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Background and Aim: HCC development in liver cirrhosis is associated with impaired autophagy leading to increased production of extracellular vesicles (EVs) including exosomes and microvesicles. The goal of the study is to determine which of these particles is primarily involved in releasing of HCC-specific biomarker glypican-3 (GPC3) when autophagy is impaired. Methods: Streptavidin-coated magnetic beads were coupled with either biotinylated CD63 or Annexin A1 antibodies. Coupled beads were incubated with EVs isolated from either HCC culture or serum. EVs captured by immuno-magnetic beads were then stained with FITC or PE fluorescent-conjugated antibodies targeting exosomes (CD81), and microvesicles (ARF6). The percentage of GPC3 enrichment in the microvesicles and exosomes was quantified by flow cytometry. The impact of autophagy modulation on GPC3 enrichment in exosomes and microvesicles was assessed by treating cells with Torin 1 and Bafilomycin A1. For clinical validation, GPC3 content was quantified in microvesicles, and exosomes were isolated from the serum of patients with a recent HCC diagnosis. Results: The immune-magnetic bead assay distinguishes membrane-derived microvesicles from endosome-derived exosomes. The GPC3 expression was only seen in the CD63 beads group but not in the Annexin A1 beads group, confirming that in HCC, GPC3 is preferentially released through exosomes. Furthermore, we found that autophagy induction by Torin1 decreased GPC3-positive exosome secretion and decreased microvesicle release. Conversely, autophagy inhibition by Bafilomycin A1 increased the secretion of GPC3-positive exosomes. Serum analysis showed CD81+ve EVs were detected in exosomes and ARF6+ve vesicles were detected in microvesicles, suggesting that immunoaffinity assay is specific. The exosomal GPC3 enrichment was confirmed in isolated EVs from the serum of patients with HCC. The frequency of GPC3-positive exosomes was higher in patients with HCC (12.4%) compared to exosomes isolated from non-cirrhotic and healthy controls (3.7% and 1.3% respectively, p<0.001). Conclusion: Our results show that GPC3 is enriched in the endolysosomal compartment and released in exosome fractions when autophagy is impaired.
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BACKGROUND: Spontaneous bacterial peritonitis (SBP) is common in hospitalized cirrhotic patients with ascites and carries high mortality. This study aimed to determine whether early diagnostic paracentesis (EDP) <12 h of hospitalization conveys an intermediate-term (6-month) survival benefit in cirrhotic patients diagnosed with SBP. METHODS: Consecutive US veterans with cirrhosis diagnosed with SBP over 13 years at a single VA medical center were reviewed retrospectively. Kaplan-Meyer analyses assessed the effects of EDP on survival. RESULTS: A total of 79 cirrhotic patients were diagnosed with SBP (61.8 ± 8.8 years, n = 77 male, n = 52 [66.8%] Caucasian, n = 23 [29.1%] African-American). Underlying liver diseases included hepatitis c viral infection (HCV) (17.5%), alcohol (28.6%), alcohol and HCV (30.1%), and cryptogenic/metabolic (15.9%). Median baseline model for end-stage liver disease (MELD) was 12 (range 6-34), and median MELD at presentation was 18. Seven subjects had a history of hepatocellular carcinoma (11.1%), and 26 (41.3%) presented with sepsis. Thirty-three (52.4%) subjects died within 6 months after the SBP admission. Of the subjects, 41 (65.1%) underwent EDP, of which 23 (56.0%) survived at least 6 months, compared to only 7 of the 22 patients (31.8%) undergoing paracentesis >12 h from presentation (P = 0.057). The maximal benefit of EDP on survival was observed beyond days 14 and 30; at these time points, no statistical difference in mortality was discernable (P = 0.55 and 0.71). In a multivariate model including age, MELD at admission, hepatocellular cancer, and sepsis criteria, EDP (p 0.034) positively impacted patient survival at 6 months. CONCLUSIONS: EDP is associated with improved 6-month mortality in cirrhotic patients with ascites. In this veteran cohort, EDP was as important as MELD as a predictor of intermediate-term survival.
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Hepatitis C virus (HCV) infection is the major risk factor for liver cirrhosis and hepatocellular carcinoma (HCC). The mechanisms of HCC initiation, growth, and metastasis appear to be highly complex due to the decade-long interactions between the virus, immune system, and overlapping bystander effects of host metabolic liver disease. The lack of a readily accessible animal model system for HCV is a significant obstacle to understand the mechanisms of viral carcinogenesis. Traditionally, the primary prevention strategy of HCC has been to eliminate infection by antiviral therapy. The success of virus elimination by antiviral treatment is determined by the SVR when the HCV is no longer detectable in serum. Interferon-alpha (IFN-α) and its analogs, pegylated IFN-α (PEG-IFN-α) alone with ribavirin (RBV), have been the primary antiviral treatment of HCV for many years with a low cure rate. The cloning and sequencing of HCV have allowed the development of cell culture models, which accelerated antiviral drug discovery. It resulted in the selection of highly effective direct-acting antiviral (DAA)-based combination therapy that now offers incredible success in curing HCV infection in more than 95% of all patients, including those with cirrhosis. However, several emerging recent publications claim that patients who have liver cirrhosis at the time of DAAs treatment face the risk of HCC occurrence and recurrence after viral cure. This remains a substantial challenge while addressing the long-term benefit of antiviral medicine. The host-related mechanisms that drive the risk of HCC in the absence of the virus are unknown. This review describes the multifaceted mechanisms that create a tumorigenic environment during chronic HCV infection. In addition to the potential oncogenic programming that drives HCC after viral clearance by DAAs, the current status of a biomarker development for early prediction of cirrhosis regression and HCC detection post viral treatment is discussed. Since DAAs treatment does not provide full protection against reinfection or viral transmission to other individuals, the recent studies for a vaccine development are also reviewed.
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BACKGROUND: Hepatitis C remains a major cause of liver disease globally and is responsible for approximately 500 000 deaths annually. Newer direct-acting antivirals achieve cure rates at or above 90% with excellent tolerability for most patients. The literature focusing on identification of predictors of efficacy and safety with specific hepatitis C therapies has been inconclusive and often conflicting. METHODS: A retrospective, single-center, case-control analysis of all veteran patients aged ≥18 through ≤89 years who completed a treatment course of 8, 12, or 24 weeks with ledipasvir and sofosbuvir (LDV/SOF) combination therapy for hepatitis C infection was conducted. Patients who were identified and met inclusion criteria were assigned to either the case group (SVR12 failure; hepatitis C viral load detectable at least 11 weeks after therapy completion) or the control group (SVR12 success; hepatitis C viral load undetectable at least 11 weeks after therapy completion). RESULTS: Twenty-nine SVR12 failures and 411 SVR12 successes were included in the analysis. The overall failure rate was consistent with the current literature, at 6.6% (29/440). Bivariate analysis identified only baseline creatinine clearance >80 mL min-1 (Cockcroft-Gault) as a possible predictor of SVR12 failure (P = .026). In the multivariate analysis, pretreatment creatinine clearance >80 mL min-1 remained independently associated with SVR12 failure (odds ratio, 2.95; 95% confidence interval, 1.17-7.46; P = .023). CONCLUSIONS: In hepatitis C patients treated with LDV/SOF, a pretreatment creatinine clearance of >80 mL min-1 was associated with SVR12 failure.