Circulating microRNAs improve bacterial infection diagnosis and overall survival prediction in acute decompensation of liver cirrhosis.

Bacterial infections are the most frequent precipitating event in patients with acute decompensation of cirrhosis (AD) and are associated with high mortality. Early diagnosis is challenging due to cirrhosis-related systemic inflammation. Here we investigated the potential of circulating microRNAs to diagnose bacterial infections and predict survival in cirrhotic patients with AD. High throughput profiling of circulating microRNAs was performed using the Nanostring technology in 57 AD patients and 24 patients with compensated cirrhosis (CC). Circulating miRs profiling showed that: (a) miRs differentially detected in AD vs. CC were mostly down-regulated; (b) a composite score including absolute neutrophil count, C reactive protein and miR-362-3p could diagnose bacterial infection with an excellent performance (AUC of 0.825 [95% CI = 0.671-0.980; p < 0.001]); (c) a composite score including miR-382-5p, miR-592 and MELD-Na improved 6-month survival prediction. Circulating miRs are strongly dysregulated in patients with AD and may help to improve bacterial infection diagnosis and survival prediction.

Performance of the cobas® HBV RNA automated investigational assay for the detection and quantification of circulating HBV RNA in chronic HBV patients.

BACKGROUND: The amount of HBV RNA in peripheral blood may reflect HBV covalently closed circular DNA (cccDNA) transcriptional activity within infected hepatocytes. Quantification of circulating HBV RNA (cirB-RNA) is thus a promising biomarker for monitoring antiviral treatment. OBJECTIVES: We evaluated the performance of an automated, prototype quantitative HBV RNA assay for use on the Roche cobas® 6800/8800 systems. STUDY DESIGN: The sensitivity, specificity, linearity, and potential interference by HBV DNA of the cobas® HBV RNA assay were assessed using synthetic HBV armored RNA and clinical specimens. RESULTS: cobas® HBV RNA results were linear between 10 and 107 copies/mL in clinical samples of several HBV genotypes, and up to 109 copies/mL with synthetic RNA. Precision and reproducibility were excellent, with standard deviation below 0.15 log10 copies/mL and coefficients of variation below 5% throughout the linear range. The presence of HBV DNA had minimal (<0.3 log10 copies/mL) impact on HBV RNA quantification at DNA:RNA ratios of up to approximately one million. In a panel of 36 untreated patient samples, cirB-RNA concentrations were approximately 200-fold lower than HBV DNA. cirB-RNA was detected in all 13 HBeAg-positive patients (mean 6.0 log10 copies/mL), and in 20 of 23 HBeAg-negative patients (mean of quantifiable samples 2.2 log10 copies/mL). Finally, cirB-RNA was detected in 12 of 20 nucleoside analog-treated patients (mean of quantifiable samples 3.4 log10 copies/mL). CONCLUSIONS: The cobas® 6800/8800 investigational HBV RNA assay is a high throughput, sensitive and inclusive assay to evaluate the clinical relevance of cirB-RNA quantification in patients with chronic hepatitis B.

Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients.

BACKGROUND & AIMS: It has been proposed that serum hepatitis B core-related antigen (HBcrAg) reflects intrahepatic covalently closed circular (ccc)DNA levels. However, the correlation of HBcrAg with serum and intrahepatic viral markers and liver histology has not been comprehensively investigated in a large sample. We aimed to determine if HBcrAg could be a useful therapeutic marker in patients with chronic hepatitis B. METHODS: HBcrAg was measured by chemiluminescent enzyme immunoassay in 130 (36 hepatitis B e antigen [HBeAg]+ and 94 HBeAg-) biopsy proven, untreated, patients with chronic hepatitis B. HBcrAg levels were correlated with: a) serum hepatitis B virus (HBV)-DNA, quantitative hepatitis B surface antigen and alanine aminotransferase levels; b) intrahepatic total (t)HBV-DNA, cccDNA, pregenomic (pg)RNA and cccDNA transcriptional activity (defined as pgRNA/cccDNA ratio); c) fibrosis and necroinflammatory activity scores. RESULTS: HBcrAg levels were significantly higher in HBeAg+ vs. HBeAg- patients and correlated with serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA and cccDNA levels, and transcriptional activity. Patients who were negative for HBcrAg (<3 LogU/ml) had less liver cccDNA and lower cccDNA activity than the HBcrAg+ group. Principal component analysis coupled with unsupervised clustering identified that in a subgroup of HBeAg- patients, higher HBcrAg levels were associated with higher serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA, cccDNA transcriptional activity and with higher fibrosis and necroinflammatory activity scores. CONCLUSIONS: Our results indicate that HBcrAg is a surrogate marker of both intrahepatic cccDNA and its transcriptional activity. HBcrAg could be useful in the evaluation of new antiviral therapies aiming at a functional cure of HBV infection either by directly or indirectly targeting the intrahepatic cccDNA pool. LAY SUMMARY: Hepatitis B virus causes a chronic infection which develops into severe liver disease and liver cancer. The viral covalently closed circular DNA (cccDNA) is responsible for the persistence of the infection in hepatocytes. To better manage patient treatment and follow-up, and to develop new antiviral treatments directly targeting the intrahepatic pool of cccDNA, serum surrogate markers reflecting the viral activity in the liver are urgently needed. In this work, we demonstrate that quantification of hepatitis B core-related antigen in serum correlates with cccDNA amount and activity and could be used to monitor disease progression.

Perspectives and limitations for nucleo(t)side analogs in future HBV therapies.

The latest generation of nucleo(t)side analogs (NAs) provide robust virus suppression with high barrier to resistance. Long term NAs treatment is associated with a partial restoration in HBV-specific T-cell functions, regression of fibrosis, no disease progression and a reduction of HCC risk but rarely lead to cure and life-long treatments is often required. New insights into the hepatitis B viral life cycle and the host immune response have expanded the potential targets for drug therapies with interesting antiviral candidates and novel immunotherapeutic approaches in early stage development. Here we review the mode of action of current drugs (NAs and PEG IFN) and new classes of anti-HBV compounds, focusing on the possible role of nucleo(t)side analogs in future combination therapies.

Interaction between Toll-Like Receptor 9-CpG Oligodeoxynucleotides and Hepatitis B Virus Virions Leads to Entry Inhibition in Hepatocytes and Reduction of Alpha Interferon Production by Plasmacytoid Dendritic Cells.

We previously reported that Toll-like receptor 9 (TLR9)-CpG oligonucleotides could inhibit the establishment of hepatitis B virus (HBV) infections in hepatocytes. Our aim was to uncover the underlying mechanisms of this inhibition. HepaRG cells, RPMI-B lymphoblastoma cells, and primary plasmacytoid dendritic cells (pDCs) exposed to HBV and TLR9 ligands/agonists in various configurations were used. We observed an inhibition of HBV infection upon TLR9 stimulations only when agonist was applied during inoculation. This inhibition was independent of interleukin-6 (IL-6)/interferon-inducible protein 10 (IP-10) production as well as of TLR9 expression in hepatocytes. We further demonstrated an entry inhibition mechanism by showing a noncovalent binding of TLR9 agonist to HBV particles. Besides inhibiting HBV entry into hepatocytes, this biophysical interaction between HBV virions and TLR9 agonist was responsible for a reduction of alpha interferon (IFN-α) expression by pDCs. Interestingly, subviral particles composed of only HBsAg were able to genuinely inhibit the TLR9 pathway, without titrating TLR9 ligands. To conclude, our data suggest that synthetic TLR9-CpG oligonucleotides can strongly inhibit HBV entry by "coating" HBV virions and thereby preventing their interaction with cellular receptor. This titration effect of TLR9 agonist is also artifactually responsible for the inhibition of TLR9 engagement in pDCs, whereas a genuine inhibition of this innate pathway was confirmed with HBsAg subviral particles.

How to improve access to therapy in hepatitis B patients.

Despite the availability of a preventive vaccine and active antiviral treatments that stop disease progression and reduce the risk of hepatocellular carcinoma, hepatitis B is still a major public health problem. Only an estimated 10% of the 257 million people living with HBV have been diagnosed and as few as 1% are being adequately treated. Barriers to diagnosis and treatment include: (i) limited awareness and lack of knowledge about HBV infection and HBV-related diseases; (ii) under-diagnosis with insufficient screening and referral to care; (iii) limited treatment due to drug availability, costs, reimbursement policies and the need for long-term or life-long therapy. These barriers and the actions needed to improve access to treatment are strongly influenced by the prevalence of infection and affect middle-high vs low-middle income countries differently, where most HBV carriers are found. In high-prevalence regions and low-to middle-income countries, the main challenges are availability and cost while in low-prevalence regions and middle-to high-income countries low screening rates, public awareness, social stigma and discrimination play an important role. Overcoming these challenges on a global scale is a complex clinical and public health challenge and multilateral commitment from pharmaceutical companies, governments, funders and the research community is lacking. The new WHO 2016 Global Health Sector Strategy on viral hepatitis targets testing and treatment, suggesting that important but strong actions are needed from advocacy groups, scientific societies and funding agencies to foster awareness and access to cure.

[Predictors of liver fibrosis in patients with non-alcoholic fatty liver disease. The role of metabolic syndrome, insulin-resistance and inflammation]. / Predittori di fibrosi epatica in pazienti con steatosi epatica non-alcolica. Il ruolo della sindrome metabolica, dell'insulino-resistenza e dell'infiammazione.

INTRODUCTION: NAFLD (non-alcoholic fatty liver disease) reaches an high prevalence in the general population, and it is closely related to metabolic syndrome (MetS). The entity of metabolic abnormalities and the chronic inflammation seem to play a main role in the development of liver fibrosis. The aim of our study is to determine whether subjects with NAFLD and MetS have higher liver fibrosis degree when compared with NAFLD subjects without MetS, and to investigate the relations between fibrosis, MetS and its single components and inflammation. MATERIALS AND METHODS: We considered 24 patients with NAFLD. Those who had viral- and alcohol- related liver disease were excluded. MetS was diagnosed according to NCEP ATP III criteria; inflammatory status was determined through C-reactive protein (PCR) assay. The peripheral insulin-resistance was assessed by calculating HOMA ir. Liver fibrosis was measured by transient elastography (Fibroscan®). RESULTS: Subjects with MetS had higher HOMA ir, PCR and Fibroscan® score (log value: 0.92±0.24 KPa vs 0.73±0.2 KPa; p=0.047). The linear correlation analysis showed that Fibroscan® score was related to MetS, number of MetS components, waist circumference, HOMA ir and PCR. However the multivariate regression analysis showed that only HOMA ir (B=0.077; 95%CI: -0.002- 0.157; p=0.05) and PCR (B=0.152; 95% CI: 0.006 - 0.299; p=0.006) were independent predictors of higher Fibroscan® score. CONCLUSION: MetS is associated to higher liver fibrosis degree in subjects with NAFLD. The insulin-resistance and inflammation seem to be the main determinants.