Cytokeratin 18-based cell death markers indicate severity of liver disease and prognosis of cirrhotic patients.

BACKGROUND & AIMS: Hepatocyte death is an important factor in development and progression of cirrhosis. Cytokeratin 18-based serum markers reflecting apoptotic (M30) and overall epithelial cell death (M65 and M65EpiDeath) have been used as prognostic parameters for survival in patients with acute liver failure. However, there has been no trial investigating M30, M65 and M65EpiDeath as survival parameters in patients with cirrhosis and acute-on-chronic liver failure. METHODS: Patients with cirrhosis were enrolled and followed until death, liver transplantation or last contact. M30, M65 and M65EpiDeath serum levels were quantified in patient's sera. RESULTS: Three hundred and thirty-one patients were screened and 211 patients could be included in this study. The median duration of follow-up was 322 days with a range of 1-1382 days. All three cell death parameters correlated with the extent of the severity of the disease. However, M65EpiDeath was the only of the three parameters which was associated with the severe complications of cirrhosis including ascites, spontaneous bacterial peritonitis and hepatorenal syndrome. Additionally, M65EpiDeath was the only cell death parameter which was independently from liver function and its surrogate parameter such as Child-Pugh score and the model of end-stage liver disease associated with overall survival. CONCLUSIONS: Epithelial cell death reflected by M65EpiDeath serum levels is an indicator for the severity of cirrhosis and a prognostic survival parameter in cirrhotic patients.

Telaprevir drug monitoring during antiviral therapy of hepatitis C graft infection after liver transplantation.

BACKGROUND & AIMS: Recurrence of hepatitis C virus (HCV) infection after orthotopical liver transplantation (OLT) is common and associated with reduced graft and patient survival. The protease inhibitor telaprevir may enhance virological response rates in patients after OLT in combination with pegylated interferon-alfa and ribavirin. Pharmacokinetic studies have shown significant drug-drug interactions between telaprevir and immunosuppression (IS), but telaprevir pharmacokinetics in OLT patients with IS are unknown. Aim of the present study was to analyse telaprevir plasma concentrations in patients with HCV genotype 1 infection after OLT in comparison to patients without OLT and IS. METHODS: Five patients with HCV genotype 1 infection after OLT and 37 HCV genotype 1-infected patients patients without prior OLT were treated with telaprevir 2250 mg daily, ribavirin 1000/1200 mg daily and pegylated interferon-alfa-2a 180 µg once weekly (triple therapy). Telaprevir plasma concentrations were analysed by liquid chromatography-electrospray-ionization-tandem mass spectrometry. HCV RNA was assessed by automatized reverse-transcription polymerase chain-reaction. RESULTS: Median (range) telaprevir plasma concentrations of TW 4, 8 and 12 were 3970 (1980-4430) ng/ml and 2520 (1870-8730) ng/ml in patients after OLT and ciclosporin- or tacrolimus-based IS, respectively, as compared to 2790 (1870-3140) in non-OLT patients (P = 0.3). In one patient with tacrolimus-based IS, telaprevir dose had to be adjusted to achieve virological response. Telaprevir plasma concentrations were steady at treatment weeks 4, 8 and 12 in patients with and without IS. CONCLUSIONS: Telaprevir drug monitoring may be necessary in patients with tacrolimus-based IS in patients with HCV graft infection after OLT.

No Distal Migration in Unfixed Versus Fixed Cell Structure Covered Self-Expanding Metal Stents for Treatment of Benign Biliary Disease.

BACKGROUND: Fully covered self-expandable metal stents (FCSEMS) are increasingly used for treatment of benign common bile duct (CBD) stricture or leakage, but dislodgement of FCSEMS is frequent. AIMS: To compare dislocation rate and clinical outcome of a standard fixed cell structure FCSEMS (S-FCSEMS) to a novel FCSEMS with an unfixed cell structure (N-FCSEMS). METHODS: We performed a retrospective analysis of all patients with FCSEMS insertion for benign biliary disease at our Hospital from 03/2008 to 03/2014. Both stent types N-FCSEMS and S-FCSEMS were applied as available unrelated to the indication. RESULTS: Twenty-nine patients (S-FCSEMS: 18, N-FCSEMS: 11) were included. Stent placement was technically successful in 28/29 (96.6 %) patients; stent removal was successful in 26/27 (96.2 %). Two patients with N-FCSEMS were excluded due to unsuccessful placement and withdrawal of consent for stent removal, respectively. Stent migration into the duodenum (distal migration) was observed in 9/18 (50 %) in the S-FCSEMS group compared to 0/9 in the N-FCSEMS (p < 0.005). FCSEMS migration into the CBD (proximal migration) was found in 2/18 (11 %, S-FCSEMS) versus 2/9 (22 %, N-FCSEMS, p = 0.514). A foreshortening of the N-FCSEMS occurred in 3/9 patients (33 %) compared to 0/18 S-FCSEMS (p = 0.08). Clinical resolution of the treated CBD-disease was observed in 5/9 (56 %, N-FCSEMS) versus 12/18 (67 %, S-FCSEMS) at the time of stent removal (p = 0.604) and in 0/9 and 10/18 (56 %) cases during follow-up, respectively (p < 0.005). CONCLUSION: An unfixed cell structure of FCSEMS seems to prevent distal migration, but proximal migration still occurs and foreshortening of the N-FCSEMS constrains clinical outcome.

Low vitamin D serum concentration is associated with high levels of hepatitis B virus replication in chronically infected patients.

UNLABELLED: Vitamin D is an important immune modulator that plays an emerging role in inflammatory and metabolic liver diseases, including infection with hepatitis C virus (HCV). In contrast, the relationship between vitamin D metabolism and chronic hepatitis B (CHB) is less well characterized. Therefore, we quantified 25(OH)D3 serum levels in a cohort of 203 treatment-naïve patients with chronic hepatitis B virus (HBV) infection and tested for their association with clinical parameters of CHB. Of 203 patients, 69 (34%), 95 (47%), and 39 (19%) had severe vitamin D deficiency (25(OH)D3 <10 ng/mL), vitamin D insufficiency (25(OH)D3 ≥10 and <20 ng/mL), or adequate vitamin D serum levels (25(OH)D3 ≥20 ng/mL), respectively. In both uni- and multivariate analyses, HBV DNA viral load (log10 IU/mL) was a strong predictor of low 25(OH)D3 serum levels (P = 0.0007 and P = 0.000048, respectively) and vice versa. Mean 25(OH)D3 serum concentrations in patients with HBV DNA <2,000 versus ≥2,000 IU/mL were 17 versus 11 ng/mL, respectively (P < 0.00001). In addition, hepatitis B early antigen (HBeAg)-positive patients had lower 25(OH)D3 serum levels than HBeAg-negative patients (P = 0.0013). Finally, 25(OH)D3 and HBV DNA serum levels showed inverse seasonal fluctuations. CONCLUSION: Low 25(OH)D3 serum levels are associated with high levels of HBV replication in patients with CHB. This represents a major difference from chronic hepatitis C, where numerous previous studies have shown a lack of correlation between HCV viral load and vitamin D serum levels. Inverse seasonal fluctuations of 25(OH)D3 and HBV DNA serum levels are suggestive of a functional relationship between both variables.

A novel, stiff-shaft, flexible-tip guidewire for cannulation of biliary stricture during endoscopic retrograde cholangiopancreatography: a randomized trial.

BACKGROUND AND STUDY AIMS: During endoscopic retrograde cholangiopancreatography (ERCP), a guidewire is used to cannulate biliary strictures and allow for therapeutic interventions. The aim of this study was to assess the success of stricture cannulation using a combination of a flexible guidewire and a stable nitinol wire vs. a novel, single, stiff-shaft, flexible-tip guidewire. PATIENTS AND METHODS: Consecutive patients who were scheduled for ERCP for biliary obstruction were randomized to undergo the procedure with either a 260-cm long, angled-tip hydrophilic wire in combination with a nitinol wire as required (standard group), or a novel, 270-cm guidewire featuring a hyperflexible, hydrophilic tip with a stiff shaft (novel group). At unsuccessful negotiation of the stricture, patients in the standard group were switched to the novel guidewire and vice versa ("crossover"). Successful cannulation (primary success: as assigned; final success: after "crossover"), procedure time, and total number of wires needed per procedure were compared. RESULTS: A total of 222 patients were randomized and 197 were included in the study (97 in the standard group and 100 in the novel group). The primary success rate was significantly higher in the novel group (94/100, 94 %) compared with the standard group (77/97, 79 %; P = 0.00041), and final success was similar. Mean time (median, interquartile range) to stricture cannulation was 11.2 minutes (6.3, 3.7 - 14.6) in the standard group and 8.1 minutes (2.5, 0.9 - 7.7) in the novel group (P < 0.0001). The mean total procedure time was 31.2 minutes (24.6, 16.5 - 40.8) vs. 24.3 minutes (16.9, 10.0 - 31.5), respectively (P = 0.0011). There were no complications observed with either of the guidewires. CONCLUSIONS: A guidewire that features a flexible tip with a stable shaft could replace the use of a combination of flexible and stable guidewires and increase the success rate of stricture cannulation while decreasing the procedure time.ClinicalTrials.gov Identifier: NCT 01382680.

Macrophage activation is a prognostic parameter for variceal bleeding and overall survival in patients with liver cirrhosis.

BACKGROUND & AIMS: Soluble CD163 (sCD163) is shed in the blood circulation by activated macrophages, correlates strongly with the hepatic venous pressure gradient (HVPG) and is thereby a good indicator of portal hypertension. It is unknown whether sCD163 correlates with the risk of variceal bleeding and overall survival (OS) in patients with liver cirrhosis. We performed a prospective study to investigate if sCD163 serum levels correlate with the risk of variceal bleeding and OS in cirrhotic patients. METHODS: Patients with liver cirrhosis were prospectively enrolled and followed until death or last contact. At the day of inclusion in the study, blood samples were taken and sCD163 serum levels were assessed by ELISA (enzyme-linked immunosorbent assay). The time until the end points death and variceal bleeding was assessed and the risks of death or variceal bleeding were calculated with uni- and multivariate Cox regression analyses. RESULTS: High sCD163 levels (>4100 ng/L) were associated with death independently of the MELD (model of end stage liver disease) score, CRP (C-reactive protein), age and gender. Furthermore, high sCD163 levels were associated with gastrointestinal bleeding independently of the variceal stage and red spots. CONCLUSIONS: The sCD163 serum level is a new independent non-invasive risk factor for death and variceal bleeding in cirrhotic patients.

Diagnostic and prognostic significance of cell death and macrophage activation markers in patients with hepatocellular carcinoma.

BACKGROUND & AIMS: The serum cell death parameters M30 and M65 and the macrophage activation marker sCD163 (soluble CD163) are elevated in patients with acute and chronic liver diseases. However, their diagnostic and prognostic potential in patients with hepatocellular carcinoma (HCC) has not yet been investigated. METHODS: Serum levels of M30, M65, and sCD163 were measured in two cohorts of HCC patients and a cohort of cirrhotic patients. The parameters were compared between patients with and without HCC and the overall survival (OS) times according to M30, M65, and sCD163 were assessed. RESULTS: M30 and M65 levels were higher in HCC patients than in cirrhotic patients (both p < 0.001). M65 was an independent parameter for non-invasive identification of HCC patients by logistic regression analysis and could supplement AFP (alpha-fetoprotein) and abdominal ultrasound in non-invasive detection of HCC patients. High M65 serum levels as well as high sCD163 concentrations were associated with an impaired prognosis in univariate Cox regression analysis. The sCD163 level was associated with OS independently of the CLIP (Cancer of the Liver Italian Program) score, the BCLC (Barcelona Clinic Liver Cancer) stage, and the CRP (C-reactive protein) level in a multivariate Cox regression model. CONCLUSIONS: Serum M65 has the potential as a new diagnostic parameter for HCC and serum sCD163 is a new prognostic parameter in HCC patients.

Interleukin-22 predicts severity and death in advanced liver cirrhosis: a prospective cohort study.

BACKGROUND: Interleukin-22 (IL-22), recently identified as a crucial parameter of pathology in experimental liver damage, may determine survival in clinical end-stage liver disease. Systematic analysis of serum IL-22 in relation to morbidity and mortality of patients with advanced liver cirrhosis has not been performed so far. METHODS: This is a prospective cohort study including 120 liver cirrhosis patients and 40 healthy donors to analyze systemic levels of IL-22 in relation to survival and hepatic complications. RESULTS: A total of 71% of patients displayed liver cirrhosis-related complications at study inclusion. A total of 23% of the patients died during a mean follow-up of 196 ± 165 days. Systemic IL-22 was detectable in 74% of patients but only in 10% of healthy donors (P < 0.001). Elevated levels of IL-22 were associated with ascites (P = 0.006), hepatorenal syndrome (P < 0.0001), and spontaneous bacterial peritonitis (P = 0.001). Patients with elevated IL-22 (>18 pg/ml, n = 57) showed significantly reduced survival compared to patients with regular (≤18 pg/ml) levels of IL-22 (321 days versus 526 days, P = 0.003). Other factors associated with reduced overall survival were high CRP (≥2.9 mg/dl, P = 0.005, hazard ratio (HR) 0.314, confidence interval (CI) (0.141 to 0.702)), elevated serum creatinine (P = 0.05, HR 0.453, CI (0.203 to 1.012)), presence of liver-related complications (P = 0.028, HR 0.258, CI (0.077 to 0.862)), model of end stage liver disease (MELD) score ≥20 (P = 0.017, HR 0.364, CI (0.159 to 0.835)) and age (P = 0.011, HR 0.955, CI (0.922 to 0.989)). Adjusted multivariate Cox proportional-hazards analysis identified elevated systemic IL-22 levels as independent predictors of reduced survival (P = 0.007, HR 0.218, CI (0.072 to 0.662)). CONCLUSIONS: In patients with liver cirrhosis, elevated systemic IL-22 levels are predictive for reduced survival independently from age, liver-related complications, CRP, creatinine and the MELD score. Thus, processes that lead to a rise in systemic interleukin-22 may be relevant for prognosis of advanced liver cirrhosis.

Portal vein thrombosis as complication of romiplostim treatment in a cirrhotic patient with hepatitis C-associated immune thrombocytopenic purpura.

BACKGROUND & AIMS: Thrombopoietin receptor agonists are a new class of compounds licenced for the treatment of immune thrombocytopenic purpura. They are currently being studied for patients with thrombopenia in advanced liver disease or under therapy for hepatitis C. There are indications that the risk for development of portal vein thrombosis in patients with advanced liver cirrhosis might be increased under therapy with thrombopoietin receptor agonists. We report a case of a patient with Child class B liver cirrhosis with concurrent immune thrombocytopenic purpura that developed portal vein thrombosis under therapy with the thrombopoietin receptor agonist romiplostim. METHODS: A 50-year-old woman with hepatitis C virus associated immune thrombocytopenic purpura and Child class B liver cirrhosis presented in our emergency with rapidly evolving hydropic decompensation and general malaise. For immune thrombocytopenic purpura, the patient was started on the thrombopoietin receptor agonist romiplostim nine months ago. RESULTS: During hospitalization, the platelet count was measured above 330,000/µl and partial portal vein thrombosis was diagnosed by imaging studies. The thrombotic event was assumed to be associated with the romiplostim treatment for immune thrombocytopenic purpura via excessive elevation of platelet count. After anticoagulation with heparin and cessation of romiplostim treatment, complete recanalisation of the portal vein was achieved. CONCLUSIONS: We conclude that romiplostim should be used with precaution in patients with hepatitis C-associated immune thrombocytopenic purpura and advanced liver cirrhosis as the risk for thrombotic complications may increase significantly.

Serum miR-122 as a biomarker of necroinflammation in patients with chronic hepatitis C virus infection.

OBJECTIVES: The liver contains large amounts of microRNA-122 (miR-122), whereas other tissues contain only marginal amounts of this miRNA. MicroRNAs have also been found to circulate in the blood in a cell-free form; their potential as readily accessible disease markers is currently evaluated. Here, we investigated if the serum levels of miR-122 might be useful as disease parameter in patients with chronic hepatitis C virus (HCV) infection. METHODS: RNA was extracted from sera of patients with chronic HCV infection (CHC) and healthy controls and was analyzed for miR-22 content by quantitative real-time reverse-transcription polymerase chain reaction. miR-122 serum levels were correlated with standard parameters of liver function. Liver biopsies from the same patients were examined for the histologic activity index (HAI) and the degree of fibrosis. RESULTS: Sera from patients with CHC contained higher levels of miR-122 than sera from healthy controls. Serum miR-122 levels correlated well with markers of liver inflammatory activity, that is, the serum levels of alanine leucine transaminase (ALT) and aspartate transaminase, and the HAI score. In patients with persistently normal ALT levels, serum miR-122 levels did not differ from healthy controls. There was no correlation of serum miR-122 levels with serum albumin, international normalized ratio, liver fibrosis, or serum HCV RNA. CONCLUSIONS: The serum level of miR-122 strongly correlates with serum ALT activity and with necroinflammatory activity in patients with CHC and elevated ALT levels, but not with fibrosis stage and functional capacity of the liver.

CD81 expression for discrimination between sustained virologic response and relapse in patients with chronic hepatitis C.

BACKGROUND AND AIM: The hepatitis C virus (HCV) receptor CD81 is overexpressed on peripheral blood mononuclear cells (PBMC) in patients chronically infected with HCV compared with healthy controls, and expression declines during antiviral therapy. The aim of this study was to prospectively investigate CD81 expression on PBMC for early discrimination between sustained virologic response (SVR) and relapse (REL) to pegylated interferon alfa-2b and ribavirin treatment. METHODS: Sixty-one patients with chronic HCV infection (genotype, GT, 1 and low baseline viremia <600,000 IU/ml, n = 30; GT 2 or 3, n = 31) were investigated. CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was measured at baseline, therapy week (TW) 4 and 12 during antiviral therapy by fluorescence-activated cell sorting (FACS) analysis. RESULTS: Baseline levels of CD81 on CD4(+), CD8(+), and CD56(+) cells were similar between patients who achieved a SVR (n = 42) and those who relapsed (n = 19). On CD19(+) cells, baseline CD81 expression was higher in patients with SVR than in patients with virologic relapse (REL) (p < 0.006). A cutoff value of 720 relative fluorescence units (RFU) discriminated correctly between SVR and REL with a sensitivity and specificity of 73.7% and 66.7%, respectively. SVR patients showed a significant decline of CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells (p < 0.01 for all) while in REL patients a significant decline of CD81 expression was observed on CD8(+) and CD56(+) cells, only (p = 0.050 and p = 0.038, respectively). CONCLUSIONS: The current study confirms significant down-regulation of CD81 expression on different lymphocyte subpopulations during pegylated interferon alfa-based antiviral therapy in patients with chronic hepatitis C. Baseline CD81 expression on CD19(+) cells was found to discriminate between SVR and REL.

Meta-analysis shows extended therapy improves response of patients with chronic hepatitis C virus genotype 1 infection.

BACKGROUND & AIMS: Clinical trials provided conflicting results about whether extended duration of treatment with pegylated interferon-alfa (pegIFN-alfa) and ribavirin (more than 48 weeks) improves rates of sustained virologic response (SVR) in patients infected with hepatitis C virus (HCV) genotype 1 and slow virologic response. We performed a meta-analysis to determine the overall impact of extended treatment, compared with standard treatment, on virologic response rates in these patients. METHODS: We performed a literature search to identify randomized controlled trials (RCTs) that included monoinfected, treatment-naive patients infected with HCV genotype 1; data were compared between slow responding patients treated with pegIFN-alfa-2a/b plus ribavirin for 48 weeks and those that received extended treatment (as much as 72 weeks). End points included SVR rates, end-of-treatment (EOT) response and relapse rates; they were calculated according to the DerSimonian-Laird estimate. RESULTS: Six RCTs assessed the benefits of extended treatment with pegIFN-alfa-2a/b and ribavirin in treatment-naive patients with HCV genotype 1 that were slow responders (n = 669). The extended treatment significantly improved SVR rates in slow responders, compared with the standard of care (14.7% increase in overall SVR; 95% confidence interval, 4%-25.5%; P = .0072). Rates of viral relapse were significantly reduced by extended treatment, but EOT response rates were similar. The frequency of voluntary treatment discontinuation, but not of serious adverse events, was significantly increased by extended therapy. CONCLUSIONS: Extending the duration of treatment with pegIFN-alfa-2a/b and ribavirin in patients with HCV genotype 1 and a slow response to therapy improves the rate of SVR.

Development and evaluation of a FACS-based medium-throughput assay for HCV entry inhibitors.

The interaction between the hepatitis C virus (HCV) envelope glycoprotein E2 and the human tetraspanin protein CD81 is one of the key events involved in HCV cell entry. Therefore, compounds that interfere with this interaction may be useful tools for basic research and potential drugs for the treatment of HCV infection. The authors describe a medium-throughput assay for ligands of the E2 binding site on the CD81 receptor. In the assay, human hepatoma cells are incubated with the test compounds and stained with a fluorescently labeled anti-CD81 antibody (JS81). Flow cytometry is used to detect the level of bound antibody, reflecting the inhibitory potencies of the compounds. Eighty percent of compounds active in the assay show efficacy in an infection assay using luciferase reporter genome in cell culture. Thus, the assay can be used as a fast screening system for inhibitors of interaction of viral E2 to host cell CD81-LELs.

Treatment of chronic hepatitis C: anticipated impact of resistance in patients treated with protease inhibitors.

A main target of specifically targeted antiviral therapy for hepatitis C (STAT-C) is the NS3-protease, which has key functions in the hepatitis C virus (HCV) replication cycle. HCV/NS3-protease inhibitors have shown high antiviral activity in vitro and in patients with chronic hepatitis C. Protease-resistant HCV variants occurred rapidly in patients receiving protease-inhibitor monotherapy. The development of resistance can be best explained by selection of preexisting resistant variants, which grow out under selective pressure. Numerous mutations associated with resistance were identified. Clinical trials showed that protease-resistant strains are sensitive to interferon and that a triple combination of protease inhibitors, peginterferon, and ribavirin may improve the sustained virologic response rate compared with standard peginterferon/ribavirin combination therapy. Overall, it can be anticipated that successful treatment with protease inhibitors will require either combination therapy with peginterferon/ribavirin or a combination of STAT-C compounds with distinct modes of action and resistance patterns.

Current and future treatment options for HCV.

Aim of antiviral therapy of patients with chronic hepatitis C is the sustained elimination of the hepatitis C virus (HCV). The standard of care (SOC) is peginterferon alfa-2a/-2b with ribavirin for 48 weeks or 24 weeks in patients infected with HCV genotype 1 or 2/3, respectively. Overall, approximately half of the patients can be cured by SOC. Based on baseline viral load and the speed of virologic response during treatment, individualization of treatment duration is possible. However, this approach is not sufficient to substantially improve the sustained virologic response (SVR) rates. This goal can be achieved with new HCV specific inhibitors against the NS3/4A polymerase and the NS5B polymerase. Recent trials reported SVR rates in the order of 67-69% and 67-75% for the combination of SOC with the protease inhibitors telaprevir and boceprevir, respectively, in patients with HCV genotype 1 infection. Several new HCV specific inhibitors such as protease inhibitors, nucleoside and non-nucleoside polymerase inhibitors as well as non HCV specific compounds with anti-HCV activity such as cyclophilin inhibitors, silibinin, and nitazoxanide are currently in clinical evaluation. The review describes recent developments and discusses limitations posed by resistance development and drug toxicity.

Novel hepatitis C drugs in current trials.

Almost half of the patients who have chronic hepatitis C cannot be cured with the current standard treatment. Recent progress in structure determination of HCV proteins and development of a subgenomic replicon system and a cell culture infectious HCV clone enabled the development of a specifically targeted antiviral therapy for hepatitis C (STAT-C). Many HCV-specific compounds are under investigation in preclinical and clinical trials. The development of agents in different classes may allow construction of antiviral combinations that enhance the effectiveness of antiviral treatment, reduce the duration of treatment, and, eventually, may even avoid the use of interferon-alfa.