Cristina Musolino

Hepatitis B virus (HBV) induces the expression of interleukin-8 that in turn reduces HBV sensitivity to interferon-alpha

High levels of serum interleukin-8 (IL-8) have been detected in chronic hepatitis B (CHB) patients during episodes of hepatitis flares. We investigated whether hepatitis B virus (HBV) may directly induce IL-8 production and whether IL-8 may antagonize interferon-alpha (IFN-α) antiviral activity against HBV. We showed that CHB patients had significantly higher IL-8 levels both in serum and in liver tissue than controls. In HBV-replicating HepG2 cells, IL-8 transcription was significantly activated. AP-1, C/EBP and NF-kB transcription factors were concurrently necessary for maximum IL-8 induction. Moreover, HBx viral protein was recruited onto the IL-8 promoter and this was paralleled by IL8-bound histone hyperacetylation and by active recruitment of transcriptional coactivators. Inhibition of IL-8 increases the antiviral activity of IFN-α against HBV. Our results indicate that HBV activates IL-8 gene expression by targeting the epigenetic regulation of the IL-8 promoter and that IL-8 may contribute to reduce HBV sensitivity to IFN-α.

Hepatitis E virus infection as a cause of acute hepatitis in Southern Italy

BACKGROUND Hepatitis E virus (HEV) is a major cause of acute hepatitis in developing countries, whereas it is not considered a major health problem in Western World. AIMS To investigate the spread of HEV and its possible role in causing acute hepatitis in Southern Italy. METHODS Four hundred and thirty patients observed from April to December 2009 were studied and grouped as follows: 55 individuals with acute hepatitis (AH), 33 of whom cryptogenic; 321 individuals with chronic liver diseases (CLD), (278 Italians and 43 immigrants); 54 individuals without liver disease (control-group). Serum samples from all cases were tested for IgG anti-HEV antibodies and those positive to this test as well as all AH cases were also tested both for IgM anti-HEV and HEV RNA. RESULTS Two of 33 (6%) cryptogenic AH cases were associated with HEV infection as shown by positive IgM anti-HEV test. Both these patients had not travelled to areas at high HEV endemicity. HEV RNA was not found in any sample tested. IgG anti-HEV antibodies were detected in 5.7% of Italians with CLD and 3.7% of the control-group. No immigrant was found positive for any HEV marker. CONCLUSION Autochthonous HEV infection is present in Southern Italy where it may cause AH.

Monocytes and lymphocytes as active participants in the pathogenesis of experimental shock.

We investigated the role played by monocytes and lymphocytes in the pathogenesis of experimental shock. Splanchnic artery occlusion (SAO) shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min followed by reperfusion. Sham operated animals were used as controls. SAO shocked rats had a decreased survival time (80±11 min, while sham shocked rats survived more than 4 h), increased serum (248±21 U/ml) and macrophage (145±15 U/ml) levels of TNF-α, enhanced myeloperoxidase (MPO) activity in the ileum (3.38±0.2 U×10−3/g tissue), decreased number of monocytes, lymphocytes and neutrophils and a profound hypotension. In addition we found an increased expression of vascular cell adhesion molecule-1 (VCAM-1) on aortic endothelium and a reduced percentage of VLA-4 positive monocytes and lymphocytes. Inhibition of TNF-α synthesis, reversed the increased endothelial expression of VCAM-1, increased the percentage of integrin VLA-4 positive leukocytes and improved monocyte, lymphocyte and neutrophil count. Furthermore a passive immunization with specific antibodies raised against VCAM-1 (2 mg/kg, i.v. 3 h before SAO) increased survival, reduced MPO activity in the ileum (0.034±0.04 U×10−3/g tissue) and improved mean arterial blood pressure. Our data suggest that monocytes and lymphocytes participate in the pathogenesis of splanchnic ischaemia-reperfusion injury and may amplify the adhesion of neutrophils to peripheral tissues.

Lack of the NS5B S282T mutation in HCV isolates from liver tissue of treatment-naive patients with HCV genotype-1b infection.

BACKGROUND Despite the availability of several direct-acting antivirals (DAAs) specifically inhibiting different HCV proteins, treatment of chronic HCV infection is still a challenge also because of the possible selection of resistant viral variants under DAA therapy. Indeed, only the emergence of viruses resistant to the nucleoside inhibitors of the HCV NS5B polymerase (Pol) has not yet been reported, in spite of the fact that in vitro studies have clearly shown that an S282T amino acid change in the Pol protein may confer resistance to these drugs. On the basis of a previous study showing that viral variants resistant to HCV protease inhibitors are largely present in the liver - but not in the serum - of untreated patients, we investigated the possible natural occurrence of viral populations with the S282T change in the Pol protein, analysing viral isolates from liver and serum of HCV genotype-1b treatment-naive patients. METHODS HCV-1b isolates from liver specimens and serum samples of 10 chronic hepatitis C patients were analysed by cloning and sequencing. RESULTS The S282T mutation was not found in any of the viral isolates from either liver or serum samples of all the cases, although an S282G mutation of unknown virological/clinical relevance was detected in 2/19 liver isolates from one patient. CONCLUSIONS Our study confirms that the natural selection of the S282T mutation is a rare event, thus explaining the lack of emergence and takeover of these variants under drug pressure.

Risk of occult hepatitis B virus infection reactivation in patients with solid tumours undergoing chemotherapy

BACKGROUND Hepatitis B virus reactivation may occur in occult-infected carriers with haematological malignancies, whereas little data are available in patients undergoing chemotherapy for solid tumours. AIMS Evaluation of cancer patients undergoing chemotherapy to investigate occult hepatitis B virus infection and its clinical-virological outcome. METHODS Forty-four patients with solid tumours and without liver disease were prospectively enrolled and sampled before starting chemotherapy and between the second and third chemotherapy cycles (time points 1 and 2, respectively); 24 were also sampled 6 months after the end of chemotherapy (time point 3). At each time point, subjects were tested for liver biochemistry, hepatitis B serology and occult infection. RESULTS No sample tested positive for virus surface antigen. Twelve subjects (27.3%) were antibody positive to hepatitis B virus. Overall, occult infection was detected in 4 cases (9%), with positive HBV DNA at time points 1 and 2 (one case), at time point 1 only (one case), only at time points 2 and 3 (two cases), respectively. No occult-infected carrier experienced liver biochemistry flares and/or viral surface antigen positivity. CONCLUSIONS Occult hepatitis B virus infection may occur in subjects with solid tumours, although the risk of its reactivation under chemotherapy appears to be very low.

NS3 genetic variability in HCV genotype-1b isolates from liver specimens and blood samples of treatment-naive patients with chronic hepatitis C.

BACKGROUND Two distinct inhibitors of the HCV protease have been approved for the treatment of patients infected with HCV genotype-1. These drugs are highly efficient in suppressing HCV replication; however, their use is limited by the emergence of viral mutants resistant to them after a very short time of treatment. By analysing blood samples, it was shown that viral strains resistant to protease inhibitors (PIs) may exist prior to treatment. The aim of this study was to investigate the presence of viral variants resistant to PIs in isolates from liver and blood of HCV patients naive to any antiviral therapy. METHODS Liver and blood HCV genotype-1b isolates from 10 patients with chronic hepatitis were analysed by cloning and sequencing procedures. RESULTS The analyses of 10–15 clones from liver isolates of each patient showed that 7/10 cases had single or multiple mutations potentially conferring resistance to PIs. However, the analysis of the corresponding blood samples excluded the presence of these mutations in all cases but one, which had the Q80R mutation in all clones from both liver and plasma samples. No PI-resistant variants were detected in isolates from either liver or plasma samples of three patients. CONCLUSIONS Naturally occurring HCV variants resistant to PIs are commonly present at the intrahepatic level and this clearly explains their usual, very early emergence under treatment; however, the identification of these variants as circulating viral populations is not unusual in untreated patients.

Flavocoxid exerts a potent antiviral effect against hepatitis B virus

IntroductionFlavocoxid is a proprietary blend of two flavonoids, baicalin and catechin, and recent evidence has shown that bioflavonoids may exert antiviral activities. The potential antiviral activity of Flavocoxid against hepatitis B virus (HBV) was evaluated. Additionally, it was investigated if Flavocoxid used in combination with Entecavir could potentiate its anti-HBV activity.Materials and methods Hepatoma cells replicating HBV were treated with Flavocoxid, or Entecavir alone or in combination for up to 5 days. Viral replicative intermediates, transcripts, and cccDNA levels were evaluated in HBV-replicating cells by real-time PCR, Southern and Northern blotting. Expression profiling was performed using TaqMan low-density arrays.ResultsFlavocoxid treatment induced a reduction of HBV replicative intermediates, the amount of transcripts, and HBsAg levels. Flavocoxid and Entecavir combination therapy further decreased the amount of HBV replicative intermediates, compared to Flavocoxid alone. Importantly, Flavocoxid alone or in combination with Entecavir also induced a reduction of cccDNA. Gene-expression analysis showed that Flavocoxid activates type I IFNs-signaling and dampens the HBV-induced inflammatory response.ConclusionsFlavocoxid inhibits HBV replication by targeting multiple steps of viral life cycle. These results indicate that the antiviral activity of Entecavir is potentiated by Flavocoxid, suggesting that this medical food might be considered as an adjuvant for anti-HBV therapy.

OC-29 Antiviral activity of flavonoid-derived compounds against hepatitis B virus

Background and Aims: The selective oral TLR7 agonist GS-9620 is being developed for the treatment of chronic viral hepatitis by safely inducing long-term immune control after finite therapy. The mechanism of GS-9620-mediated anti-viral effects involves secretion of type I interferons, e.g. IFN-a. The goal of GS-9620 therapy is to induce a local hepatic anti-viral response in the absence of systemic IFN-a to avoid interferon-mediated adverse effects. In this in vivo study we investigated whether a low oral dose of GS-9620 can induce a hepatic anti-viral gene expression signature in the absence of detectable serum levels of IFN-a. Methods: Nine weeks old male CD-1 mice received vehicle or a single dose of 0.3mg/kg GS-9620 via oral gavage and were sacrificed at 2, 4, 8, 12, and 24h post-dose (n =5 per time point). Serum levels of IFN-a were assessed together with the liver expression of antiviral genes MX1, OAS1, and ISG15. Results: GS-9620 induced transient expression of hepatic OAS1, MX1, and ISG15 in all treated animals. Undetectable serum IFN-a (<8pg/ml) was observed in 2/5 to 5/5 animals at each terminal time point. At 2 to 12h post-dose, all animals with undetectable serum IFN-a had concurrent two-fold or higher elevation of the hepatic expression of OAS1, MX1, or ISG15 (Figure 1). At 24h, the hepatic anti-viral gene expression was comparable to vehicle-treated and naive control animals (not shown). Conclusions: Low dose (0.3mg/kg) treatment of mice with GS-9620 induced hepatic expression of MX1, OAS1, and ISG15 in animals with undetectable IFN-a in serum. These results suggest that a low oral dose of selective TLR7 agonist can achieve immune activation in the liver in the absence of high systemic levels of IFN-a.

NS3 Variability in Hepatitis C Virus Genotype 1A Isolates from Liver Tissue and Serum Samples of Treatment-Naïve Patients with Chronic Hepatitis C

Background: Hepatitis C virus (HCV) NS3 resistance-associated substitutions (RASs) reduce HCV susceptibility to protease inhibitors. Little is known about NS3 RASs in viral isolates from the liver of chronic hepatitis C (CHC) patients infected with HCV genotype-1a (G1a). Aim: The objective of this work was to study NS3 variability in isolates from the serum and liver of HCV-G1a-infected patients naïve to direct-acting antivirals (DAAs). Methods: NS3 variability of HCV-G1a isolates from the serum and liver of 11 naïve CHC patients, and from sera of an additional 20 naïve CHC patients, was investigated by next-generation sequencing. Results: At a cutoff of 1%, NS3 RASs were detected in all the samples examined. At a cutoff of 15%, they were found in 54.5% (6/11) and 27.3% (3/11) of the paired liver and serum samples, respectively, and in 22.5% (7/31) of the overall serum samples examined. Twenty-six out of thirty-one (84%) patients showed NS3 variants with multiple RASs. Phylogenetic analysis showed that NS3 sequences clustered within 2 clades, with 10/31 (32.2%) patients infected by clade I, 15/31 (48.8%) by clade II, and 6/31 (19.3%) by both clades. Conclusions: Though the number of patients examined was limited, NS3 variants with RASs appear to be major components of both intrahepatic and circulating viral quasispecies populations in DAA-naïve patients.