Hepatitis: Prieto J

Berasain C, Castillo J, Perugorria MJ, Latasa MU, Prieto J, Avila MA. · Division of Hepatology and Gene Therapy, CIMA-Universidad de Navarra, Pamplona, Spain. · Ann N Y Acad Sci. · Pubmed #19250206 No free full text.

Abstract: A connection between inflammation and cancer has been long suspected. Epidemiological studies have established that many tumors occur in association with chronic infectious diseases, and it is also known that persistent inflammation in the absence of infections increases the risk and accelerates the development of cancer. One clear example of inflammation-related cancer is hepatocellular carcinoma (HCC). HCC is a type tumor that slowly unfolds on a background of chronic inflammation mainly triggered by exposure to infectious agents (hepatotropic viruses) or to toxic compounds (ethanol). The molecular links that connect inflammation and cancer are not completely known, but evidences gathered over the past few years are beginning to define the precise mechanisms. In this article we review the most compelling evidences on the role of transcription factors such as NF-kappaB and STAT3, cytokines like IL-6 and IL-1alpha, ligands of the EGF receptor and other inflammatory mediators in cancer development, with special emphasis in HCC. The molecular dissection of the pathways connecting the inflammatory reaction and neoplasia will pave the way for better therapies to treat cancers.

Berasain C, Castillo J, Prieto J, Avila MA. · Division of Hepatology and Gene Therapy, CIMA, Universidad de Navarra, Pamplona, Spain. · Liver Int. · Pubmed #17311611 No free full text.

Abstract: Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths. This malignancy is often diagnosed at an advanced state, when most potentially curative therapies are of limited efficacy. In addition, HCC is a type of tumor highly resistant to available chemotherapeutic agents, which leaves HCC patients with no effective therapeutic options and a poor prognosis. From a molecular perspective, HCC is a heterogeneous type of tumor. However, in most cases, HCC emerges on a background of persistent liver injury, inflammation and hepatocellular proliferation, which is characteristic of chronic hepatitis and cirrhosis. Recent studies have revealed that dysregulation of a limited number of growth and survival-related pathways can play a key role in HCC development. The epidermal growth factor receptor (ErbB1) can be bound and activated by a broad family of ligands, and can also engage in extensive cross talk with other signaling pathways. This system is considered as an important defense mechanism for the liver during acute tissue injury; however, accumulating evidences suggest that its chronic stimulation can participate in the neoplastic conversion of the liver. Agents that target the ErbB1 receptor have shown antineoplastic activity in other types of tumors, but their efficacy either alone or in combination with other compounds has just started to be tested in experimental and human HCC. Here, we review the evidences that support the involvement of the ErbB1 in HCC development and that provide a rationale for ErbB1 targeting in HCC prevention and treatment.

Gil-Guerrero L, Sarobe P, Prieto J. · Clínica Universitaria y Centro de Investigación Médica Aplicada, Universidad de Navarra, Pamplona, Navarra, España. · Med Clin (Barc). · Pubmed #16828002 No free full text.

Abstract: Hepatitis C virus is an important public health threat, not only because of the high prevalence of this infection in western and third world countries, but also because of the high rate of resistance to the available antiviral therapy that consists on the use of pegylated interferon plus ribavirin. Currently, new forms of therapy are being developed based on a more precise knowledge of the structure and function of the viral proteins and of the strategies used by the virus to escape the immune and interferon systems. The new therapeutic approaches aim at different objectives: a) the inhibition of viral replication by blocking the viral protease and/or replicase; b) the use of other types of interferon with more potent antiviral effect, c) the induction of a specific anti-viral immune response by means of immunomodulatory compounds or therapeutic vaccination, d) the blockade of "de novo" infection of other cells with neutralizing antibodies, e) the induction of a antiviral state in the liver by transferring to this organ the gene of interferon and/or immunostimulating cytokines.

Prieto J, Qian C, Hernandez-Alcoceba R, Gonzalez-Aseguinolaza G, Mazzolini G, Sangro B, Kramer MG. · Department of Internal Medicine, Clinica Universitaria de Navarra, Avda. Pio XII 36, 31008 Pamplona, Spain. · Expert Opin Biol Ther. · Pubmed #15268675 No free full text.

Abstract: Many liver diseases lack satisfactory treatment and alternative therapeutic options are urgently needed. Gene therapy is a new mode of treatment for both inherited and acquired diseases, based on the transfer of genetic material to the tissues. Genes are incorporated into appropriate vectors in order to facilitate their entrance and function inside the target cells. Gene therapy vectors can be constructed on the basis of viral or non-viral molecular structures. Viral vectors are frequently used, due to their higher transduction efficiency. Both the type of vector and the expression cassette determine the duration, specificity and inducibility of gene expression. A considerable number of preclinical studies indicate that a great variety of liver diseases, including inherited metabolic defects, chronic viral hepatitis, liver cirrhosis and primary and metastatic liver cancer, are amenable to gene therapy. Gene transfer to the liver can also be used to convert this organ into a factory of secreted proteins needed to treat conditions that do not affect the liver itself. Clinical trials of gene therapy for the treatment of inherited diseases and liver cancer have been initiated but human gene therapy is still in its infancy. Recent progress in vector technology and imaging techniques, allowing in vivo assessment of gene expression, will facilitate the development of clinical applications of gene therapy.

Prieto J, Herraiz M, Sangro B, Qian C, Mazzolini G, Melero I, Ruiz J. · Division of Hepatology and Gene Therapy, Department of Medicine, Clinica Universitaria, University of Navarra, Pamplona, Spain. · Gut. · Pubmed #12651882 links to  free full text

Abstract: Gene therapy consists of the transfer of genetic material to cells to achieve a therapeutic goal. In the field of gastroenterology and hepatology gene therapy has produced considerable expectation as a potential tool in the management of conditions that lack effective therapy including non-resectable neoplasms of the liver, pancreas and gastrointestinal tract, chronic viral hepatitis unresponsive to interferon therapy, liver cirrhosis, and inflammatory bowel disease.

Schmitz V, Qian C, Ruiz J, Sangro B, Melero I, Mazzolini G, Narvaiza I, Prieto J. · Gene Therapy Unit, Department of Internal Medicine, Clinica Universitaria, Faculty of Medicine, Universidad de Navarra, Pamplona Spain. · Gut. · Pubmed #11772981 links to  free full text

Abstract: Gene therapy has emerged as a powerful and very plastic tool to regulate biological functions in diseased tissues with application in virtually all medical fields. An increasing number of experimental and clinical studies underline the importance of genes as curative agents in the future. However, intense research is needed to evaluate the potential of gene therapy to improve efficacy and minimise the toxicity of the procedure.

Ruiz J, Qian C, Drozdzik M, Prieto J. · Department of Medicine, Medical School, University of Navarra, Pamplona, Spain. · J Viral Hepat. · Pubmed #10847127 No free full text.

Abstract: Gene therapy represents an attractive approach to treat a great variety of diseases, both inherited and acquired, and it is moving slowly from a proof-of-principle phase to a wide application in most medical fields. Liver cancer and viral hepatitis are natural targets for this new therapeutic alternative due to the lack of success of conventional antitumoral and antiviral treatments and the ominous prognosis related with liver tumours. Gene therapy for viral hepatitis is aimed to boost the patient immune response against viral antigens or to make cells resistant to infection by blocking the viral life cycle. Gene transfer techniques applied to the treatment of hepatocellular carcinoma include drug sensitization by suicide genes, genetic immunotherapy, normal tissue protection by transfer of the multidrug resistance gene, replacement of tumour suppressor genes, inhibition of oncogenes and modifications of the biology of the tumour (antiangiogenesis). However, major advances in our understanding of the regulation of gene expression, design of the expression cassettes and development of more efficient gene transfer vectors are mandatory before gene therapy can become a widely used therapeutic modality.

Civeira MP, Prieto J. · Department of Medicine and Liver Unit, Clinica Universitaria and Medical School, University of Navarra, Pamplona, Spain. · J Hepatol. · Pubmed #10622595 No free full text.

Abstract: The term sustained response should be applied to patients with negative serum HCV-RNA and normal values of serum transaminases 6 months after interferon withdrawal. To investigate which factors identify sustained responders early during treatment we analysed 18 reports which used the definition sustained response. Eight reports, comprising 988 patients, have studied the value of early clearance of viraemia as a predictor of sustained response using multivariate analysis and all of them found that this was the strongest predictor of a sustained response. Determination of HCV-RNA 4 or 12 weeks after initiation of IFN therapy predicts treatment outcome more accurately than baseline viral load (the best pre-treatment predictor). ALT levels during the first 12 weeks of treatment have lower predictive value than early viral clearance. The sensitivity of a negative HCV-RNA test is similar at week 4 and at week 12 of therapy while the specificity and the accuracy is higher at week 4. The value of persistent viraemia for early prediction of no response is higher than 97%, with similar values at weeks 4 and 12. Persistence of HCV-RNA in serum at week 4 strongly indicates that the patient will not respond to treatment and in these cases interruption of treatment or other therapeutic options could be considered.

Grant PR, Black A, Garcia N, Prieto J, Garson JA. · Department of Virology, Royal Free and University College Medical School, London, United Kingdom. · J Med Virol. · Pubmed #10897061 No free full text.

Abstract: A small pilot study in patients with chronic hepatitis C (HCV) infection suggested that antiviral treatment with interferon (IFN) plus N-acetyl cysteine (NAC) was more effective than treatment with interferon alone [Beloqui et al. (1993) Journal of Interferon Research 13:279-282]. An attempt was made to confirm this by performing a placebo-controlled double-blind study at 8 medical centres in Spain and Italy. One-hundred forty-seven patients with chronic HCV infection were investigated, 73 received 3MU IFN-alpha thrice weekly plus NAC 1800 mg daily and 74 received IFN alone. Treatment was continued for 6 months and patients were followed up for a further 6 months. Amongst patients receiving IFN plus NAC, sustained virological responses were observed in 5.5%, transient responses in 26% and non-response in 68.5%. The figures for patients receiving IFN only were 4.1%, 24.3% and 71.6% respectively. Sustained virological response was significantly associated with non-type 1 genotypes (P = 0.045) and with low pre-treatment viraemia levels (P = 0.034). Biochemical response (serum ALT concentrations) correlated with virological outcome in 97% (n = 139) of cases. Patients who experienced a sustained virological response also showed reduction in the Knodell histological activity index. It is concluded that patients with chronic HCV infection are very unlikely to benefit from the addition of N-acetyl cysteine to conventional therapy with interferon-alpha.

Benedicto I, Molina-Jiménez F, Bartosch B, Cosset FL, Lavillette D, Prieto J, Moreno-Otero R, Valenzuela-Fernández A, Aldabe R, López-Cabrera M, Majano PL. · Hospital Universitario de la Princesa, Madrid, Spain. · J Virol. · Pubmed #19515778 No free full text.

Abstract: The precise mechanisms regulating hepatitis C virus (HCV) entry into hepatic cells remain unknown. However, several cell surface proteins have been identified as entry factors for this virus. Of these molecules, claudin-1, a tight junction (TJ) component, is considered a coreceptor required for HCV entry. Recently, we have demonstrated that HCV envelope glycoproteins (HCVgp) promote structural and functional TJ alterations. Additionally, we have shown that the intracellular interaction between viral E2 glycoprotein and occludin, another TJ-associated protein, could be the cause of the mislocalization of TJ proteins. Herein we demonstrated, by using cell culture-derived HCV particles (HCVcc), that interference of occludin expression markedly reduced HCV infection. Furthermore, our results with HCV pseudotyped particles indicated that occludin, but not other TJ-associated proteins, such as junctional adhesion molecule A or zonula occludens protein 1, was required for HCV entry. Using HCVcc, we demonstrated that occludin did not play an essential role in the initial attachment of HCV to target cells. Surface protein labeling experiments showed that both expression levels and cell surface localization of HCV (co)receptors CD81, scavenger receptor class B type I, and claudin-1 were not affected upon occludin knockdown. In addition, immunofluorescence confocal analysis showed that occludin interference did not affect subcellular distribution of the HCV (co)receptors analyzed. However, HCVgp fusion-associated events were altered after occludin silencing. In summary, we propose that occludin plays an essential role in HCV infection and probably affects late entry events. This observation may provide new insights into HCV infection and related pathogenesis.

Berraondo P, Prieto J, Gonzalez-Aseguinolaza G. · Division of Hepatology and Gene Therapy, Center for Investigation in Applied Medicine (CIMA), University of Navarra, 31008 Pamplona, Spain. · Curr Gene Ther. · Pubmed #19355864 No free full text.

Abstract: Interleukin-12 (IL-12) is a multifunctional cytokine that stimulates both innate and adaptive immunity, acting as a key regulator of cell-mediated immune responses. The immunomodulating and antiangiogenic functions of IL-12 have provided the rationale for exploiting this cytokine as an anticancer and antiviral agent. The promising data obtained by the administration of IL-12 recombinant protein in preclinical animal models of cancer and chronic viral hepatitis raised hopes that recombinant IL-12 could be a powerful therapeutic agent against both pathologies. However, clinical trials revealed a modest clinical response that was limited by the development of an adaptive response that down-regulated IL-12 activity and by severe toxicity when high doses of this cytokine were used. Gene therapy can significantly increase cytokine expression in the target organ without excessively elevating systemic cytokine levels, which leads to an increased efficacy/toxicity ratio. Early clinical trials with short-term IL-12 expression vectors have set the proof-of-concept that local production of IL-12 inside a tumor can stimulate tumor infiltration by effector immune cells, sometimes followed by tumor regression. Recent advances in long-term expression vectors for the delivery of IL-12 or lytic viruses armed with this cytokine may be key to unlocking the therapeutic potential of IL-12. However, the new generation of IL-12 gene therapy protocols should cope with two major limitations. First, promoter silencing induced by IL-12 may abrogate long-term production of this cytokine. Second, regulatory immune systems induced by IL-12 should be blocked to maximize antitumor and antiviral activity.

Larrea E, Aldabe R, Gonzalez I, Segura V, Sarobe P, Echeverria I, Prieto J. · Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain. · J Virol. · Pubmed #19158240 No free full text.

Abstract: Oncostatin M (OSM) is released together with type I interferon (IFN) by activated dendritic cells, suggesting a concerted action of these cytokines in the biological response against infection. We found that OSM increases the antiviral effect of IFN-alpha in Huh7 hepatoma cells infected with hepatitis A or hepatitis C virus and synergizes with IFN-alpha in the induction of antiviral genes. The combination of OSM and IFN-alpha led to upregulation of both STAT1 and STAT3 together with intense and prolonged activation of STAT1, STAT3, and Jak1. OSM with or without IFN-alpha also activated p38 mitogen-activated protein kinase, which is known to enhance transcription of IFN-alpha-inducible genes. Interestingly, OSM combined with IFN-alpha strongly induced immunoproteasome genes and other genes involved in antigen processing and presentation. Moreover, OSM, alone or in combination with IFN-alpha, upregulated relevant innate immunity molecules and increased the expression of intracellular adhesion molecule 1 and interleukin-15 receptor alpha (IL-15Ralpha) in liver cells. Hepatoma cells transfected with a plasmid encoding a viral antigen were able to activate effector T cells when pretreated with IFN-alpha plus OSM but not with each cytokine separately. Also, OSM, more than IFN-alpha, augmented the ability of Huh7 cells to transpresent IL-15 to responding lymphocytes and increased the immunostimulatory activity of liver epithelial cells by presenting a short viral peptide to sensitized cytotoxic T cells. In conclusion, OSM enhances the antiviral effects of type I interferon and cooperates with it in the induction of adaptive immune responses to pathogens. These findings may have therapeutic implications.

Crettaz J, Otano I, Ochoa L, Benito A, Paneda A, Aurrekoetxea I, Berraondo P, Rodríguez-Madoz JR, Astudillo A, Kreppel F, Kochanek S, Ruiz J, Menne S, Prieto J, Gonzalez-Aseguinolaza G. · Department of Gene Therapy and Hepatology, Center for Investigation in Applied Medicine (CIMA), Avda Pio XII 55, 31008 Pamplona, Spain. · J Virol. · Pubmed #19116251 No free full text.

Abstract: Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.

Benedicto I, Molina-Jiménez F, Barreiro O, Maldonado-Rodríguez A, Prieto J, Moreno-Otero R, Aldabe R, López-Cabrera M, Majano PL. · Molecular Biology Unit, Hospital Universitario de la Princesa, Madrid, Spain. · Hepatology. · Pubmed #18802961 No free full text.

Abstract: Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells. CONCLUSION: We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis.

Echeverría I, Zabaleta A, Silva L, Díaz-Valdés N, Riezu-Boj JI, Lasarte JJ, Borrás-Cuesta F, Civeira MP, Prieto J, Sarobe P. · Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), Pamplona, Spain. · J Viral Hepat. · Pubmed #18637068 No free full text.

Abstract: Dendritic cells (DC) transfected with an adenovirus encoding hepatitis C virus (HCV) NS3 protein (AdNS3) induce potent antiviral immune responses when used to immunize mice. However, in HCV infected patients, controversial results have been reported regarding the functional properties of monocyte-derived DC (MoDC), a cell population commonly used in DC vaccination protocols. Thus, with the aim of future vaccination studies we decided to characterize MoDC from HCV patients transfected with AdNS3 and stimulated with the TLR3 ligand poly(I:C). Phenotypic and functional properties of these cells were compared with those from MoDC obtained from uninfected individuals. PCR analysis showed that HCV RNA was negative in MoDC from patients after the culture period. Also, phenotypic analysis of these cells showed lower expression of CD80, CD86, and CD40, but similar expression of HLA-DR molecules as compared to MoDC from uninfected individuals. Functional assays of MoDC obtained from patients and controls showed a similar ability to activate allogeneic lymphocytes or to produce IL-12 and IL-10, although lower IFN-alpha levels were produced by cells from HCV patients after poly(I:C) stimulation. Moreover, both groups of MoDC induced similar profiles of IFN-gamma and IL-5 after stimulation of allogeneic T-cells. Finally, migration assays did not reveal any difference in their ability to respond to CCL21 chemokine. In conclusion, MoDC from HCV patients are functional after transduction with AdNS3 and stimulation with poly(I:C). These findings suggest that these cells may be useful for therapeutic vaccination in chronic HCV infection.

Zabaleta A, Llopiz D, Arribillaga L, Silva L, Riezu-Boj JI, Lasarte JJ, Borrás-Cuesta F, Prieto J, Sarobe P. · Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain. · Mol Ther. · Pubmed #17923840 No free full text.

Abstract: Chronic infection by hepatitis C virus (HCV) is characterized by the absence of efficient antiviral T-cell responses. Thus, vaccination strategies to induce strong anti-HCV T-cell responses are of paramount importance for prophylactic and therapeutic purposes. Dendritic cells (DCs) are the most potent antigen presenting cells; therefore, immunization with these cells loaded with viral antigens offers a new approach for induction of antiviral immunity. Here we show that immunization with DCs transfected with an adenovirus encoding non-structural 3 protein, from HCV (AdNS3), induced multiepitopic CD4 T helper cell 1 (Th1) and CD8 T-cell responses in different mouse strains. These responses prevented the growth of a tumorexpressing HCV proteins, in short- and long-term experiments. Moreover, immunization with AdNS3-transfected DCs did not induce anti-adenoviral antibodies, as compared to direct immunization with AdNS3, but elicited T-cell responses even in the presence of pre-existing anti-adenoviral antibodies. Finally, responses induced by this protocol down-regulated the expression of HCV RNA in the liver. In conclusion, DCs transfected with AdNS3 may prove to be an efficient anti-HCV vaccine.

Luquin E, Larrea E, Civeira MP, Prieto J, Aldabe R. · Division of Hepatology and Gene Therapy (CIMA), University of Navarra, Pamplona, Spain. · Antiviral Res. · Pubmed #17675168 No free full text.

Abstract: Hepatitis C virus (HCV) is remarkably efficient at establishing persistent infection. The current treatment with IFN-alpha given alone or in combination with ribavirin is ineffective in eliminating the virus in a large proportion of individuals with chronic hepatitis C. Recent data suggest that HCV blocks IFN-alpha signalling, an effect that facilitates viral persistence. We have used the HCV genomic and subgenomic replicon system to analyze the effect of structural and non-structural viral proteins on the activation of the Jak/STAT pathway and induction of antiviral activity by IFN-alpha. Our results show that IFN-alpha-mediated STAT activation (but not IFN-gamma-stimulated STAT phosphorylation) is blocked in Huh7 cell line containing the genomic replicon, while this is not observed in cells with the subgenomic replicon. In agreement with these findings, the transcriptional activity and the antiviral effect of IFN-alpha were significantly lower in cells harboring the genomic replicon than in cells with the subgenomic replicon. These results indicate that HCV structural proteins play an important role in the escape of HCV from the interferon system.

Ochoa-Callejero L, Berraondo P, Crettaz J, Olagüe C, Vales A, Ruiz J, Prieto J, Tennant BC, Menne S, González-Aseguinolaza G. · Laboratory of Gene Therapy of Viral Hepatitis, Division of Gene Therapy and Hepatology, Center for Applied Medical Research (CIMA), Pamplona, Navarra, Spain. · J Med Virol. · Pubmed #17385694 No free full text.

Abstract: Woodchucks infected with the woodchuck hepatitis virus (WHV) is the best available animal model for testing the immunotherapeutic effects of dendritic cells (DCs) in the setting of a chronic infection, as woodchucks develop a persistent infection resembling that seen in humans infected with the hepatitis B virus. In the present study, DCs were generated from woodchuck peripheral blood mononuclear cells (wDCs) in the presence of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and human interleukin 4 (hIL-4). After 7 days of culture, cells with morphology similar to DCs were stained positively with a cross-reactive anti-human CD86 antibody. Functional analysis showed that uptake of FITC-dextran by wDCs was very efficient and was partially inhibited after LPS-induced maturation. Furthermore, wDCs stimulated allogenic lymphocytes and induced proliferation. Moreover, wDCs were transduced efficiently with a human adenovirus serotype 5 for the expression of beta-galactosidase. Following transduction and in vivo administration of such DCs into woodchucks, an antigen-specific cellular immune response was induced. These results demonstrate that wDCs can be generated from the peripheral blood. Following transfection with a recombinant adenovirus wDCs can be used as a feasible and effective tool for eliciting WHV-specific T-cell responses indicating their potential to serve as prophylactic and therapeutic vaccines.

Zabaleta A, Arribillaga L, Llopiz D, Dotor J, Lasarte JJ, Prieto J, Borrás-Cuesta F, Esteban JI, Quer J, Vayreda F, Sarobe P. · University of Navarra, Center for Applied Medical Research (CIMA), Division of Hepatology and Gene Therapy, 31008 Pamplona, Spain. · Antiviral Res. · Pubmed #17275104 No free full text.

Abstract: Development of vaccination strategies against hepatitis C virus (HCV) is of paramount importance. With this aim, we tested the ability of dendritic cell-activating reagents polyinosinic-polycytidylic acid (poly(I:C)) and anti-CD40, as adjuvants to induce T-cell responses against HCV. Immunization of mice with these adjuvants induced dendritic cell maturation in vivo. Also, joint administration of poly(I:C) and anti-CD40 plus HCV antigens had a synergistic effect on the induction of anti-HCV T-cell responses. CD4 responses displayed a Th1 cytokine profile, and CD8 responses could be induced by immunization with a minimal CD8 epitope. Addition of a low amount of NS3 protein (as a source of Th epitopes) to the immunization mixture enhanced CD8 responses, whereas immunization with higher doses of NS3 induced both CD4 and CD8 responses. Surprisingly, immunization with NS3 protein but not with CD8 epitopes was able to induce CD8 responses and able to recognize cells expressing HCV antigens endogenously. Moreover, immunization with these adjuvants activated NK cells, which in turn helped to induce Th1 responses. Finally, this combined immunization protocol afforded long-lasting T-cell responses, suggesting that this strategy may prove to be useful in vaccination and/or treatment of HCV infection.

Larrea E, Riezu-Boj JI, Gil-Guerrero L, Casares N, Aldabe R, Sarobe P, Civeira MP, Heeney JL, Rollier C, Verstrepen B, Wakita T, Borrás-Cuesta F, Lasarte JJ, Prieto J. · Division of Gene Therapy and Hepatology, Center for Applied Medical Research, CIMA, Avenida Pío XII 55, 31008 Pamplona, Spain. · J Virol. · Pubmed #17229698 links to  free full text

Abstract: Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.

Vilasco M, Larrea E, Vitour D, Dabo S, Breiman A, Regnault B, Riezu JI, Eid P, Prieto J, Meurs EF. · Institut Pasteur, Unit Hepacivirus, Paris, France. · Hepatology. · Pubmed #17133498 No free full text.

Abstract: During a viral infection, binding of viral double-stranded RNAs (dsRNAs) to the cytosolic RNA helicase RIG-1 leads to recruitment of the mitochondria-associated Cardif protein, involved in activation of the IRF3-phosphorylating IKKepsilon/TBK1 kinases, interferon (IFN) induction, and development of the innate immune response. The hepatitis C virus (HCV) NS3/4A protease cleaves Cardif and abrogates both IKKepsilon/TBK1 activation and IFN induction. By using an HCV replicon model, we previously showed that ectopic overexpression of IKKepsilon can inhibit HCV expression. Here, analysis of the IKKepsilon transcriptome profile in these HCV replicon cells showed induction of several genes associated with the antiviral action of IFN. Interestingly, IKKepsilon still inhibits HCV expression in the presence of neutralizing antibodies to IFN receptors or in the presence of a dominant negative STAT1alpha mutant. This suggests that good IKKepsilon expression levels are important for rapid activation of the cellular antiviral response in HCV-infected cells, in addition to provoking IFN induction. To determine the physiological importance of IKKepsilon in HCV infection, we then analyzed its expression levels in liver biopsy specimens from HCV-infected patients. This analysis also included genes of the IFN induction pathway (RIG-I, MDA5, LGP2, Cardif, TBK1), and three IKKepsilon-induced genes (IFN-beta, CCL3, and ISG15). The results show significant inhibition of expression of IKKepsilon and of the RNA helicases RIG-I/MDA5/LGP2 in the HCV-infected patients, whereas expression of TBK1 and Cardif was not significantly altered. In conclusion, given the antiviral potential of IKKepsilon and of the RNA helicases, these in vivo data strongly support an important role for these genes in the control of HCV infection.

Berraondo P, Crettaz J, Ochoa L, Pañeda A, Prieto J, Trocóniz IF, González-Aseguinolaza G. · Laboratory of Gene Therapy of Viral Hepatitis, Division of Gene Therapy and Hepatology, Center for Applied Medical Research (CIMA), University of Navarra, 31080 Pamplona, Navarra, Spain. · Hum Gene Ther. · Pubmed #16776569 No free full text.

Abstract: The liver is an attractive organ for gene therapy because of its important role in many inherited and acquired diseases. Recombinant adeno-associated viruses (rAAVs) have been shown to be good candidates for liver gene delivery, leading to long-term gene expression. We evaluated the influence of the route of administration on rAAV-mediated liver transduction by comparing levels of luciferase expression in the livers of male and female mice after injection of rAAV serotype 2, using three different routes of administration: intravenous (IV), intraportal (IP), or direct intrahepatic (IH) injection. To determine transgene expression we used a noninvasive optical bioluminescence imaging system that allowed long-term in vivo analysis. After IV injection dramatic differences in liver transgene expression were observed, depending on gender. When IP injection was used the differences were reduced although they were still significant. Interestingly, direct intrahepatic injection of rAAV vectors was associated with the fastest and strongest onset of luciferase expression. Moreover, no gender differences in liver transduction were observed and luciferase expression was confined to the site of injection. Thus, direct intrahepatic injection of rAAV offers specific advantages, which support the potential of this route of administration for future clinical applications.

Sarobe P, Lasarte JJ, García N, Civeira MP, Borrás-Cuesta F, Prieto J. · Division of Hepatology and Gene Therapy, Clínica Universitaria/School of Medicine, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain. · J Viral Hepat. · Pubmed #16364082 No free full text.

Abstract: Successful clearance of hepatitis C virus (HCV) infection has been associated with strong cellular immune responses against viral antigens. However, although the magnitude of these responses is clearly important for viral eradication, more studies are needed to unravel the fine specificity of the protective anti-HCV immunity in infected patients. This was the aim of the present study. Overlapping peptides spanning the sequence of HCV E2 and NS4a proteins were used to stimulate T cells from patients with chronic hepatitis C divided into three groups: naïve patients, patients who exhibited sustained response to interferon (IFN)-alpha therapy and patients who failed to respond to the treatment. Interleukin-2 production by stimulated cells was measured in each case. Patients with sustained response to therapy had stronger responses to E2 peptides than nonresponders, whereas naïve patients demonstrated intermediate reactivity. In the case of NS4a, responses against peptides where similar in all groups of patients. Analysis of the peptides recognized by T cells showed that responses were broad and heterogeneous, and some immunodominant epitopes, preferentially recognized by patients exhibiting sustained response to treatment, were found. These results confirm the role of cellular immune responses in viral clearance, and stress the importance of immunodominant regions within HCV antigens. These viral sequences may represent valuable immunogens for preparation of therapeutic or prophylactic vaccines.

Fernández-Irigoyen J, Santamaría E, Sesma L, Muñoz J, Riezu JI, Caballería J, Lu SC, Prieto J, Mato JM, Avila MA, Corrales FJ. · Division of Hepatology and Gene Therapy, CIMA, Universidad de Navarra, 31008 Pamplona, Spain. · Proteomics. · Pubmed #16252306 No free full text.

Abstract: Hepatocellular carcinoma (HCC) is the fifth most common neoplasm with more than 500 000 new cases diagnosed yearly. Although major risk factors of HCC are currently known, the identification of biological targets leading to an early diagnosis of the disease is considered one of the priorities of clinical hepatology. In this work we have used a proteomic approach to identify markers of hepatocarcinogenesis in the serum of a knockout mice deficient in hepatic AdoMet synthesis (MAT1A(-/-)), as well as in patients with HCC. Three isoforms of apolipoprotein A-I (Apo A-I) with different pI were identified in murine serum. Isoform 1 is up-regulated in the serum of MAT1A(-/-) mice much earlier than any histological manifestation of liver disease. Further characterization of the differential isoform by electrospray MS/MS revealed specific oxidation of methionine 85 and 216 to methionine sulfoxide while the sequence of the analogous peptides on isoforms 2 and 3 showed the nonoxidized methionine residues. Enrichment of an acidic isoform of Apo A-I was also assessed in the serum of hepatitis B virus patients who developed HCC. Specific oxidation of methionine 112 to methionine sulfoxide and tryptophans 50 and 108 to formylkinurenine were identified selectively in the up-regulated isoform. Although it is not clear at present whether the occurrence of these modifications has a causal role or simply reflects secondary epiphenomena, this selectively oxidized Apo A-I isoform may be considered as a pathological hallmark that may help to the understanding of the molecular pathogenesis of HCC.

Guitart A, Riezu-Boj JI, Elizalde E, Larrea E, Berasain C, Aldabe R, Civeira MP, Prieto J. · Division of Hepatology and Gene Therapy, Clinica Universitaria and School of Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain. · J Gen Virol. · Pubmed #16227229 links to  free full text

Abstract: Systems for in vitro culture of Hepatitis C virus (HCV) are essential tools to analyse virus-cell interactions and to investigate relevant pathophysiological aspects of HCV infection. Although the HCV replicon methodology has increased our understanding of HCV biology, this system does not reproduce the natural infection. Recently, tupaia (Tupaia belangeri chinensis) hepatocytes have been utilized for in vitro culture of HCV. In the present work, primary tupaia hepatocytes infected in vitro with HCV were used to analyse the evolution of HCV quasispecies in infected cells and the ability of the virus to influence antiviral and proinflammatory responses in cells sustaining virus replication. The results confirmed the potential of tupaia hepatocytes as a model for HCV infection, although this system is limited by rapid loss of differentiated cell phenotype in culture. These findings revealed an extraordinary plasticity of HCV quasispecies, which underwent rapid evolution to tupaia-tropic variants as early as 24 h after infection. It was also shown that HCV could activate interferon-sensitive genes, albeit modestly in comparison with other viruses such as Semliki Forest virus. Importantly, HCV activated NF-kappaB in primary hepatocytes and upregulated NF-kappaB-responsive genes including the chemokines MCP-1 and CXCL2 (MIP-2). This effect may play a role in induction of the hepatic inflammatory reaction in vivo. In summary, HCV quasispecies adapt rapidly to the specific biology of the host and HCV stimulates a blunted interferon response while inducing a proinflammatory phenotype in the infected cell.