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Ongoing liver inflammation in patients with chronic hepatitis C and sustained virological response
(2017)
Background: Novel direct-acting antiviral DAA combination therapies tremendously improved sustained virologic response (SVR) rates in patients with chronic HCV infection. SVR is typically accompanied by normalization of liver enzymes, however, hepatic inflammation, i.e. persistently elevated aminotransferase levels may persist despite HCV eradication. Aim: To investigate prevalence and risk factors for ongoing hepatic inflammation after SVR in two large patient cohorts.
Methods: This post-hoc analysis was based on prospectively collected demographic and clinical data from 834 patients with SVR after HCV treatment with either PegIFN- or DAA-based treatment regimens from the PRAMA trial (n = 341) or patients treated at our outpatient clinic (n = 493).
Results: We observed an unexpected high prevalence of post-SVR inflammation, including patients who received novel IFN-free DAA-based therapies. Up to 10% of patients had ongoing elevation of aminotransferase levels and another 25% showed aminotransferase activity above the so-called healthy range. Several baseline factors were independently associated with post-SVR aminotransferase elevation. Among those, particularly male gender, advanced liver disease and markers for liver steatosis were strongly predictive for persistent ALT elevation. The use of IFN-based antiviral treatment was independently correlated with post-SVR inflammation, further supporting the overall benefit of IFN-free combination regimens.
Conclusion: This is the first comprehensive study on a large patient cohort investigating the prevalence and risk factors for ongoing liver inflammation after eradication of HCV. Our data show a high proportion of patients with ongoing hepatic inflammation despite HCV eradication with potential implications for the management of approximately one third of all patients upon SVR.
Background: Advanced liver diseases are associated with profound alterations of the coagulation system increasing the risk not only of bleeding, but also of thromboembolic complications. A recent milestone study has shown that prophylactic anticoagulation in liver cirrhosis patients results in a reduced frequency of hepatic decompensation. Yet, INR measurement, one of the most widely applied tests to assess liver function, only inaccurately predicts the risk of hepatic decompensation related to alterations of the coagulation system. To assess the relationship between selected coagulation factors / natural anticoagulants with INR, MELD score, and hepatic decompensation, we performed the present pilot study. A total number of 92 patients with various stages of liver cirrhosis were included and prospectively followed for at least 6 months. We found that important natural anticoagulants, namely antithrombin and protein C, as well as factor XI (which may also serve as an anticoagulant) decreased earlier and by a larger magnitude than one would expect from classical coagulation test results. The correlation between these factors and INR was only moderate. Importantly, reduced plasma activities of natural anticoagulants but not INR or MELD score were independent predictors of hepatic encephalopathy (P = 0.013 and 0.003 for antithrombin and protein C, respectively).
Conclusion: In patients with liver cirrhosis plasma activities of several natural anticoagulants are earlier and stronger affected than routine coagulation tests. Reduced activities of natural anticoagulants may be predictive for the development of hepatic encephalopathy.
Background: IL28B gene polymorphism is the best baseline predictor of response to interferon alfa-based antiviral therapies in chronic hepatitis C. Recently, a new IFN-L4 polymorphism was identified as first potential functional variant for induction of IL28B expression. Individualization of interferon alfa-based therapies based on a combination of IL28B/IFN-L4 polymorphisms may help to optimize virologic outcome and economic resources.
Methods: Optimization of treatment outcome prediction was assessed by combination of different IL28B and IFN-L4 polymorphisms in patients with chronic HCV genotype 1 (n = 385), 2/3 (n = 267), and 4 (n = 220) infection treated with pegylated interferon alfa (PEG-IFN) and ribavirin with (n = 79) or without telaprevir. Healthy people from Germany (n = 283) and Egypt (n = 96) served as controls.
Results: Frequencies of beneficial IL28B rs12979860 C/C genotypes were lower in HCV genotype 1/4 infected patients in comparison to controls (20–35% vs. 46–47%) this was also true for ss469415590 TT/TT (20–35% vs. 45–47%). Single interferon-lambda SNPs (rs12979860, rs8099917, ss469415590) correlated with sustained virologic response (SVR) in genotype 1, 3, and 4 infected patients while no association was observed for genotype 2. Interestingly, in genotype 3 infected patients, best SVR prediction was based on IFN-L4 genotype. Prediction of SVR with high accuracy (71–96%) was possible in genotype 1, 2, 3 and 4 infected patients who received PEG-IFN/ribavirin combination therapy by selection of beneficial IL28B rs12979860 C/C and/or ss469415590 TT/TT genotypes (p<0.001). For triple therapy with first generation protease inhibitors (PIs) (boceprevir, telaprevir) prediction of high SVR (90%) rates was based on the presence of at least one beneficial genotype of the 3 IFN-lambda SNPs.
Conclusion: IFN-L4 seems to be the best single predictor of SVR in genotype 3 infected patients. For optimized prediction of SVR by treatment with dual combination or first generation PI triple therapies, grouping of interferon-lambda haplotypes may be helpful with positive predictive values of 71–96%.
Platelets participate in the development of liver fibrosis in animal models, but little is known about the benefit of antiplatelet agents in preventing liver fibrosis in humans. We therefore explored the relationship between the use of antiplatelet agents and liver fibrosis in a prospective cohort study of patients at high risk of liver fibrosis and cardiovascular events. Consecutive patients undergoing elective coronary angiography at the University Hospital Frankfurt were prospectively included in the present study. Associations between use of antiplatelet agents (acetyl salicylic acid, P2Y12 receptor antagonists) and liver fibrosis were assessed in regression models, and the relationship between platelet‐derived growth factor beta (PDGF‐β) serum concentration, platelets, liver fibrosis, and use of antiplatelet agents was characterized. Out of 505 included patients, 337 (67%) received antiplatelet agents and 134 (27%) had liver fibrosis defined as a FibroScan transient elastography (TE) value ≥7.9 kPa. Use of antiplatelet agents was inversely associated with the presence of liver fibrosis in univariate and multivariate analyses (multivariate odds ratio [OR], 0.67; 95% confidence interval [CI], 0.51‐0.89; P = 0.006). Use of antiplatelet agents was also inversely associated with FibroTest values (beta, –0.38; SD beta, 0.15; P = 0.02). Furthermore, there was a significant correlation between platelet counts and PDGF‐β serum concentration (rho, 0.33; P < 0.0001), but PDGF‐β serum levels were not affected by antiplatelet agents. Conclusion: There is a protective association between the use of antiplatelet agents and occurrence of liver fibrosis. A randomized controlled trial is needed to explore causality and the potential of antiplatelet agents as antifibrotic therapy in patients at risk for liver fibrosis progression.
Hepatitis C virus (HCV) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the HCV life cycle on key molecules in these metabolic pathways. Yet, little is known on the role in the HCV life cycle of glycogen synthase kinase 3 (GSK3), one of the most important kinases in cellular metabolism. Therefore, the impact of GSK3 on the HCV life cycle was assessed in human hepatoma cell lines harboring subgenomic genotype 1b and 2a replicons or producing cell culture-derived HCV genotype 2a by exposure to synthetic GSK3 inhibitors, GSK3 gene silencing, overexpression of GSK3 constructs and immunofluorescence analyses. In addition, the role of GSK3 in hepatitis E virus (HEV) replication was investigated to assess virus specificity of the observed findings. We found that both inhibition of GSK3 function by synthetic inhibitors as well as silencing of GSK3β gene expression resulted in a decrease of HCV replication and infectious particle production, whereas silencing of the GSK3α isoform had no relevant effect on the HCV life cycle. Conversely, overexpression of GSK3β resulted in enhanced HCV replication. In contrast, GSK3β had no effect on replication of subgenomic HEV replicon. The pro-viral effect of GSK3β on HCV replication was mediated by supporting expression of microRNA-122 (miR-122), a micro-RNA which is mandatory for wild-type HCV replication, as GSK3 inhibitors suppressed miR-122 levels and as inhibitors of GSK3 had no antiviral effect on a miR-122-independent HCV mutant. In conclusion, we have identified GSK3β is a novel host factor supporting HCV replication by maintaining high levels of hepatic miR-122 expression.
Single nucleotide polymorphism (SNP) rs738409 C>G in the patatin‐like phospholipase domain containing 3 (PNPLA3) gene results in an amino acid exchange from isoleucin to methionine at position I148M of PNPLA3. The expression of this loss‐of‐function mutation leads to impaired hepatocellular triglyceride hydrolysis and is associated with the development of liver steatosis, fibrosis, and hepatocellular carcinoma. In contrast to these well‐established associations, the relationship of the PNPLA3 rs738409 variant with other metabolic traits is incompletely understood. We therefore assessed the association of the PNPLA3 rs738409 genotype with relevant metabolic traits in a prospective study of patients at high risk for cardiovascular events, i.e., patients undergoing coronary angiography. In a total of 270 patients, known associations of the PNPLA3 rs738409 GG genotype with nonalcoholic steatohepatitis and liver fibrosis were confirmed. In addition, we found an association of the PNPLA3 rs738409 G allele with the presence of diabetes (22% versus 28% versus 58% for CC versus CG versus GG genotype, respectively; P = 0.02). In contrast to its association with nonalcoholic fatty liver disease, liver fibrosis, and diabetes, the minor G allele of PNPLA3 rs738409 was inversely associated with total serum cholesterol and low‐density lipoprotein serum levels (P = 0.003 and P = 0.02, respectively). Finally, there was a trend toward an inverse association between the presence of the PNPLA3 rs738409 G allele and significant coronary heart disease. Comparable trends were observed for the transmembrane 6 superfamily member 2 (TM6SF2) 167 K variant, but the sample size was too small to evaluate this rarer variant. Conclusion: The PNPLA3 rs738409 G allele is associated with liver disease but also with a relatively benign cardiovascular risk profile.
Pathophysiological role of prostanoids in coagulation of the portal venous system in liver cirrhosis
(2019)
Background: Prostanoids are important regulators of platelet aggregation and thrombotic arterial diseases. Their involvement in the development of portal vein thrombosis, frequent in decompensated liver cirrhosis, is still not investigated.
Methods: Therefore, we used pro-thrombotic venous milieu generation by bare metal stent transjugular intrahepatic portosystemic shunt insertion, to study the role of prostanoids in decompensated liver cirrhosis. Here, 89 patients receiving transjugular intrahepatic portosystemic shunt insertion were included in the study, and baseline levels of thromboxane B2, prostaglandin D2 and prostaglandin E2 were measured in the portal and the hepatic vein.
Results: While the hepatic vein contained higher levels of thromboxane B2 than the portal vein, levels of prostaglandin E2 and D2 were higher in the portal vein (all P<0.0001). Baseline concentrations of thromboxane B2 in the portal vein were independently associated with an increase of portal hepatic venous pressure gradient during short term follow-up, as an indirect sign of thrombogenic potential (multivariable P = 0.004). Moreover, severity of liver disease was inversely correlated with portal as well as hepatic vein levels of prostaglandin D2 and E2 (all P<0.0001).
Conclusions: Elevated portal venous thromboxane B2 concentrations are possibly associated with the extent of thrombogenic potential in patients with decompensated liver cirrhosis.
Trial registration: ClinicalTrials.gov identifier: NCT03584204.
Alcoholism is one of the leading and increasingly prevalent reasons of liver associated morbidity and mortality worldwide. Alcoholic hepatitis (AH) constitutes a severe disease with currently no satisfying treatment options. Lipoxin A4 (LXA4), a 15-lipoxygenase (ALOX15)-dependent lipid mediator involved in resolution of inflammation, showed promising pre-clinical results in the therapy of several inflammatory diseases. Since inflammation is a main driver of disease progression in alcoholic hepatitis, we investigated the impact of endogenous ALOX15-dependent lipid mediators and exogenously applied LXA4 on AH development. A mouse model for alcoholic steatohepatitis (NIAAA model) was tested in Alox12/15+/+ and Alox12/15−/− mice, with or without supplementation of LXA4. Absence of Alox12/15 aggravated parameters of liver disease, increased hepatic immune cell infiltration in AH, and elevated systemic neutrophils as a marker for systemic inflammation. Interestingly, i.p. injections of LXA4 significantly lowered transaminase levels only in Alox12/15−/− mice and reduced hepatic immune cell infiltration as well as systemic inflammatory cytokine expression in both genotypes, even though steatosis progressed. Thus, while LXA4 injection attenuated selected parameters of disease progression in Alox12/15−/− mice, its beneficial impact on immunity was also apparent in Alox12/15+/+ mice. In conclusion, pro-resolving lipid mediators may be beneficial to reduce inflammation in alcoholic hepatitis.
Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein–protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor σN (σ54) are not conserved in archaea suggesting a novel mechanism of transcriptional control.
Bacteria are generally assumed to be monoploid (haploid). This assumption is mainly based on generalization of the results obtained with the most intensely studied model bacterium, Escherichia coli (a gamma-proteobacterium), which is monoploid during very slow growth. However, several species of proteobacteria are oligo- or polyploid, respectively. To get a better overview of the distribution of ploidy levels, genome copy numbers were quantified in four species of three different groups of proteobacteria. A recently developed Real Time PCR approach, which had been used to determine the ploidy levels of halophilic archaea, was optimized for the quantification of genome copy numbers of bacteria. Slow-growing (doubling time 103 minutes) and fast-growing (doubling time 25 minutes) E. coli cultures were used as a positive control. The copy numbers of the origin and terminus region of the chromosome were determined and the results were in excellent agreement with published data. The approach was also used to determine the ploidy levels of Caulobacter crescentus (an alpha-proteobacterium) and Wolinella succinogenes (an epsilon-proteobacterium), both of which are monoploid. In contrast, Pseudomonas putida (a gamma-proteobacterium) contains 20 genome copies and is thus polyploid. A survey of the proteobacteria with experimentally-determined genome copy numbers revealed that only three to four of 11 species are monoploid and thus monoploidy is not typical for proteobacteria. The ploidy level is not conserved within the groups of proteobacteria, and there are no obvious correlations between the ploidy levels with other parameters like genome size, optimal growth temperature or mode of life.