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Sandalova E.,Singapore Institute for Clinical science | Laccabue D.,Unit of Infectious Diseases and Hepatology | Boni C.,Unit of Infectious Diseases and Hepatology | Watanabe T.,Nagoya City University | And 5 more authors.
Gastroenterology | Year: 2012

Background & Aims: During viral infection, the activities of virus-specific CD8 + T cells are carefully regulated to prevent severe damage of the infected organs. We investigated the mechanisms that control the functions of activated T cells. Methods: We measured the size of the population of activated and proliferating CD8 + T cells and the functional pattern of CD8 + T cells specific for the entire hepatitis B virus proteome and for selected heterologous virus (Epstein-Barr virus, human cytomegalovirus, and influenza virus) using blood samples from 18 patients with acute hepatitis B. We analyzed the effects of different modulatory mechanisms, such as inhibitory molecules, suppressive cytokines (interleukin-10), and arginase, on the activities of CD8 + T cells. Results: In patients with acute hepatitis B, the expansion of activated and proliferating (HLA-DR/CD38 +, Ki-67 +/Bcl-2 low) CD8 + T cells did not quantitatively match their specific functions ex vivo; virus-specific CD8 + T cells had functional impairments that were temporally restricted to the acute phase of viral hepatitis. These impairments in function were not limited to HBV-specific CD8 + T cells but were also observed in CD8 + T cells with specificities for other viruses. We investigated possible causes of antigen-independent CD8 + T cell inhibition and found that the increased levels of arginase observed in patients with acute hepatitis could suppress the function of activated, but not resting, CD8 + T cells. Conclusions: The increased level of arginase in patients with acute hepatitis B suppresses the functions of activated CD8 + T cells. This mechanism might limit the amount of liver damage caused by activated CD8 + T cells in patients with acute HBV infection. © 2012 AGA Institute.


Lampertico P.,University of Milan | Di Costanzo G.G.,Liver Unit | Sagnelli E.,Infectious Disease Unit | Fasano M.,University of Bari | And 11 more authors.
Gut | Year: 2013

Objective: Treatment with peginterferon α-2a (PegIFN) for 48 weeks is the standard of care for selected HBeAg-negative patients chronically infected with hepatitis B virus (HBV), but with limited treatment efficacy. A study was undertaken to investigate whether treatment extension to 96 weeks improves the outcome in this patient population. Methods: 128 HBeAg-negative patients (120 genotype D) were randomised to weekly 180 mg PegIFN for 48 weeks (group A, n=51), 180 μg PegIFN for 48 weeks followed by 135 mg weekly for an additional 48 weeks (group B, n=52) or 180 μg PegIFN plus lamivudine (100 mg/day) for 48 weeks then 135 μg PegIFN for 48 weeks (group C, n=25). Endpoints were alanine aminotransferase normalisation plus HBV DNA <3400 IU/ml (primary), HBV DNA <2000 IU/ml and HBsAg clearance at 48 weeks after treatment. Results: Forty-eight weeks after treatment, six patients in group A and 13 in group B achieved alanine aminotransferase normalisation plus HBV DNA <3400 IU/ml (11.8% vs 25.0%, p=0.08), 6 vs 15 patients had HBV DNA <2000 IU/ml (11.8% vs 28.8%, p=0.03), 0 vs 3 achieved HBsAg clearance (0% vs 5.8%, p=0.24) and 0 vs 5 had HBsAg <10 IU/ml (0% vs 9.6%, p=0.06). While extended PegIFN treatment was the strongest independent predictor of response, the combination with lamivudine did not improve responses. Discontinuation rates were similar among the groups (19.6%, 23.1%, 32.0%, p=0.81) and were mostly due to PegIFN-related adverse events. Conclusions: In HBeAg-negative genotype D patients with chronic hepatitis B, PegIFN treatment for 96 weeks was well tolerated and the post-treatment virological response improved significantly compared with 48 weeks of treatment. Trial registration number http://ClinicalTrials.gov registration number: NCT01095835.


Mangia A.,IRCCS Casa Sollievo della Sofferenza Hospital | Santoro R.,IRCCS Casa Sollievo della Sofferenza Hospital | Copetti M.,IRCCS Casa Sollievo della Sofferenza Hospital | Massari M.,Infectious Disease Division IRCCS Hospital Reggio Emilia | And 11 more authors.
Journal of Hepatology | Year: 2013

Background & Aims The lack of consensus on the optimal timing, regimen, and duration of treatment, in patients with acute HCV infection, stimulates the research on both favourable outcome predictors and individualized treatment regimens. This study aimed at investigating the impact of IL28B SNP rs12979860 alone or in combination with HLA class II alleles in both predicting spontaneous viral clearance and individualizing treatment strategies for patients with HCV persistence, after acute HCV exposure. Methods 178 patients with AHC, consecutively treated with interferon alone or in combination with ribavirin, starting within or after 48 weeks from the diagnosis of AHC, were tested for IL28B SNPs and HLA class II alleles. Results Spontaneous viral clearance was achieved in 28% of 169 patients available for genetic testing. Factors associated with HCV elimination were jaundice (OR 2.75, 95% CI 1.31-5.77) and IL28B CC (OR 3.87, CI 1.71-8.51), but not HLA alleles. In CT/TT patients without jaundice, NPV for virus persistence was 98%. In patients with IL28B CT/TT, starting treatment 48 weeks after the onset was significantly associated with lower rates of response (28% vs. 100%, p = 0.027). By contrast, no significant differences in the rate of SVR were observed for CC carriers who started treatment later (65% vs. 85%, p = 1.0). Conclusions In patients with acute HCV hepatitis, lack of viral clearance may be predicted by absence of jaundice and IL28B CT/TT genotype; in patients with these characteristics, treatment needs to be started immediately. © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.


Bertoletti A.,Agency for Science, Technology and Research Singapore | Bertoletti A.,National University of Singapore | Maini M.K.,University College London | Ferrari C.,Unit of Infectious Diseases and Hepatology
Antiviral Therapy | Year: 2010

HBV is a hepatotropic and non-cytopathic virus that causes more than one million deaths annually from liver cirrhosis and hepatocellular carcinoma. As the virus itself is non-cytopathic, it is widely accepted that both viral control and liver pathology are mediated by the host immune system. Until recently, the focus has been on the crucial role of adaptive immune responses in controlling HBV infection, but the potential contribution of the innate system is now an important area of controversy. Unanswered questions include whether and when HBV can trigger components of innate immunity, and whether HBV can actively suppress the induction of innate immunity. We discuss the data available from animal models and human HBV infection addressing the role of innate immunity in the first part of this review. In the second part, we address the immunopathogenesis of the inflammatory events that characterize chronic hepatitis B. The mechanisms thought to be responsible for liver inflammation, namely the intrahepatic recruitment of inflammatory cells, which is orchestrated by chemokines, have been described; however, the underlying immunological triggers are much less clear. The prevailing idea is that liver inflammation results from a recovery of HBV-specific T-cells directly causing liver injury, but this scenario is supported by scanty experimental data. By contrast, recent findings raise the possibility of a contribution from innate components, such as natural killer cells. ©2010 International Medical Press.


Sandalova E.,Singapore Institute for Clinical science | Laccabue D.,Unit of Infectious Diseases and Hepatology | Boni C.,Unit of Infectious Diseases and Hepatology | Tan A.T.,Singapore Institute for Clinical science | And 7 more authors.
PLoS Pathogens | Year: 2010

Herpesviruses infect most humans. Their infections can be associated with pathological conditions and significant changes in T cell repertoire but evidences of symbiotic effects of herpesvirus latency have never been demonstrated. We tested the hypothesis that HCMV and EBV-specific CD8 T cells contribute to the heterologous anti-viral immune response. Volume of activated/proliferating virus-specific and total CD8 T cells was evaluated in 50 patients with acute viral infections: 20 with HBV, 12 with Dengue, 12 with Influenza, 3 with Adenovirus infection and 3 with fevers of unknown etiology. Virus-specific (EBV, HCMV, Influenza) pentamer+ and total CD8 T cells were analyzed for activation (CD38/HLA-DR), proliferation (Ki- 7/Bcl- 2low) and cytokine production. We observed that all acute viral infections trigger an expansion of activated/proliferating CD8 T cells, which differs in size depending on the infection but is invariably inflated by CD8 T cells specific for persistent herpesviruses (HCMV/EBV). CD8 T cells specific for other non-related non persistent viral infection (i.e. Influenza) were not activated. IL-15, which is produced during acute viral infections, is the likely contributing mechanism driving the selective activation of herpesvirus specific CD8 T cells. In addition we were able to show that herpesvirus specific CD8 T cells displayed an increased ability to produce the anti-viral cytokine interferon-c during the acute phase of heterologous viral infection. Taken together, these data demonstrated that activated herpesvirus specific CD8 T cells inflate the activated/ proliferating CD8 T cells population present during acute viral infections in human and can contribute to the heterologous anti-viral T cell response. © 2010 Sandalova et al.


Zerbini A.,Unit of Infectious Diseases and Hepatology | Pilli M.,Unit of Infectious Diseases and Hepatology | Laccabue D.,Unit of Infectious Diseases and Hepatology | Pelosi G.,Unit of Infectious Diseases and Hepatology | And 7 more authors.
Gastroenterology | Year: 2010

Background & Aims: Radiofrequency thermal ablation (RFA) is a minimally invasive technique used as standard local therapy of hepatocellular carcinoma and second-line treatment for metastatic liver tumors. Studies in preclinical models and in patients have shown that thermal destruction of tumor tissue can enhance anti-tumor cellular responses, but our knowledge of its impact on natural killer (NK) cells is still very limited. Methods: Thirty-seven patients undergoing RFA for hepatocellular carcinoma were studied for peripheral blood lymphocytes counts followed by phenotypic and functional characterization of NK-cell population. Results: Peripheral blood lymphocytes kinetics revealed an increased frequency and absolute number of NK cells expressing higher levels of activatory along with reduced levels of inhibitory NK receptors, and increased functional NK-cell activity. A prevalent expansion of the CD3-CD56dim NK subset was observed compared to the CD3-CD56bright counterpart. Interferon-γ production, anti-K562 cell cytotoxicity, and antibody-dependent cell cytotoxicity, appeared consistently increased in terms of both absolute activity and killing efficiency at 4 weeks after RFA, as compared to baseline. Interestingly, when recurrence-free survival was assessed in 2 groups of patients separated according to higher vs lower enhancement of cytotoxicity and/or interferon-γ production, a significant difference was observed, thus suggesting a potential predictive role of NK functional assays on efficacy of RFA. Conclusions: RFA can lead to stimulation of NK cells with a more differentiated and proactivatory phenotypic profile with general increase of functional activities. This observation may be relevant for development of adjuvant immunotherapeutic strategies aimed at enhancing NK-cell responses against primary and metastatic liver tumors. © 2010 AGA Institute.


Alisi A.,Confocal Microscopy Facility of Bambino Gesu Childrens Hospital | Arciello M.,University of Rome La Sapienza | Arciello M.,University of L'Aquila | Petrini S.,Confocal Microscopy Facility of Bambino Gesu Childrens Hospital | And 5 more authors.
PLoS ONE | Year: 2012

Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs. © 2012 Alisi et al.


Chiu M.,University of Parma | Tardito S.,University of Parma | Pillozzi S.,University of Florence | Arcangeli A.,University of Florence | And 11 more authors.
British journal of cancer | Year: 2014

A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO). Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1. Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro. The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.


Penna A.,Laboratory of Viral Immunopathology | Laccabue D.,Laboratory of Viral Immunopathology | Libri I.,Laboratory of Viral Immunopathology | Giuberti T.,Unit of Infectious Diseases and Hepatology | And 10 more authors.
Journal of Hepatology | Year: 2012

Background & Aims: The effect of IFN-α therapy on HBV-specific T-cell responses in HBeAg-negative, genotype D, chronic hepatitis B is largely undefined. Understanding to what extent IFN-α can modulate HBV-specific T-cells is important to define strategies to optimize IFN efficacy and to identify immunological parameters to predict response to therapy. Methods: HBV-specific T-cell responses were analyzed longitudinally ex vivo and after expansion in vitro in 15 patients with genotype D, HBeAg-negative chronic hepatitis B treated with peginterferon-α-2a. HBV proteins and synthetic peptides were used to stimulate T-cell responses. Analysis of the CD4 and CD8 T-cell functions was performed by ELISPOT, intracellular cytokine and tetramer staining. The effect of anti-PD-L1 on T-cell functions was also analyzed. Results: Ex vivo IFN-γ production by total HBV-specific T-cells was significantly greater before therapy in patients who showed HBV DNA <50 IU/ml at weeks 24 and/or 48 of therapy. No significant improvement of T-cell proliferation, Th1 cytokine production and cytotoxicity was observed during IFN therapy by both ex vivo and in vitro analysis. PD-1/PD-L1 blockade showed a modest improvement of cytokine production in a total of 15% of T-cell lines. Conclusions: IFN-α did not improve peripheral blood HBV-specific T-cell responses in the first 24 weeks of treatment, consistent either with a predominant antiviral/antiproliferative effect or with an immunomodulatory activity on other arms of the immune system which were not analyzed in our study. A better pre-treatment ex vivo IFN-γ production was associated with better chances to control HBV replication during therapy and represents a promising predictor of IFN efficacy. © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.


PubMed | University of Navarra, Unit of Infectious Diseases and Hepatology, Institute Pasteur du Maroc and IdiSNA
Type: Journal Article | Journal: Journal of medical virology | Year: 2016

Viral clearance during acute hepatitis C virus (HCV) infection is associated with the induction of potent antiviral T-cell responses. Since dendritic cells (DC) are essential in the activation of primary T-cell responses, gene expression was analyzed in DC from patients during acute HCV infection. By using microarrays, gene expression was compared in resting and activated peripheral blood plasmacytoid (pDC) and myeloid (mDC) DC from acute HCV resolving patients (AR) and from patients who become chronically infected (ANR), as well as in healthy individuals (CTRL) and chronically-infected patients (CHR). For pDC, a high number of upregulated genes was found in AR patients, irrespective of DC stimulation. However, for mDC, most evident differences were detected after DC stimulation, again corresponding to upregulated genes in AR patients. Divergent behavior of ANR was also observed when analyzing DC from CTRL and CHR, with ANR patients clustering again apart from these groups. These differences corresponded to metabolism-associated genes and genes belonging to pathways relevant for DC activation and cytokine responses. Thus, upregulation of relevant genes in DC during acute HCV infection may determine viral clearance, suggesting that dysfunctional DC may be responsible for the lack of efficient T-cell responses which lead to chronic HCV infection.

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