BackgroundPathogenesis and factors for determining progression of alcoholic and non-alcoholic steatosis to steatohepatitis with risk of further progression to liver cirrhosis and cancer are poorly understood. In the present study, we aimed to identify potential molecular signatures for discrimination of steatohepatitis from steatosis.Methodology and ResultsGlobal microarray gene expression analysis was applied to unravel differentially expressed genes between steatohepatitis compared to steatosis and control samples. For functional annotation as well as the identification of disease-relevant biological processes of the differentially expressed genes the gene ontology (GO) database was used. Selected candidate genes (n = 46) were validated in 87 human liver samples from two sample cohorts by quantitative real-time PCR (qRT-PCR). The GO analysis revealed that genes down-regulated in steatohepatitis were mainly involved in metabolic processes. Genes up-regulated in steatohepatitis samples were associated with cancer progression and proliferation. In surgical liver resection samples, 39 genes and in percutaneous liver biopsies, 30 genes were significantly up-regulated in steatohepatitis. Furthermore, immunohistochemical investigation of human liver tissue revealed a significant increase of AKR1B10 protein expression in steatohepatitis.ConclusionsThe development of steatohepatitis is characterized by distinct molecular changes. The most striking examples in this respect were KRT23 and AKR1B10, which we found to be highly differentially expressed in steatohepatitis compared to steatosis and normal liver. We propose that KRT23 and AKR1B10 may serve as future potential biomarkers for steatohepatitis as well as markers for progression to HCC.

Bacterial infection and sepsis are common complications of chronic kidney disease (CKD). A vicious cycle of increased gut permeability, endotoxemia, inadequate activation of the innate immune system and resulting innate immune dysfunction is hypothesized. We assessed endotoxemia, neutrophil function and its relation to oxidative stress, inflammation and gut permeability in patients with CKD grade 3–5 without renal replacement therapy (CKD group, n = 57), patients with CKD stage 5 undergoing haemodialysis (HD, n = 32) or peritoneal dialysis (PD, n = 28) and patients after kidney transplantation (KT, n = 67) in a cross-sectional observational study. In HD patients, endotoxin serum levels were elevated and neutrophil phagocytic capacity was decreased compared to all other groups. Patients on HD had a significantly higher mortality, due to infections during follow up, compared to PD (p = 0.022). Oxidative stress, neutrophil energy charge, systemic inflammation and gut permeability could not completely explain these differences. Our findings suggest that dialysis modality and not renal function per se determine the development of neutrophil dysfunction and endotoxemia in CKD-patients. HD patients are particularly prone to neutrophil dysfunction and endotoxemia whereas neutrophil function seems to improve after KT. Multi-target approaches are therefore warranted to improve neutrophil function and potentially reduce the rate of infections with patients undergoing haemodialysis.

Chronic hepatitis C virus infection is characterized by multiple extra-hepatic manifestations. Innate immune dysfunction and hemolysis are symptoms which might be associated with each other. We investigated the impact of direct acting antivirals on neutrophil function and its connection to hemolysis. In this prospective study, 85 patients with or without cirrhosis and 21 healthy controls were included. Patients' blood samples were taken at baseline, at the end of therapy and at follow-up 12 weeks after end of therapy. Neutrophil phagocytosis, oxidative burst, and hemolysis parameters were studied. Multivariate analysis was performed to decipher the relationship between hemolysis and neutrophil function. Ex vivo cross-incubation experiments with neutrophils and serum fractions were done. Impaired neutrophil phagocytosis and mild hemolysis were observed in patients with and without cirrhosis. A proteome approach revealed different expression of hemolysis-related serum proteins in patients and controls. Direct acting antiviral therapy restored neutrophil function irrespective of severity of liver disease, achievement of sustained virologic response or type of drug and reduced hemolysis. Treatment with ribavirin delayed the improvement of neutrophil function. Statistical analysis revealed associations of haptoglobin with neutrophil phagocytic capacity. Neutrophil dysfunction could be transferred to healthy cells by incubation with patients' serum fractions (>30 kDa) ex vivo. Neutrophil dysfunction and hemolysis represent extrahepatic manifestations of chronic hepatitis C virus infection and simultaneously improve during direct acting antiviral therapy independently of therapy-related liver function recovery. Therefore, large-scale treatment would not only drive viral eradication but also improve patients' immune system and may reduce susceptibility to infections.

Secondary sclerosing cholangitis in critically ill patients (SC-CIP) occurs after long-term intensive care treatment. This study aimed to assess the gut–liver axis in SC-CIP. Stool microbiome composition, gut permeability, bacterial translocation and serum bile acid profiles of 18 SC-CIP patients compared to 11 patients after critical illness without liver disease (CIP controls), 21 patients with cirrhosis and 21 healthy controls were studied. 16S rDNA was isolated from stool and sequenced using the Illumina technique. Diamine oxidase, zonulin, soluble CD14 (sCD14) and lipopolysaccharide binding protein were measured in serum and calprotectin in stool. Serum bile acids were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Reduced microbiome alpha diversity and altered beta diversity were seen in SC-CIP, CIP controls and cirrhosis compared to healthy controls. SC-CIP patients showed a shift towards pathogenic taxa and an oralization. SC-CIP, CIP controls and cirrhotic patients presented with impaired gut permeability, and biomarkers of bacterial translocation were increased in SC-CIP and cirrhosis. Total serum bile acids were elevated in SC-CIP and cirrhosis and the bile acid profile was altered in SC-CIP, CIP controls and cirrhosis. In conclusions, observed alterations of the gut–liver axis in SC-CIP cannot solely be attributed to liver disease, but may also be secondary to long-term intensive care treatment.

Sclerosing cholangitis in critically ill patients (SC-CIP) is a progressive cholestatic disease of unknown aetiology characterized by chronic biliary infections. Hence we hypothesized that common NOD2 (nucleotide-binding oligomerisation domain containing 2) gene variants, known risk factors for Crohn’s disease and bacterial translocation in liver cirrhosis, increase the odds of developing SC-CIP. Screening of 4,641 endoscopic retrograde cholangiography procedures identified 17 patients with SC-CIP, who were then genotyped for the three common NOD2 mutations (Cohort 1, discovery cohort). To validate the association, we subsequently tested these NOD2 variants in 29 patients from SC-CIP cohorts of three additional medical centers (Cohort 2, replication cohort). In Cohort 1, the NOD2 variants were present in 5 of 17 SC-CIP patients (29.4%), which is twice the frequency of the general population. These results were replicated in Cohort 2 with 8 patients (27.6%) showing NOD2 mutations. In contrast, polymorphisms of hepatocanalicular transporter genes did not have major impact on SC-CIP risk. This first study on genetic susceptibility in SC-CIP patients shows an extraordinary high frequency of NOD2 variation, pointing to a critical role of inherited impaired anti-bacterial defense in the development of this devastating biliary disease.