiRNA expression profile in liver was highly altered in AH and distinctive from alcohol-induced cirrhotic livers. Moreover, we identified a set of 18 miRNAs predominantly expressed in AH as compared with other chronic liver conditions. Integrative miRNA-mRNA functional analysis revealed the association of AH-altered miRNAs with nuclear receptors, IGF-1 signalling and cholestasis. Interestingly, miR-182 was the most highly expressed miRNA in AH, which correlated with degree of ductular reaction, disease severity and short-term mortality. MiR-182 mimic induced an upregulation of inflammatory mediators in biliary cells. At experimental level, miR-182 was increased in biliary cells in mice fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet but not upregulated by alcohol intake or fibrosis. Inhibition of miR-182 in DDC-fed mice reduced liver damage, bile acid accumulation and inflammatory response. CONCLUSIONS: AH is characterised by a deregulated miRNA profile, including miR-182, which is associated with disease severity and liver injury. These results highlight the potential of miRNAs as therapeutic targets and biomarkers in AH.

Kinase analysis in alcoholic hepatitis identifies p90RSK as a potential mediator of liver fibrogenesis.

OBJECTIVE: Alcoholic hepatitis (AH) is often associated with advanced fibrosis, which negatively impacts survival. We aimed at identifying kinases deregulated in livers from patients with AH and advanced fibrosis in order to discover novel molecular targets. DESIGN: Extensive phosphoprotein analysis by reverse phase protein microarrays was performed in AH (n=12) and normal human livers (n=7). Ribosomal S6 kinase (p90RSK) hepatic expression was assessed by qPCR, Western blot and immunohistochemistry. Kaempferol was used as a selective pharmacological inhibitor of the p90RSK pathway to assess the regulation of experimentally-induced liver fibrosis and injury, using in vivo and in vitro approaches. RESULTS: Proteomic analysis identified p90RSK as one of the most deregulated kinases in AH. Hepatic p90RSK gene and protein expression was also upregulated in livers with chronic liver disease. Immunohistochemistry studies showed increased p90RSK staining in areas of active fibrogenesis in cirrhotic livers. Therapeutic administration of kaempferol to carbon tetrachloride-treated mice resulted in decreased hepatic collagen deposition, and expression of profibrogenic and proinflammatory genes, compared to vehicle administration. In addition, kaempferol reduced the extent of hepatocellular injury and degree of apoptosis. In primary hepatic stellate cells, kaempferol and small interfering RNA decreased activation of p90RSK, which in turn regulated key profibrogenic actions. In primary hepatocytes, kaempferol attenuated proapoptotic signalling. CONCLUSIONS: p90RSK is upregulated in patients with chronic liver disease and mediates liver fibrogenesis in vivo and in vitro. These results suggest that the p90RSK pathway could be a new therapeutic approach for liver diseases characterised by advanced fibrosis.

Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis.

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.

Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells.

Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.

The biliary epithelium gives rise to liver progenitor cells.

UNLABELLED: Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1ß). However, HNF1ß expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)-positive hepatocytes. In order to investigate the role of HNF1ß-expressing cells we used a tamoxifen-inducible Hnf1ßCreER/R26R(Yfp/LacZ) mouse to lineage-trace Hnf1ß(+) biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1ß(+) cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1ß(+) cells to hepatocyte was detected. We next assessed the contribution of Hnf1ß(+) -derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl-1,4-dihydro-collidin (DDC)-diet and a choline-deficient ethionine-supplemented (CDE)-diet. The contribution of Hnf1ß(+) cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC-diet treatment, mice fed with a CDE-diet showed a small population of hepatocytes derived from Hnf1ß(+) cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome-wide expression profile of Hnf1ß(+) -derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways. CONCLUSION: HNF1ß(+) biliary duct cells are the origin of LPC. HNF1ß(+) cells do not contribute to hepatocyte turnover in the healthy liver, but after certain liver injury, they can differentiate to hepatocytes contributing to liver regeneration.

CCL20 mediates lipopolysaccharide induced liver injury and is a potential driver of inflammation and fibrosis in alcoholic hepatitis.

OBJECTIVE: Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury. DESIGN: CCL20 gene expression and serum levels and their correlation with disease severity were assessed in patients with AH. Cellular sources of CCL20 and its biological effects were evaluated in vitro and in vivo in chronic, acute and acute-on-chronic experimental models of carbon tetrachloride and LPS induced liver injury. RNA interference technology was used to knockdown CCL20 in vivo. RESULTS: CCL20 hepatic and serum levels were increased in patients with AH and correlated with the degree of fibrosis, portal hypertension, endotoxaemia, disease severity scores and short term mortality. Moreover, CCL20 expression was increased in animal models of liver injury and particularly under acute-on-chronic conditions. Macrophages and hepatic stellate cells (HSCs) were identified as the main CCL20 producing cell types. Silencing CCL20 in vivo reduced LPS induced aspartate aminotransferase and lactate dehydrogenase serum levels and hepatic proinflammatory and profibrogenic genes. CCL20 induced proinflammatory and profibrogenic effects in cultured primary HSCs. CONCLUSIONS: Our results suggest that CCL20 upregulation is strongly associated with LPS and may not only represent a new potential biomarker to predict outcome in patients with AH but also an important mediator linking hepatic inflammation, injury and fibrosis in AH.

Transcriptome analysis identifies TNF superfamily receptors as potential therapeutic targets in alcoholic hepatitis.

OBJECTIVE: Alcoholic hepatitis (AH) is a severe clinical condition that needs novel therapies. The identification of targets for therapy is hampered by the lack of animal models of advanced AH. The authors performed a translational study through a transcriptome analysis in patients with AH to identify new molecular targets. DESIGN: Hepatic gene expression profiling was assessed by DNA microarray in patients with AH (n=15) and normal livers (n=7). Functional analysis was assessed by gene set enrichment analysis. Quantitative PCR was performed in patients with AH (n=40), hepatitis C (n=18), non-alcoholic steatohepatitis (n=20) and in mouse models of acute and chronic liver injury. Protein expression was assessed by immunohistochemistry and western blotting. RESULTS: Gene expression analysis showed 207 genes >5-fold differentially expressed in patients with AH and revealed seven pathways differentially regulated including 'cytokine-cytokine receptor interaction'. Several tumour necrosis factor (TNF) superfamily receptors, but not ligands, were overexpressed in AH. Importantly, Fn14 was the only TNF superfamily receptor exclusively upregulated in AH compared with other liver diseases and correlated with both 90-day mortality and severity of portal hypertension. Fn14 protein expression was detected in areas of fibrogenesis and in a population of hepatocytes. Fn14 expression was increased in experimental models of liver injury and was detected in progenitor cells. CONCLUSION: Translational research revealed that TNF superfamily receptors are overexpressed in AH. Fn14, the receptor for TNF-like weak inducer of apoptosis, is selectively upregulated in patients with AH. TNF superfamily receptors could represent a potential target for therapy.

Liver progenitor cell markers correlate with liver damage and predict short-term mortality in patients with alcoholic hepatitis.

UNLABELLED: Alcoholic hepatitis (AH) is a severe condition developed in patients with underlying alcoholic liver disease. Ductular reaction has been associated with chronic alcohol consumption but there is no information regarding the extent of liver progenitor cell (LPC) proliferation in AH. The aim of this study was to investigate LPC markers in AH and its correlation with disease severity. Fifty-nine patients with clinical and histological diagnosis of AH were included in the study. LPC markers were assessed by real-time polymerase chain reaction (PCR) and immunohistochemistry. Standard logistic regression analysis and classification and regression trees (CART) analysis were used for statistical analysis. A microarray analysis showed an up-regulation of LPC markers in patients with AH. Real-time PCR demonstrated that epithelial cell adhesion molecule (EpCAM), Prominin-1, and Keratin7 were significantly increased in patients with AH compared with normal livers (P ≤ 0.01), chronic hepatitis C (P ≤ 0.01), and HCV-induced cirrhosis (P ≤ 0.01). Immunohistochemistry scores generated for Keratin7 and EpCAM demonstrated a good correlation with gene expression. Keratin7 gene expression correlated with liver failure as assessed by model for endstage liver disease score (r = 0.41, P = 0.006) and Maddrey's discriminant function (r = 0.43, P = 0.004). Moreover, Keratin7 (OR1.14, P = 0.004) and Prominin-1 (OR1.14, P = 0.002), but not EpCAM (OR1.16, P = 0.06), were identified as independent predictors of 90-day mortality. CART analysis generated an algorithm based on the combination of Keratin7 and EpCAM gene expression that stratified three groups of patients with high, intermediate, and low short-term mortality (89%, 33%, and 6%, respectively; area under the receiver operating curve 0.73, 95% confidence interval 0.60-0.87). Keratin7 expression provided additional discrimination potential to the age, bilirubin, international normalization ratio, creatinine (ABIC) score. CONCLUSION: LPC markers correlate positively with severity of liver disease and short-term mortality in AH patients. This study suggests that LPC proliferation may be an important feature of AH pathophysiology.