Civeira, M.P. (María Pilar)
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- Risk factors for recurrence of hepatitis C after liver transplantation.(Wiley-Blackwell, 1998) Civeira, M.P. (María Pilar); Peña, A. (Andrés) de la; Álvarez-Cienfuegos, J. (Javier); Sola, J. (Josu); Sangro, B. (Bruno); Prieto, J. (Jesús); Herrero, J.I. (José Ignacio); Garcia, N. (Nicolás); Quiroga, J. (Jorge)Recurrent hepatitis C is a frequent complication after liver transplantation for hepatitis C virus– related cirrhosis, but risk factors related to its development remain ill defined. Twenty-three patients receiving a primary liver graft for hepatitis C virus–related cirrhosis and with an assessable biopsy performed at least 6 months after liver transplantation were studied retrospectively. The end point of this study was to look for risk factors associated with the development of histologic hepatitis C in the graft. Thirty-six major variables were studied, and those reaching significance by univariate analysis were included in a multivariate analysis. Eighteen patients (78%) developed posttransplant hepatitis C. On univariate analysis, six variables showed significant predictive value: increased immunosuppression for treatment of acute rejection; pretransplant hepatocellular carcinoma; cumulative doses of prednisone at 3, 6, and 12 months after transplantation; and mean blood trough levels of cyclosporine in the first 6 months posttransplantation. On multivariate analysis, two variables retained independent statistical significance as predictors of hepatitis C recurrence, namely receipt of antirejection therapy (P 5 .0087) and lower mean cyclosporine levels in the first 6 months after transplantation (P 5 .0134). Therefore, recurrence of hepatitis C after liver transplantation seems to be at least partially related to posttransplantation immunosuppressive therapy.
- The stilbene disulfonic acid DIDS stimulates the production of TNF-alpha in human lymphocytes(Elsevier, 1992) Civeira, M.P. (María Pilar); Arrazola, A. (Arantxa); Larrea, E. (Esther); Medina, J.F. (Juan Francisco); Diez-Martinez, J. (Javier); Qian, C. (Cheng); Prieto, J. (Jesús); Garciandia, A. (Ana)Exposure of human peripheral blood mononuclear cells (PBMC) to the stilbene derivative DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) (60 microM and above) significantly increased the release of tumor necrosis factor-alpha (TNF-alpha), as determined by TNF-alpha activity in the incubation media. When the TNF-alpha message was analyzed in PBMC by a reverse transcription/polymerase chain reaction (RT/PCR)-based procedure, it was found that incubation with DIDS (60 microM) was followed by a time-dependent accumulation of TNF-alpha mRNA. Measurements of intracellular pH showed that the presence of increasing concentrations of DIDS resulted in a progressive intracellular alkalinization of PBMC. It is suggested that the known DIDS effect of inhibiting transmembrane anion exchange, i.e., chloride/bicarbonate exchange, might play a role in the stimulation of TNF-alpha production by PBMC exposed to DIDS.
- IFN-alpha5 mediates stronger Tyk2-stat-dependent activation and higher expression of 2',5'-oligoadenylate synthetase than IFN-alpha2 in liver cells(Mary Ann Liebert, 2004) Civeira, M.P. (María Pilar); Riezu-Boj, J.I. (José Ignacio); Guitart, A. (Anunciata); Baixeras, E. (Elena); Larrea, E. (Esther); Aldabe, R. (Rafael); Prieto, J. (Jesús)Interferon-alpha5 (IFN-alpha5) is the main IFN-alpha subtype expressed in the liver. Hepatitis C virus (HCV) infection is associated with low IFN-alpha5 mRNA levels, possibly reflecting an escape mechanism of the virus. In this work, we sought to compare IFN-alpha2 and IFN-alpha5 with respect to activation of early cell signaling cascades and induction of antiviral genes in the human hepatoma HepG2 and Huh7 cell lines. We found that the Tyr701 phosphorylation kinetics of Stat1 mediated by IFN stimulation was higher when cells were incubated with IFN-alpha5 than when using IFN-alpha2. Similarly, Tyr(1054/1055) phosphorylation kinetics of Tyk2 were more intense after exposure to IFN-alpha5 than when using IFN-alpha2. Concomitantly, Tyr705 phosphorylation of Stat3 was higher after stimulation with IFN-alpha5 than with IFN-alpha2. In parallel to these findings, the mRNA levels of the antiviral IFN-inducible gene 2',5'-oligoadenylate synthetase were higher in cell samples treated with IFN-alpha5 than with IFN-alpha2. These findings suggest that interaction of IFN-alpha5 and IFN-alpha2 subtypes with IFN type I receptor occurs differently, and this affects the intensity of expression of antiviral genes. In conclusion, our data show that in hepatocytic cells, IFN-alpha5 induces stronger signaling and higher expression of antiviral genes than IFN-alpha2. These data warrant clinical trials to evaluate the efficacy of IFN-alpha5 in chronic viral hepatitis.
- Detección del DNA del virus de la hepatitis B en suero mediante amplificación génica en pacientes con hepatitis crónica B y en pacientes con hepatitis crónica C(Elsevier, 1992) Civeira, M.P. (María Pilar); Riezu-Boj, J.I. (José Ignacio); Serrano, M. (Manuel); Camps, J. (J.); Jauregui, J.I. (Javier Ignacio); Castilla, A. (Alberto); Prieto, J. (Jesús)
- Transmission of hepatitis C virus infection to tree shrews(Elsevier, 1998) Civeira, M.P. (María Pilar); Riezu-Boj, J.I. (José Ignacio); Xie, Z.C. (Zhi-Chun); Su, J.H. (Jie-Han); Guillen, J. (Javier); Prieto, J. (Jesús); Lasarte, J.J. (Juan José)Although hepatitis C virus (HCV) infection can be reproduced in chimpanzees, these animals are rare and expensive. Tree shrews (tupaias) are small animals, closely related to primates, which adapt easily to a laboratory environment. In this work we have investigated the susceptibility of Tupaia belangeri chinensis to HCV infection. Tupaias caught in the wild in Yunnan (China) were inoculated in China with HCV genotype 1b (study A) and in Spain with a mixture of genotypes 1b, 1a, and 3 (study B). In study B tupaias were divided into three groups: group I was inoculated without previous manipulation, group II received 750 cGy of X-ray whole-body irradiation before inoculation, and group III was used as control. Transient or intermittent viremia occurred in 34.8% (8/23) and anti-HCV in 30.4% (7/23) of tupaias in study A. In study B a transient viremia was detected in 20% (2/10) in group I and in 50% (2/4) in group II. Anti-HCV was found in 1 tupaia from group I and in 3 from group II: Viremia lasted for longer and anti-HCV tended to reach higher titers in animals which received total body irradiation. ALT elevations and nonspecific pathological changes occurred in inoculated tupaias; however, the wild nature of the animals precludes the interpretation of these changes as solely due to HCV infection. In summary our results show that T.b. chinensis are susceptible to HCV and that whole-body irradiation may possibly increase the efficiency of the infection. These animals may serve as an in vivo system for culturing HCV and addressing pathophysiological and therapeutic issues of HCV infection.
- Hepatitis C virus infection of primary tupaia hepatocytes leads to selection of quasispecies variants, induction of interferon-stimulated genes and NF-kappaB nuclear translocation(Society for General Microbiology, 2005) Civeira, M.P. (María Pilar); Riezu-Boj, J.I. (José Ignacio); Berasain, C. (Carmen); Guitart, A. (Anunciata); Larrea, E. (Esther); Aldabe, R. (Rafael); Prieto, J. (Jesús); Elizalde, E. (Edurne)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.
- Hepatic and extrahepatic HCV RNA strands in chronic hepatitis C: different patterns of response to interferon treatment(Wiley-Blackwell, 1993) Civeira, M.P. (María Pilar); Riezu-Boj, J.I. (José Ignacio); Qian, C. (Cheng); Gil, B. (Beatriz); Prieto, J. (Jesús)We investigated the presence of positive (genomic) and negative (replicative intermediate) hepatitis C virus RNA strands in liver, peripheral mononuclear cells and serum from patients with chronic hepatitis C using a selective and semiquantitative polymerase chain reaction procedure. Negative and positive hepatitis C virus RNA strands were present in liver, serum and lymphoid cells in all untreated patients and in all those who did not respond to interferon therapy. In the latter group of patients, the titers of RNA strands in the liver and peripheral mononuclear cells at the end of the treatment were similar to those encountered in untreated patients, but the serum titers were about 100 times lower than pretreatment values. In patients who responded to interferon with normalization of serum aminotransferase levels (n = 10), the rate of detection and the titer of the two viral strands in liver, serum and mononuclear cells were markedly decreased at the end of the therapy. In the six responders who did not relapse after interferon withdrawal, both hepatitis C virus RNA strands were absent from the liver, serum and lymphoid cells. By contrast, the positive RNA strand was present in liver cells, mononuclear cells or both at the end of therapy in all patients who experienced posttherapy relapse. In conclusion, our results indicate that interferon can clear hepatitis C virus from hepatic and extrahepatic sites only in responder patients. Disappearance of genomic hepatitis C virus RNA from the liver and from mononuclear cells may predict complete response without posttherapy relapse.
- Altered expression and activation of signal transducers and activators of transcription (STATs) in hepatitis C virus infection: in vivo and in vitro studies(BMJ Publishing Group, 2006) Civeira, M.P. (María Pilar); Ametzazurra, A. (Amagoia); Larrea, E. (Esther); Aldabe, R. (Rafael); Prieto, J. (Jesús); Fernandez-Rodriguez, C.M. (Conrado M.); Molano, E. (Eduardo)BACKGROUND: Signal transducers and activators of transcription (STATs) play a critical role in antiviral defence. STAT3 is also important in cell protection against inflammatory damage. STAT proteins are activated by interferons and by hepatoprotective cytokines of the interleukin 6 superfamily, including cardiotrophin 1. METHODS: We analysed the status of STATs in hepatitis C virus (HCV) infected livers and the relationship between expression and activation of STATs and HCV replication in Huh7 cells transfected with HCV genomic replicon. RESULTS: STAT3alpha expression was reduced in HCV infected livers showing an inverse correlation with serum alanine aminotransferase. In patients with HCV infection, nuclear staining for phosphorylated STAT3 was faint in parenchymal cells (although conspicuous in infiltrating leucocytes), in contrast with strong nuclear staining in hepatocytes from control livers. Expression and activation of STAT1 (a factor activated by both interferon (IFN)-alpha and IFN-gamma) were increased in HCV infected livers, particularly in those with high inflammatory activity. Conversely, phosphorylated STAT2 (a factor selectively activated by IFN-alpha) was undetectable in livers with HCV infection, a finding that was associated with marked downregulation of the two functional subunits of the IFN-alpha receptor. HCV replication in Huh7 cells caused STAT3alpha downregulation and blocked STAT3 phosphorylation by either IFN-alpha or cardiotrophin 1. HCV replication in Huh7 cells also inhibited STAT1 and STAT2 activation by IFN-alpha while there was no impairment of STAT1 phosphorylation by the proinflammatory cytokine IFN-gamma. CONCLUSIONS: STAT3 is downregulated in HCV infected livers and in Huh7 cells bearing the full length HCV replicon. HCV replication is associated with impaired Jak-STAT signalling by antiviral and cytoprotective cytokines. These effects may favour viral replication while facilitating the progression of liver disease
- Upregulation of indoleamine 2,3-dioxygenase in hepatitis C virus infection(American Society for Microbiology, 2007) Civeira, M.P. (María Pilar); Riezu-Boj, J.I. (José Ignacio); Gil-Guerrero, L. (Lucía); Larrea, E. (Esther); Rollier, C.S. (Christine S.); Aldabe, R. (Rafael); Casares, N. (Noelia); Verstrepen, B.E. (Babs E.); Sarobe, P. (Pablo); Heeney, J.L. (Jonathan L.)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
- Detection of hepatitis C virus antibodies with new recombinant antigens: assessment in chronic liver diseases(Elsevier, 1992) Civeira, M.P. (María Pilar); Riezu-Boj, J.I. (José Ignacio); Camps, J. (J.); Corbishley, T.P. (T.P.); Castilla, A. (Alberto); Parker, D. (D.); Prieto, J. (Jesús); Phippard, D. (D.)A new serological assay to detect antibodies against hepatitis C, based on a recombinant protein (BHC10) which incorporates structural and non-structural viral antigens, was tested in 67 healthy subjects and 409 patients with various forms of liver disease. Results were compared with the current assay based on the recombinant non-structural viral antigen c100 and with the recently introduced second-generation assay, Ortho2. None of the healthy subjects was positive by any of the assays. In patients with chronic non-A, non-B hepatitis the prevalence of anti-BHC10 was 96.8%, higher than anti-c100 (83.3%, p less than 0.001) and similar to Ortho2 (94.3%). False-positive results were less frequently found when BHC10 was used. These findings show that assays incorporating structural and non-structural antigens provide higher sensitivity to detect hepatitis C virus infection and they define an almost exclusive role of hepatitis C virus in the genesis of chronic non-A, non-B hepatitis.