Molecular hepatology

Field of Interest

Hepatitis C virus is a leading cause of chronic liver diseases, cirrhosis and hepatocellular carcinoma in the western world. Approximately 170-200 millions of people in the world and at least 1.5-2 millions in Italy are infected with hepatitis C virus (HCV).

The research interest of Alberti's group is focused on the pathogenesis and treatment of most common forms of chronic liver disease such as viral hepatitis B and C and non alcoholic fatty liver disease (NAFLD). The research program of the group is aim to define 1) the role of viral products and proteins in damage and in modulating the innate and acquired immune response; 2) host genetic factors associated with disease progression; 3) viral and host factors influencing the response to currently available and experimental antiviral therapies, with special interest in defining causes and consequences of the emergence of resistance virus mutants.

Other studies are dedicated to the development and clinical validation of new and innovative molecular tools for the diagnosis of viral hepatitis and for monitoring antiviral therapies.

Summary of recent research activities and of main results achieved

HEPATITIS C

CK2 dependent phosphorylation of the NS5A viral protein

We have proven that the NS5A protein of the hepatitis C virus (HCV) can be phosphorylated by CK2, a pleiotropic cellular kinase, involved in regulation of several cellular functions, including replication of many viruses. The number of potential CK2 phosphorylation sites was highly variable among different HCV genotypes and also within isolates of the same virus genotype. A correlation was found between CK2 phosphorylation sites and virological parameters of clinical relevance such as replication activity and sensitivity to antiviral therapy, leading to the hypothesis that heterogeneity in CK2 sites of NS5A may have pathogenetic and clinical implications. These studies were carried out in collaboration with Prof Pinna's group at the VIMM. (Dal Pero et al., J Hepatol. 2007 Dec;47(6):768-776).

Role of the Microsomal Triglyceride Transfer Protein (MTP) in liver steatosis

Figure 1: Localization of HCV Core protein to lipid droplets: Huh-7 cells were transfected with a plasmid expressing the capsid (Core) protein from hepatitis C virus genotype 1 or genotype 3. 1A) Merged image demonstrating colocalization of HCV core protein (green) and neutral lipids (red) as intracellular lipid droplets 1B) FITC localized genotype 3 core protein and 1C) genotype 1.

Figure 1: Localization of HCV Core protein to lipid droplets: Huh-7 cells were transfected with a plasmid expressing the capsid (Core) protein from hepatitis C virus genotype 1 or genotype 3. 1A) Merged image demonstrating colocalization of HCV core protein (green) and neutral lipids (red) as intracellular lipid droplets 1B) FITC localized genotype 3 core protein and 1C) genotype 1.
[click image to enlarge]

An important hallmark of chronic hepatitis C virus (HCV) infection is hepatic steatosis that may be seen also in the absence of any known steatogenic cofactors and may be extensive and severe. Hepatic steatosis in hepatitis C is of great clinical importance as it associates with more severe disease progression and poor response to antiviral therapy. We have been able to demonstrate that HCV is causing hepatic steatosis by direct interference with the microsomal triglyceride transfer protein (MTP) intracellular machinery. By measuring MTP gene expression and activity directly in liver biopsies of patients with HCV, we have proven that hepatic steatosis is associated with reduced MTP gene expression and activity, likely as consequence of direct interference by viral proteins. Indeed, the HCV core protein was shown to localize on lipid droplets (Fig. 1), in the presence of abnormal intracellular VLDL accumulation. These results led us to propose the hypothesis that liver steatosis in hepatitis C is a storage disease due to interference by the core protein with the assembly and secretion of VLDL. This is in agreement with results indicating that HCV has profound effect on intracellular lipid metabolism. In line with these findings and concepts, we were also able to demonstrate the influence of MTP genetic polymorphisms on the phenotypic expression of liver steatosis in HCV infected patients.
This project was in collaboration with the research groups of Prof. M.M. Hussain of the SUNY Downstate Medical Center Brooklyn, NY and of Dr Christian Datz of Obendorf Hospital, Salzburg, Austria (Mirandola et al., Gastroenterology, 2006;130:1661-1669). (Mirandola et al., Liver Intern., 2008)

Genetics and fibrosis progression

Progression of liver fibrosis and development of cirrhosis is highly heterogeneous and often unpredictable in hepatitis C. Although genetic factors are most likely involved, no specific genetic signature has been identified until recently. We have therefore focused our interest on a set of 7 single-nucleotide polymorphisms (SNPs) recently described by Investigators at Celera as possibly linked to liver cirrhosis. Our results, obtained in a large cohort of HCV infected patients followed prospectively for many years with sequential liver biopsies, clearly demonstrate that this seven gene variant signature, originally used to developed a Cirrhosis Risk Score (CRS), is indeed associated with more rapid fibrosis progression in patients infected with HCV. More recently, we have been able to further dissect the individual role of different SNPs and have identified some novel haplotypes in the TLR4 and STXBP5L genes that have a highly significant association with the clinical outcome of chronic hepatitis C. This project is carried out in collaboration with the research group of Celera, California, USA (Marcolongo et al., Hepatology, 2009)

Pathogenesis and clinical consequences of insulin resistance

Insulin resistance (IR) is a frequent features of chronic HCV infection, and it is suspected to be directly caused by HCV itself. IR associates with poor prognosis of liver damage and reduced response to interferon-alpha based therapy, but the underlying machanisms are still largely unknown. We have recently shown that the early decay of HCV replication during the first 24 hour after initiation of alpha interferon therapy is influenced by serum insulin levels, the higher the insulin levels, the lower the inhibition of virus replication by IFN (Bortoletto et al., J Viral Hepatitis 2009). These results led us to suggest direct interference by insulin on intracellular IFN signalling in hepatocytes, and this has been most recently confirmed by experiments conducted in hepatic cell lines co-stimulated with insulin and interferon alpha (Franceschini et al., J Hepatol 2009).

STANDARDISATION AND VALIDATION OF NEW NON INVASIVE MARKERS OF LIVER FIBROSIS

Staging of liver fibrosis is of paramount clinical importance in all etiological forms of chronic liver disease, and dictates prognosis and therapeutic interventions. Liver biopsy has been traditionally used , but this procedure is invasive and costly ; there is therefore increasing interest for the development of non-invasive methods. On this line, we have developed and validated in an International multicenter study a new diagnostic approach , called SAFE (Sequential Algorithm for Fibrosis Evaluation) - Biopsy, based on sequential used of biochemical markers of liver fibrosis (Sebastiani et al., Hepatology 2009). Other studies have been conducted for the standardisation and validation of new non-invasive approaches of liver fibrosis staging (VIRTUAL BIOPSY) based on combination of transient elastography and biochemical markers .

HEPATITIS B

The interest of the lab is currently focused on the characterisation of mutations in the polymerase protein of HBV causing resistance during antiviral therapy. Primary and compensatory mutations, identified by direct sequencing and phenotypic analysis, are currently investigated on a prospective basis in all the patients undergoing therapy in 15 Clinical Centers of the Triveneto region.

Synoptic CV

Honours

Selected VIMM Publications

• Bortoletto G, Scribano L, Realdon S, Marcolongo M, Mirandola S, Franceschini L, Bonisegna S, Noventa F, Plebani M, Martines D, Alberti A (2010) Hyperinsulinaemia reduces the 24-h virological response to PEG-interferon therapy in patients with chronic hepatitis C and insulin resistance. J. Viral Hepat. 17:475-80.
• Marcolongo M, Young B, Dal Pero F, Fattovich G, Peraro L, Guido M, Sebastiani G, Palù G, Alberti A (2009) A seven-gene signature (cirrhosis risk score) predicts liver fibrosis progression in patients with initially mild chronic hepatitis C. Hepatology 50:1038-44.
• Sebastiani G, Halfon P, Castera L, Pol S, Thomas DL, Mangia A, Di Marco V, Pirisi M, Voiculescu M, Guido M, Bourliere M, Noventa F, Alberti A (2009) SAFE biopsy: a validated method for large-scale staging of liver fibrosis in chronic hepatitis C. Hepatology 49:1821-7.
• Mirandola S, Osterreicher CH, Marcolongo M, Datz C, Aigner E, Schlabrakowski A, Realdon S, Gerotto M, Alberti A, Stickel F (2009) Microsomal triglyceride transfer protein polymorphism (-493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C. Liver Int. 29:557-65.
• Mirandola S, Realdon S, Iqbal J, Gerotto M, Dal Pero F, Bortoletto G, Marcolongo M, Vario A, Datz C, Hussain MM, Alberti A (2006) Liver microsomal triglyceride transfer protein is involved in hepatitis C liver steatosis. Gastroenterology 130:1661-9.

VIMM Publications

• Castéra L, Sebastiani G, Le Bail B, de Lédinghen V, Couzigou P, Alberti A (2010) Prospective comparison of two algorithms combining non-invasive methods for staging liver fibrosis in chronic hepatitis C. J. Hepatol. 52:191-8.
• Castera L, Forns X, Alberti A (2008) Non-invasive evaluation of liver fibrosis using transient elastography. J. Hepatol. 48:835-47.
• Sebastiani G, Alberti A (2008) Implementing non-invasive markers for liver fibrosis in clinical practice. J. Hepatol. 48:880-1; author reply .
• Sebastiani G, Vario A, Guido M, Alberti A (2008) Performance of noninvasive markers for liver fibrosis is reduced in chronic hepatitis C with normal transaminases. J. Viral Hepat. 15:212-8.
• Dal Pero F, Di Maira G, Marin O, Bortoletto G, Pinna LA, Alberti A, Ruzzene M, Gerotto M (2007) Heterogeneity of CK2 phosphorylation sites in the NS5A protein of different hepatitis C virus genotypes. J. Hepatol. 47:768-76.
• Dal Pero F, Tang KH, Gerotto M, Bortoletto G, Paulon E, Herrmann E, Zeuzem S, Alberti A, Naoumov NV (2007) Impact of NS5A sequences of Hepatitis C virus genotype 1a on early viral kinetics during treatment with peginterferon- alpha 2a plus ribavirin. J. Infect. Dis. 196:998-1005.
• Bortoletto G, Gerotto M, Pigozzi B, Michelotto A, Alberti A, Vezzi A, Alaibac M (2007) Analysis of immunoglobulin variable kappa gene mutations in cutaneous B-cell lymphoma. J. Dermatol. Sci. 47:248-52.
• Sebastiani G, Vario A, Guido M, Alberti A (2007) Sequential algorithms combining non-invasive markers and biopsy for the assessment of liver fibrosis in chronic hepatitis B. World J. Gastroenterol. 13:525-31.
• Sebastiani G, Alberti A (2006) Non invasive fibrosis biomarkers reduce but not substitute the need for liver biopsy. World J. Gastroenterol. 12:3682-94.
• Gerotto M, Resti M, Dal Pero F, Migliorato I, Alberti A, Bortolotti F (2006) Evolution of hepatitis C virus quasispecies in children with chronic hepatitis C. Infection 34:62-5.
• Ferri S, Dal Pero F, Bortoletto G, Bianchi FB, Lenzi M, Alberti A, Gerotto M (2006) Detailed analysis of the E2-IgM complex in hepatitis C-related type II mixed cryoglobulinaemia. J. Viral Hepat. 13:166-76.
• Gerotto M, Dal Pero F, Bortoletto G, Ferrari A, Pistis R, Sebastiani G, Fagiuoli S, Realdon S, Alberti A (2006) Hepatitis C minimal residual viremia (MRV) detected by TMA at the end of Peg-IFN plus ribavirin therapy predicts post-treatment relapse. J. Hepatol. 44:83-7.
• Gerotto M, Dal Pero F, Bortoletto G, Realdon S, Ferrari A, Boccato S, Alberti A (2004) PKR gene expression and response to pegylated interferon plus ribavirin therapy in chronic hepatitis C. Antivir. Ther. (Lond.) 9:763-70.

Selected Seminars

Contact

Alfredo Alberti Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Tel.:  (+39) 049 7923 224 Fax:  (+39) 049 7923 250

Last updated: 28/03/2010, AA ·