Quantitative trace element mapping in liver tissue from patients with Wilson`s disease determined by micro X-ray fluorescence.

AIMS: of this investigation were to quantify copper (Cu), iron (Fe) and zinc (Zn) along with sulphur (S) and phosphorus (P) in hepatocytes and connective tissue in liver section from patients with Wilson´s disease (WD) by micro Synchrotron X-ray fluorescence (µ-SRXRF). Secondly to establish two-dimensional µ-SRXRF element mappings for comparison with histologically prepared slices, and thirdly to assess whether elemental distributions are associated. METHODS: Archival liver tissues from twelve patients with end-stage cirrhosis or fulminant WD were investigated. Mutations in ATP7B have been classified before. For control seven archived normal liver tissues were investigated. µ-SRXRF measurements were performed at the DORIS III storage ring at HASYLAB/DESY (Hamburg, Germany). Two-dimensional element distribution were compared with histologically prepared slices about 20-30 µm apart from those investigated by µ-SRXRF. RESULTS: Elementary copper (Cu) could be demonstrated in all investigated liver sections simultaneously with Fe, Zn, P and S. In WD mean Cu was 20 fold increased in hepatocytes and threefold in fibrotic areas in comparison with controls. In regeneration nodules Cu was strikingly inhomogeneous distributed. Cu concentrations measured by µ-SRXRF correlated with those measured by atom absorption spectroscopy. Strong associations in their regional distribution existed between Zn and Cu or Fe and S. Moreover, differences in Cu/S were found between hepatocytes and fibrotic areas. An increase of Fe could only be documented in hepatocytes compared to fibrotic areas. With a beam size of 15 x 15 µm two-dimensional distributions of these elements are morphologically comparable with histological section with a magnification of about 25x optic microscope. CONCLUSIONS: µ-SRXRF investigations are a valuable tool for quantifying element concentrations in biological tissues and further provide 2-dimensional information of element distribution and elemental association in a biological tissues, thus speeding up basic knowledge in a synopsis with biological and clinical data.

Impact of patatin-like phospholipase domain containing 3 rs738409 G/G genotype on hepatic decompensation and mortality in patients with portal hypertension.

BACKGROUND: The rs738409 C>G p.I148M variant in the patatin-like phospholipase domain containing 3 (PNPLA3)-gene promotes triglyceride accumulation in hepatocytes and hepatic stellate cell activation and has previously been linked to hepatic steatosis/liver fibrosis. AIM: To investigate its impact on hepatic decompensation and (liver-related) mortality in patients who had already developed portal hypertension. Moreover, we assessed its link with hepatic steatosis as evaluated by controlled attenuation parameter. METHODS: We performed a retrospective analysis in prospectively characterised patients with viral hepatitis/fatty liver disease-induced portal hypertension (hepatic venous pressure gradient [HVPG] ≥ 6 mm Hg) diagnosed at the Medical University of Vienna who underwent HVPG measurement (until 2013; n = 372; longitudinal study) or simultaneous HVPG and controlled attenuation parameter measurement (2014-2017; n = 153; cross-sectional study). RESULTS: While survival was similar between PNPLA3-C/C and -C/G patients, we observed substantially increased mortality in PNPLA3-G/G patients. PNPLA3-G/G had no impact on mortality in the subgroup of patients with viral hepatitis; however, we observed a strong independent association between PNPLA3-G/G and hepatic decompensation (adjusted subdistribution hazard ratio [aSHR]: 2.1, 95% confidence interval [95% CI]: 1.1-4; P = 0.024) as well as mortality (overall: aSHR: 2.2, 95% CI: 1.22-3.98; P = 0.009; liver-related: aSHR: 2.2, 95% CI: 1.08-4.46; P = 0.029) in patients with fatty liver disease. Interestingly, even in the subgroup of patients who had already progressed to clinically significant portal hypertension (HVPG ≥ 10 mm Hg), PNPLA3-G/G substantially increased mortality (aSHR: 2.33, 95% CI: 1.27-4.29; P = 0.006). PNPLA3-genotype had no influence on controlled attenuation parameter or the prevalence of values ≥248 dB/m. CONCLUSION: PNPLA3-G/G-genotype seems to double the risks of hepatic decompensation and (liver-related) mortality in patients with portal hypertension due to fatty liver disease. Further studies are warranted to investigate potential underlying pathophysiological mechanisms unrelated to hepatic steatosis.

Time to viral suppression is not related to achievement of SVR12 in HCV GT1-infected patients treated with ombitasvir/paritaprevir/ritonavir and dasabuvir with or without ribavirin.

High rates of sustained virologic response at post-treatment week 12 (SVR12) were achieved in six phase 3 trials of ombitasvir (OBV, an NS5A inhibitor), paritaprevir (an NS3/4A protease inhibitor) co-dosed with ritonavir (PTV/r) + dasabuvir (DSV, an NS5B RNA polymerase inhibitor) (ie, 3D regimen) with or without ribavirin (RBV) in adults with chronic genotype (GT) 1 hepatitis C virus (HCV) infection. We assessed whether time to first HCV RNA value below the lower limit of quantification in patients with and without cirrhosis was associated with achievement of SVR12. Data were analysed from GT1-infected patients enrolled in six phase 3 studies of 3D ± RBV. Patients who experienced non-virologic failure were excluded from analysis. HCV RNA was determined using the Roche COBAS TaqMan RT-PCR assay (lower limit of quantification, LLOQ =25 IU/mL). SVR12 was analysed by week of first HCV RNA suppression, defined as HCV RNA

Open-label study of faldaprevir plus peginterferon and ribavirin in hepatitis C virus genotype 1-infected patients who failed placebo plus peginterferon and ribavirin.

Faldaprevir, a hepatitis C virus (HCV) NS3/4A protease inhibitor, was evaluated in HCV genotype 1-infected patients who failed peginterferon and ribavirin (PegIFN/RBV) treatment during one of three prior faldaprevir trials. Patients who received placebo plus PegIFN/RBV and had virological failure during a prior trial were enrolled and treated in two cohorts: prior relapsers (n = 43) and prior nonresponders (null responders, partial responders and patients with breakthrough; n = 75). Both cohorts received faldaprevir 240 mg once daily plus PegIFN/RBV for 24 weeks. Prior relapsers with early treatment success (ETS; HCV RNA <25 IU/mL detectable or undetectable at week 4 and <25 IU/mL undetectable at week 8) stopped treatment at week 24. Others received PegIFN/RBV through week 48. The primary efficacy endpoint was sustained virological response (HCV RNA <25 IU/mL undetectable) 12 weeks post treatment (SVR12). More prior nonresponders than prior relapsers had baseline HCV RNA ≥ 800,000 IU/mL (80% vs 58%) and a non-CC IL28B genotype (91% vs 70%). Rates of SVR12 (95% CI) were 95.3% (89.1, 100.0) among prior relapsers and 54.7% (43.4, 65.9) among prior nonresponders; corresponding ETS rates were 97.7% and 65.3%. Adverse events led to faldaprevir discontinuations in 3% of patients. The most common Division of AIDS Grade ≥ 2 adverse events were anaemia (13%), nausea (10%) and hyperbilirubinaemia (9%). In conclusion, faldaprevir plus PegIFN/RBV achieved clinically meaningful SVR12 rates in patients who failed PegIFN/RBV in a prior trial, with response rates higher among prior relapsers than among prior nonresponders. The adverse event profile was consistent with the known safety profile of faldaprevir.

Interferon-free regimens for chronic hepatitis C overcome the effects of portal hypertension on virological responses.

BACKGROUND: Portal hypertension is the strongest predictor of virological response to pegylated interferon (IFN)/ribavirin in patients with chronic hepatitis C (CHC)-related cirrhosis. AIM: To investigate the effects of portal pressure assessed by hepatic venous pressure gradient (HVPG) measurement on virological responses in patients treated with IFN-free regimens outside of clinical trials. METHODS: Fifty-six patients with CHC and cirrhosis who underwent HVPG measurement before starting an IFN-free therapy were retrospectively studied. Patients were treated with sofosbuvir in combination with daclatasvir (n = 32), ribavirin (n = 12) or simeprevir (n = 11), or the combination of simeprevir/daclatasvir (n = 1), for 12-24 weeks. RESULTS: Hepatic venous pressure gradient values ≥10 mmHg and ≥16 mmHg were observed in 41 (73%) and 31 (55%) patients respectively. The distributions of treatment regimens and durations were comparable between patients with or without portal hypertension. Patients with portal hypertension had lower platelet counts and albumin levels, while bilirubin levels, INR, MELD and Child-Pugh scores were higher than in patients without portal hypertension. Importantly, rates of on-treatment virological response and viral kinetics, as well as the rates of sustained virological response 12 weeks after the end of therapy [96% (54/56)] were not affected by portal hypertension. Anti-viral therapy improved liver stiffness, platelet count, serum albumin and bilirubin levels, as well as prothrombin time. CONCLUSIONS: This is the first study to demonstrate that IFN-free regimens overcome the negative effect of portal hypertension on virological responses and viral kinetics. Improvements in liver stiffness and platelet count might reflect an anti-portal hypertensive effect of IFN-free treatments.

Successful anti-viral treatment improves survival of patients with advanced liver disease due to chronic hepatitis C.

BACKGROUND: Long-term outcome of chronic hepatitis C patients with successful viral eradication seems to be promising. AIM: To evaluate mortality, incidence of hepatocellular carcinoma (HCC), liver failure and liver transplantation in sustained virological responders (SVR) and non-SVR patients with different stages of fibrosis. METHODS: Seven hundred and fourteen patients with a follow-up of 7.2 (1-21.1) years (age: 51.4 ± 12.0 years, 276 female, IFN-monotherapy: n = 19, IFN/RBV: n = 122, peg-IFN/RBV: n = 573, SVR: 551, non-SVR: 163) were studied. Two hundred and ten of 540 patients with a liver biopsy prior to treatment had advanced stages of fibrosis (Metavir F3/F4). RESULTS: Forty-eight patients died during follow-up, 15 with SVR and 33 without (P < 0.001). Five- and 10-year mortality rates were 1.8% (10/551) and 2.7% (15/551) in the SVR group and 8.6% (14/163) and 19.1% (31/163) in the non-SVR patients (P < 0.001). In 29 patients, decompensation of liver disease [SVR: 9 (1.6%) vs. non-SVR: 20 (12.3%); P < 0.001] occurred and in 29 patients, HCC developed during follow-up [SVR: 10 (1.8%) vs. non-SVR: 19 (11.7%); P < 0.001]. Non-SVR was an independent predictor for developing (i) HCC [HR: 2.36 (95% CI: 1.07-5.23; P = 0.034], (ii) liver-related complications [HR: 2.62; (95% CI: 1.18-5.81; P = 0.018] and (iii) mortality (HR: 3.46; 95% CI: 1.91-6.29; P < 0.001). For patients with early stages of fibrosis (F0-F2), a survival benefit of SVR patients could not be demonstrated. CONCLUSIONS: Successful anti-viral therapy decreases mortality, incidence of hepatocellular carcinoma and liver failure in patients with advanced fibrosis. However, hepatocellular carcinoma development or liver failure are not prevented completely, and further follow-up of patients is advisable.

Oral anti-CD3 immunotherapy for HCV-nonresponders is safe, promotes regulatory T cells and decreases viral load and liver enzyme levels: results of a phase-2a placebo-controlled trial.

UNLABELLED: Orally administered anti-CD3 antibodies are biologically active in the gut through induction of regulatory T cells, exert an immune-modulatory effect, and alleviate insulin resistance and liver damage in patients with NASH. AIMS: To determine the safety of oral anti-CD3 monoclonal antibody (MAb) immunotherapy in chronic HCV patients with associated immune dysfunction. METHODS: Four groups (n = 9) of chronic HCV patients who were nonresponders to interferon plus ribavirin therapy received oral placebo (group A) or anti-CD3 MAb at one of three dosage levels for 30 days. Patients were followed for safety parameters and serum levels of liver enzymes, virus, cytokines and regulatory T cells. RESULTS: Oral anti-CD3 immunotherapy was safe and well tolerated; no treatment-related adverse events were noted. The following improvements were noted relative to pretreatment levels: HCV viral load and AST and ALT levels decreased in the low- and high-dose groups following 30 days of therapy. In two of the treated groups, an increase in regulatory T cells (CD4(+) CD25(+) ) was noted. The positive effects were somewhat more apparent in subjects with initially elevated liver enzyme levels. CONCLUSIONS: Oral anti-CD3 MAb immunotherapy for nonresponder HCV patients was safe and well tolerated. Trends and statistically significant improvements were observed as reductions in viral load and liver enzyme levels, along with an increase in regulatory T-cell levels. These data support a role for the immune system in the pathogenesis of HCV infection and suggest that this immunotherapy is worthy of evaluation in combination with HCV antiviral drugs.

Polymorphisms of HLA-DP are associated with response to peginterferon in Caucasian patients with chronic hepatitis B.

BACKGROUND: Polymorphisms of the HLA-DP gene are associated with the natural clearance of the hepatitis B virus in Asian patients. AIM: To investigate the association of HLA-DP polymorphisms with response to peginterferon (PEG-IFN) in Caucasian chronic hepatitis B (CHB) patients. METHODS: We studied 262 Caucasian chronic hepatitis B patients infected with HBV genotype A or D, treated with PEG-IFN for 1 year in two randomised controlled trials (HBV 99-01 and PARC study). Response was defined as an HBV DNA <2000 IU/mL at 6 months post-treatment. Variations at HLA-DPA1 and HLA-DPB1 were genotyped. RESULTS: Of the 262 patients, 58% was HBeAg-positive and HBV genotype A and D was observed in 32% and 68%, respectively. At 6 months post-treatment, 57 (22%) patients had achieved an HBV DNA <2000 IU/mL. HLA-DPB1 was independently associated with virological response [adjusted odds ratio (OR) 1.8, 95% confidence interval (CI):1.1-3.0, P = 0.025], and with an undetectable HBV DNA (adjusted OR 2.4 95% CI: 1.2-4.7, P = 0.015) when adjusted for HBeAg status and other known response modifiers. In HBeAg-positive patients, combined HBeAg seroconversion with HBV DNA <2000 IU/mL was increasingly observed with each addition of an HLA-DPB1 G-allele (adjusted OR 2.7, 95% CI: 1.2-5.9, P = 0.012). Furthermore, HLA-DPA1 and HLA-DPB1 haplotype block GG showed comparable results for virological and combined response. CONCLUSION: In this large cohort of Caucasian chronic hepatitis B patients infected with HBV genotypes A or D, polymorphisms of HLA-DP are independently associated with both virological and serological response to PEG-IFN therapy at 6 months post-treatment.

Review article: genetic factors that modify the outcome of viral hepatitis.

BACKGROUND: Genetic factors can play an important role for treatment response and disease progression in chronic viral hepatitis. AIM: To review the influence of host genetic factors on the clinical course as well as on treatment response in patients with viral hepatitis. METHODS: Review of the literature. RESULTS: A landmark genome-wide association study (GWAS) identified polymorphisms in the IL28B gene on chromosome 19 (19q13.13) associated with response to therapy with pegylated interferon-α (PEG-IFN) and ribavirin (RBV) and spontaneous viral clearance in acute hepatitis C. Furthermore, IL28B genotype is associated with changes of lipid metabolism and insulin resistance. A further GWAS demonstrated that ITPA genetic variants protect HCV genotype 1 patients from RBV-induced anaemia. Another polymorphism in the patatin-like phospholipase domain containing 3 (PNPLA3) is associated with hepatic steatosis. Difficult-to-treat hepatitis C patients homozygous for GG had an up to five-fold lower chance of viral clearance on PEG/RBV than non-GG patients. In chronic hepatitis B patients treated with PEG-IFN several retrospective analyses of IL28B rs12980275 and rs12979860 genotypes yielded conflicting results which can be explained by the heterogeneity between the study populations. Some variants of the HLA-DP locus (HLA-DPA1 A allele and HLA-DPB1) protect against progression of chronic hepatitis B infection. CONCLUSIONS: The determination of IL28B polymorphisms may be useful to individualise treatment options when using PEG/RBV based therapies for chronic hepatitis C infection. In contrast, so far identified genetic factors play only a minor role in chronic hepatitis B infection.

Loss of intrahepatic HBsAg expression predicts sustained response to peginterferon and is reflected by pronounced serum HBsAg decline.

There is a lack of knowledge regarding the effect of peginterferon (PEG-IFN) on the expression of intrahepatic hepatitis B core and surface antigen (HBcAg and HBsAg) in chronic hepatitis B (CHB) and its relation with response to therapy. Fifty-two HBeAg-positive and 67 HBeAg-negative CHB patients with paired liver biopsies taken at baseline and after 1 year of PEG-IFN therapy were studied. After PEG-IFN therapy, HBeAg-negative patients showed a significant reduction in both intrahepatic HBcAg (P = 0.04) and HBsAg expression (P < 0.001). In contrast, a reduction in intrahepatic HBcAg expression was not observed in HBeAg-positive patients, while a trend in reduction of intrahepatic HBsAg staining was found (P = 0.09). Post-treatment, 7 (13%) HBeAg-positive and 9 (14%) HBeAg-negative patients had no expression of intrahepatic HBsAg. Patients without any intrahepatic HBsAg expression post-treatment were more likely to achieve a combined response (HBeAg loss with hepatitis B virus (HBV) DNA <2000 IU/mL for HBeAg -positive and HBV DNA <2000 IU/mL and normal alanine aminotransferase for HBeAg-negative CHB): 71% vs 5% for HBeAg-positive (P < 0.001) and 60% vs 16% for HBeAg-negative patients (P = 0.004), respectively. Moreover, a more profound decline of serum HBsAg was observed in patients with absence of intrahepatic HBsAg staining (3.1 vs 0.4 log IU/mL, P < 0.001 and 1.7 vs 0.4 log IU/mL, P = 0.005 for HBeAg-positive and HBeAg-negative CHB, respectively). In conclusion, PEG-IFN reduces expression of intrahepatic HBsAg. Loss of HBsAg as assessed by immunohistochemistry from the liver predicts a sustained response and is reflected in a pronounced serum HBsAg decline.

Von Willebrand Factor as a new marker for non-invasive assessment of liver fibrosis and cirrhosis in patients with chronic hepatitis C.

BACKGROUND: Staging of liver fibrosis in patients with chronic hepatitis C (CHC) is recommended prior to anti-viral therapy. As vWF-Ag was shown as a predictor of portal hypertension, decompensation and mortality in patients with liver cirrhosis, we performed this study to investigate if vWF-Ag is able to predict different fibrosis stages and if it is comparable to other fibrosis scores. AIM: To investigate if vWF-Ag is able to predict different fibrosis stages and if it is comparable to other fibrosis scores. METHODS: We analysed 294 patients with chronic hepatitis C who underwent biopsy. We assessed stage of liver fibrosis according to Metavir, measured vWF-Ag and calculated different fibrosis scores (APRI, FCI, FORNS, FI, Fib-4) and compared them by AUCs. We also calculated a new score: vWF-Ag/thrombocytes (VITRO score) for prediction of fibrosis. RESULTS: vWF-Ag levels were increasing with stage of fibrosis: F0: vWF-Ag was median 136.5%, FI 140.6%, FII 157.5%, FIII 171.0%, FIV 252.0%; P < 0.001. vWF-Ag and VITRO score produced AUCs of 0.7 and 0.72 for ≥F2, comparable to the AUCs of APRI, Fib-4, FORNS with 0.75, 0.65 and 0.64 (P > 0.05). For ≥F3 AUCs were 0.79 and 0.86 for vWF-Ag and VITRO score, comparable with AUCs of 0.79, 0.86 and 0.87 for APRI, Fib-4 and FORNS. Cirrhosis shows AUCs of 0.84 and 0.89 for vWF-Ag and VITRO score, APRI, Fib-4 and FORNS showed similar results with AUCs of 0.82, 0.88 and 0.87. CONCLUSIONS: vWF-Ag and VITRO score offer an easy possibility to evaluate the stage of fibrosis to diagnose subclinical cirrhosis in patients with chronic hepatitis C. Both vWF-Ag and VITRO score show equal performance in comparison to other fibrosis scores assessed in our study.

Polymorphisms of interferon-λ4 and IL28B - effects on treatment response to interferon/ribavirin in patients with chronic hepatitis C.

BACKGROUND: The IL28B genotype in rs12979860 predicts success of peginterferon/ribavirin (PEG/RBV) therapy in patients with chronic hepatitis C (CHC). Recently, a dinucleotide frame shift variant in ss469415590 (TT or ΔG) was described, which generates the novel interferon lambda 4 protein (IFNL4). IFNL4 ss469415590 (ΔG) allele carriers have an impaired clearance of HCV infection and response to IFN-α therapy. In this study, we compared the role of IFNL4 polymorphism with the two commonly used IL28B SNPs rs12979860 and rs8099917 on response to PEG/RBV in patients with CHC. AIM: To compare the role of IFNL4 polymorphism with the two commonly used IL28B SNPs rs12979860 and rs8099917 on response to PEG/RBV in patients with CHC. METHODS: A total of 754 PEG/RBV patients treated (male/female = 484/270; Caucasians: 98.8%; mean age: 42.8 [CI 95%: 42.0-43.6] y; genotype (GT)1: n = 435, GT2: n = 23, GT3: n = 185, GT4: n = 114) were investigated. Liver fibrosis was assessed by liver biopsy in 456 patients. Single nucleotide polymorphisms (SNPs) in ss469415590, rs12979860 and rs8099917 were analysed by RT-PCR system. RESULTS: Of the patients, 12.9% (n = 97) had the ss469415590 ΔG/ΔG genotype (IFNL4), 51.3% (n = 387) were heterozygous (TT/ΔG) and 35.8% (n = 270) had TT/TT. IFNL4 polymorphism was independently associated with SVR in GT1 (OR: 2.539, CI 95%: 1.629-3.021, P < 0.001) and GT4 (OR: 12.573, CI 95%: 3.427-46.133, P < 0.001), but not in GT3 (OR: 1.514, CI 95%: 0.933-2.458, P = 0.093). IFNL4 correlated strongly with rs12979860 (ρ = 0.988, P < 0.001), but only moderately with rs8099917 (ρ = 0.598, P < 0.001). CONCLUSIONS: These findings underscore the role of IFNL4 for treatment response in patients with CHC genotypes 1 and 4. However, due to its strong correlation with rs12979860 in IL28B, there is no benefit in additional testing for IFNL4 for treatment prediction in Caucasian patients. By contrast, IFNL4 improves prediction of response to interferon-based therapies, if SNP rs8099917 is used.