The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.

OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

A retrospective analysis of 1.011 percutaneous liver biopsies performed in patients with liver transplantation or liver disease: ultrasonography can reduce complications?

OBJECTIVE: In the last decades, liver biopsy was the reference procedure for the diagnosis and follow-up of liver disease. Aim of present retrospective analysis was to assess the prevalence of complications and risk factors after Percutaneous Liver Biopsy (PLB) performed for diagnosis and staging in patients with chronic liver disease and for monitoring the graft in liver transplanted patients PATIENTS AND METHODS: Data were collected from a total of 1.011 PLB performed with the Menghini technique between January 2004 and December 2014 at the Hepatology and Transplant Units of the University of Rome Tor Vergata. The indications for biopsy were: follow-up of liver transplantation, chronic Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV), with or without Human Immunodeficiency Virus (HIV) and alcohol-related liver disease. Our patients were divided into two groups according to the biopsy indication: follow-up of liver transplantation (Group A) and chronic liver disease (Group B). All the procedures were performed in Day Hospital regimen. After the biopsy, patients remained in bed for about 4-6 hours. In the absence of complications, they were then discharged on the same day. RESULTS: The most frequent complication after biopsy was pain (Group A n. 57, 8.8%; Group B n. 105, 29.0%), hypotension as a result of a vasovagal reaction resolved spontaneously (Group A n. 7, 1.1%; Group B n. 6, 1.7%), and intrahepatic bleeding resolved with conservative therapy (Group A n. 1, 0.2%; Group B n. 6, 1.7%). Two cases of pneumothorax in the Group A (0.3%) were treated with a chest tube. Other complications did not have a significant impact. Also, we did not observe statistically significant differences in patients who underwent PLB without and with ultrasound guidance. CONCLUSIONS: Liver biopsy is not a replaceable tool in diagnosis and follow-up of several chronic liver diseases. The Menghini technique with the percutaneous trans costal approach, might be preferred because less traumatic and related with a low occurrence of minor and major complications. According to our case load and comparing our findings with the previous published data, we speculate that ultrasound guidance is not crucial in the prevention of major complications.

HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels.

OBJECTIVES: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen. METHODS: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays. RESULTS: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11). CONCLUSIONS: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

Identification and characterisation of local aerosol sources using high temporal resolution measurements.

Aerosol and gaseous pollution measurements were carried out at an urban background site in the south of Italy located near an industrial complex. Collection of 24 h samples of PM10 and PM2.5 and successive chemical quantification of metals were performed. Data were compared with measurements taken at a suburban background site, located at 25 km distance. The comparison showed the presence of an industrial contribution with a well defined chemical emission profile, similar, in terms of metals content, to urban emissions. As this made difficult the quantitative characterisation of the contribution of the two sources to atmospheric PM, a statistical method based on the treatment of data arising from high temporal resolution measurements was developed. Data were taken with a micrometeorological station based on an integrating nephelometer (Mie pDR-1200) for optical detection of PM2.5 concentration, with successive evaluation of vertical turbulent fluxes using the eddy-correlation method. Results show that the contribution from the two sources (urban emissions and industrial releases) have a very different behaviour, with the industrial contribution being present at high wind velocity with short concentration peaks (average duration 4 min) associated to strong positive and negative vertical fluxes. The estimated contribution to PM2.5 is 2.3% over long-term averages. The urban emissions are mainly present at low wind velocity, with longer concentration peaks in the morning and late evening hours, generally associated to small positive vertical fluxes. The characterisation of the contribution was performed using deposition velocity V(d) that is on average -3.5 mm s(-1) and has a diurnal pattern, with negligible values during the night and a minimum value of around -9 mm s(-1) late in the afternoon. Results show a correlation between V(d), friction velocity and wind velocity that could be the basis for a parameterisation of V(d) to be used in dispersion codes.

Clinical trial: low plasma cholesterol and oxidative stress predict rapid virological response to standard therapy with peginterferon and ribavirin in HCV patients.

BACKGROUND: Rapid virological response (RVR) is the best predictor of sustained response to standard HCV treatment. AIM: To evaluate predictive factors of RVR. METHODS: Sixty-five patients (mean age 52.6 +/- 13.8; 37 genotype-1, and 28 genotypes-2/3) were consecutively treated with pegIFN-alpha 2a or 2b once weekly plus daily ribavirin based on body weight for 24 or 48 weeks, according to genotype. RVR was defined as undetectable HCV-RNA at week 4. RESULTS: Twenty-seven percent of patients achieved RVR in genotypes 1 and 60.7% in genotypes 2/3 (P < 0.01). Rapid responders had higher mean serum baseline total and LDL-cholesterol levels (P < 0.01). RVR was 20.0% in the bottom tertile of total cholesterol and 63.6% in the top tertile (P < 0.01). HCV-RNA levels at week 4 were positively correlated with baseline serum insulin (P < 0.01), HOMA-IR (P < 0.01), body mass index (P < 0.05) and number of components of metabolic syndrome (P < 0.01) and negatively correlated with cholesterol levels (P < 0.05). At multivariate analysis, age, LDL-cholesterol, HCV genotype and serum 8-iso-PGF 2 alpha, a marker of oxidative stress, were independent predictors of RVR. CONCLUSIONS: Our prospective study supports a role of low serum total and LDL-cholesterol and of oxidative stress as positive independent predictive factors of poor RVR in HCV patients.

Recurrent myocardial ischaemia during combination antiviral therapy in a patient with chronic hepatitis C and normal aminotransferase levels.

A 63-year-old man with chronic hepatitis C and persistently normal alanine aminotransferase levels was treated with peginterferon alpha-2a (180 microg weekly) and ribavirin (800 mg daily). Hepatitis C virus-ribonucleic acid was negative at week 4. After 12 weeks of therapy, haemoglobin levels had decreased by 3.5mg/dL, and he developed a syncope episode with electrocardiographic signs of myocardial ischaemia. Antiviral treatment was stopped and the ischaemia-like electrocardiographic changes resolved completely within 2 months. Eight months later, because of the previous rapid virological response and patient motivation, he was treated again with peginterferon and ribavirin. Baseline and weekly electrocardiographic recordings were obtained during treatment. At week 4 hepatitis C virus-ribonucleic acid was negative. At week 8, when the haemoglobin levels had decreased by 3.4 mg/dL, the patient developed the same ischaemia-like changes that occurred during the previous treatment. Antiviral therapy was stopped and the electrocardiographic ischaemia-like changes disappeared after 1 month. The patient neither had a history of previous cardiovascular diseases, nor evidence of current disease at myocardial scintigraphy. However, a coronary microvessel spasm, possibly related to drug-toxicity and/or anaemia could not be excluded. This case indicates the need of strict electrocardiographic monitoring in elderly patients undergoing treatment with peginterferon and ribavirin.

Insulin resistance, the metabolic syndrome, and nonalcoholic fatty liver disease.

BACKGROUND/AIMS: An association of nonalcoholic fatty liver disease with the insulin-resistant metabolic syndrome has been suggested. The aim of the study was to assess the association of fatty liver to different degrees of insulin resistance and secretion. METHODS AND RESULTS: The study was performed in 308 alcohol- and virus-negative consecutive patients attending a metabolic clinic, who underwent a complete clinical and biochemical work-up including oral glucose tolerance test and routine liver ultrasonography. Steatosis was graded as absent/mild, moderate, and severe. In nondiabetic subjects, a progressive (P < 0.05) increase in mean homeostasis model of insulin resistance was recorded from the group without steatosis to the groups with mild/moderate and severe steatosis. Severe steatosis was associated with the clustering of the five clinical and biochemical features proposed for the clinical diagnosis of the metabolic syndrome. Subjects with the metabolic syndrome with a more pronounced insulin resistance had a higher prevalence of severe steatosis (P < 0.01) compared with those with homeostasis model of insulin resistance below the median. CONCLUSIONS: The findings stress the heterogeneous presentation of patients with the metabolic syndrome when the diagnosis is based on the broad Adult Treatment Panel III clinical criteria and demonstrate that those who are more insulin resistant have a higher prevalence of severe steatosis.

A randomized controlled trial of amantadine plus interferon-alpha2a vs. interferon-alpha2a alone in naive patients with chronic hepatitis C randomized according to the early virological response to interferon-alpha2a monotherapy.

BACKGROUND: An early virological response to interferon-alpha treatment is a strong predictor of sustained response, but it has never been exploited to stratify patients in clinical trials. AIM: To evaluate the efficacy of amantadine plus interferon-alpha compared with interferon-alpha alone in naive patients with chronic hepatitis C who were randomized on the basis of the early virological response to interferon-alpha. METHODS: One hundred and eighty-one patients received recombinant interferon-alpha2a (3 MU three times weekly) for 2 months and 164 were evaluated for early (i.e. month 2) virological response. Hepatitis C virus (HCV) RNA-negative patients (n = 66) were randomized to receive 3 MU of interferon-alpha three times weekly, with or without amantadine (200 mg/day); HCV RNA-positive patients (n = 98) were randomized to receive 6 MU of interferon-alpha three times weekly, with or without amantadine (200 mg/day). HCV RNA-positive patients at 6 months discontinued treatment, and all others completed 12 months. RESULTS: At month 6, HCV RNA-negative patients made up 54.2% of the interferon + amantadine group and 42.0% of the monotherapy group (P = 0.07). At month 12, HCV RNA-negative patients made up 38.5% of the interferon + amantadine group and 28.4% of the monotherapy group (N.S.). The sustained virological response rates were 21.6% and 20.9%, respectively (N.S.). CONCLUSION: The addition of amantadine does not enhance the sustained virological response to interferon-alpha in naive patients with chronic hepatitis C; however, an additive effect of amantadine occurs in the first 6 months, mainly in patients without an early response to monotherapy. Early response to interferon-alpha is a strong predictor of sustained virological response.

Treatment with ribavirin and interferon-alpha reduces interferon-gamma expression in patients with chronic hepatitis C.

Recent studies in vitro and in animals have suggested that ribavirin may potentiate the antihepatitis C virus (HCV) activity of interferon-alpha (IFN-alpha) by up-modulating the production of T cell-derived cytokines, such as interleukin (IL)-2 and IFN-gamma, which play a key role in the cellular immune response against HCV. To study the immune-modulatory mechanisms of ribavirin further, cytokine production by activated T cells and circulating cytokine levels were studied by FACS analysis and ELISA testing in 25 patients with chronic hepatitis C unresponsive to IFN-alpha, before and after treatment with either ribavirin plus IFN-alpha or IFN-alpha alone. After 16 weeks of treatment, both the expression of IFN-gamma by activated T cells and the blood levels of IFN-gamma, were significantly reduced with respect to pretreatment values in patients treated with ribavirin and IFN-alpha but not in those undergoing treatment with IFN-alpha alone. The expression of IFN-gamma was significantly lower in patients that gained normal ALT levels with respect to those that did not. No modification of the expression of IL-2, IL-4 and IL-10 was found before and after treatment in either group of patients. In conclusion, the results of this study do not support up-modulation of IFN-gamma and IL-2 production as the mechanism by which ribavirin potentiates IFN-alpha anti HCV activity. In addition, our findings suggest that ribavirin may exert an anti-inflammatory effect and may help reducing IFN-gamma-driven T cell activation and liver damage.

Cholesterol levels in Italian school children: results of an opportunistic survey.

BACKGROUND AND AIMS: A high prevalence of elevated serum total cholesterol (TC) levels has been described among Italian children. Universal screenings for TC have been suggested, though present recommendations are in favour of more selective and opportunistic surveys. Aim of the study was to assess TC distribution among 13-year-old schoolchildren in Central Italy. Further aim was to evaluate the indications for universal, selective or opportunistic screenings for TC. METHODS AND RESULTS: Children were examined opportunistically within a permanent screening programme for Mediterranean anemia carried out in the Lazio Region. TC was measured by dry chemistry in 3734 boys and 3644 girls aged 13 years from 77 schools in 37 municipalities. Results were grouped for 8 geographically and demographically similar areas. Mean TC was 137.6 mg/dl in boys and 144.3 mg/dl in girls. Desirable levels (< 170 mg/dl) were observed in about 90% of the children and fewer than 2% displayed levels indicative of genetic hypercholesterolemia (> 200 mg/dl). However, in two areas mainly populated by descendants from Northern Italy mean TC was remarkably high (158.7 and 152.2 mg/dl in boys and 164.5 and 160.0 in girls) and the percentage with desirable levels dropped to 68.6% and 74.7%. CONCLUSIONS: Our results show average good TC levels among Italian schoolchildren in Central Italy. This is probably due to their traditional Mediterranean diet. It can thus be suggested that only selective and/or opportunistic screenings for TC are indicated. However, in some areas where fewer children have desirable levels and almost 30% require dietary education, large-scale screenings are highly recommended.