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Expression of interferon-alpha subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon-alpha5

2012-10-10T00:11:12Z

Title: Expression of interferon-alpha subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon-alpha5 Author(s) : Larrea, E. (Esther); Alberdi, A. (A.); Castelruiz, Y. (Yurdana); Boya, P. (Patricia); Civeira, M.P. (María Pilar); Prieto, J. (Jesús) Abstract: Interferon (IFN)-alpha is a family of antiviral proteins encoded by different genes. The biological significance of the existence of various IFN-alpha subtypes is not clear. We have investigated the interferon system in chronic hepatitis C virus (HCV) infection, a disease that responds to interferon-alpha2 therapy in only a limited proportion of cases. We analysed the expression of interferon regulatory factor (IRF)-1, IRF-2, and IFN-alpha subtypes in nonstimulated and Sendai virus-stimulated peripheral blood mononuclear cells (PBMC) from HCV infected patients and healthy controls. We observed that the IRF-1 mRNA and IRF-1/IRF-2 ratios were increased in PBMC from hepatitis C patients with respect to normal subjects. Sendai virus stimulation of PBMC led to a significant increase in the levels of IRF-1, IRF-2 and IFN-alpha mRNAs and in the production of IFN-alpha protein with respect to basal values in healthy controls as well as in patients with HCV infection. In addition, we found that while natural HCV infection induced increased IFN-alpha5 expression in PBMC, in vitro infection of these cells with Sendai virus caused a raise in the expression of IFN-alpha8 in both patients and normal controls. In summary, our results indicate that virus-induced activation of the IFN system in human PBMC is associated with selective expression of individual IFN-alpha subtypes, IFN-alpha5 being the specific subtype induced in PBMC from patients with chronic HCV infection.

Altered expression and activation of signal transducers and activators of transcription (STATs) in hepatitis C virus infection: in vivo and in vitro studies

2012-10-10T00:11:12Z

Title: Altered expression and activation of signal transducers and activators of transcription (STATs) in hepatitis C virus infection: in vivo and in vitro studies Author(s) : Larrea, E. (Esther); Aldabe, R. (Rafael); Molano, E. (Eduardo); Fernandez-Rodriguez, C.M. (Conrado M.); Ametzazurra, A. (Amagoia); Civeira, M.P. (María Pilar); Prieto, J. (Jesús) Abstract: 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

Characterization of the human Nalpha-terminal acetyltransferase B enzymatic complex

2012-10-08T11:12:22Z

Title: Characterization of the human Nalpha-terminal acetyltransferase B enzymatic complex Author(s) : Ametzazurra, A. (Amagoia); Gazquez, C. (Cristina); Lasa, M. (Marta); Larrea, E. (Esther); Prieto, J. (Jesús); Aldabe, R. (Rafael) Abstract: BACKGROUND: Human Nalpha-acetyltransferase complex B (hNatB) is integrated by hNaa20p (hNAT5/hNAT3) and hNaa25p (hMDM20) proteins. Previous data have shown that this enzymatic complex is implicated in cell cycle progression and carcinogenesis. In yeast this enzyme acetylates peptides composed by methionine and aspartic acid or glutamic acid in their first two positions respectively and it has been shown the same specificity in human cells. METHODS: We have silenced hNAA20 expression in hepatic cell lines using recombinant adenoviruses that express specific siRNAs against this gene and analyzed cell cycle progression and apoptosis induction after this treatment. Immunopurified hNatB enzymatic complexes from human cell lines were used for analyzing hNatB in vitro enzymatic activity using as substrate peptides predicted to be acetylated by NatB. RESULTS: hNAA20 silencing in hepatic cell lines reduces cell proliferation in a p53 dependent and independent manner. At the same time this treatment sensitizes the cells to a proapototic stimulus. We have observed that the hNatB complex isolated from human cell lines can acetylate in vitro peptides that present an aspartic or glutamic acid in their second position as has been described in yeast. CONCLUSION: hNatB enzymatic complex is implicated in cell cycle progression but it exerts its effects through different mechanisms depending on the cellular characteristics. This is achievable because it can acetylate a great number of peptides composed by an aspartic or glutamic acid at their second residue and therefore it can regulate the activity of a great number of proteins.

The protein kinase IKKepsilon can inhibit HCV expression independently of IFN and its own expression is downregulated in HCV-infected livers

2012-10-09T00:12:01Z

Title: The protein kinase IKKepsilon can inhibit HCV expression independently of IFN and its own expression is downregulated in HCV-infected livers Author(s) : Velasco, M. (Myriam); Larrea, E. (Esther); Vitour, D. (Damien); Dabo, S. (Stephanie); Breiman, A. (Adrien); Regnault, B. (Béatrice); Riezu-Boj, J.I. (José Ignacio); Eid, P. (Pierre); Prieto, J. (Jesús); Meurs, E.F. (Eliane F.) 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.

The stilbene disulfonic acid DIDS stimulates the production of TNF-alpha in human lymphocytes

2012-10-09T00:11:59Z

Title: The stilbene disulfonic acid DIDS stimulates the production of TNF-alpha in human lymphocytes Author(s) : Qian, C. (Cheng); Diez, J. (Javier); Larrea, E. (Esther); Garciandia, A. (Ana); Arrazola, A. (Arantxa); Civeira, M.P. (María Pilar); Medina, J.F. (Juan Francisco); Prieto, J. (Jesús) Abstract: 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.

Interferon alfa subtypes and levels of type I interferons in the liver and peripheral mononuclear cells in patients with chronic hepatitis C and controls

2012-10-09T00:12:01Z

Title: Interferon alfa subtypes and levels of type I interferons in the liver and peripheral mononuclear cells in patients with chronic hepatitis C and controls Author(s) : Castelruiz, Y. (Yurdana); Larrea, E. (Esther); Boya, P. (Patricia); Civeira, M.P. (María Pilar); Prieto, J. (Jesús) Abstract: Viral infections stimulate the transcription of interferon type I, which includes IFN-alfa (IFN-alpha) (13 subtypes) and IFN-beta (a single substance). Hepatitis C virus (HCV) infection is remarkable by its ability to evade host antiviral defenses; however, there is little information as to whether endogenous IFN is activated or not in this disease. Additionally, despite the fact that the various IFN-alpha subtypes may differ in biological activity, there are no data concerning the IFN-alpha subtypes specifically expressed in normal and diseased liver tissue. Thus, we have analyzed the IFN-alpha subtypes and the mRNA levels of type I IFNs in samples of normal liver tissue and in liver from patients with chronic hepatitis C. Similar studies were performed in peripheral blood mononuclear cells (PBMC) from patients and controls. After amplification and cloning of IFN-alpha cDNA, we observed that 98 of the 100 clones from normal liver tissue corresponded to the IFN-alpha5 subtype. However, in livers with chronic hepatitis C and in PBMC from controls and patients, a variety of subtypes, in addition to IFN-alpha5, were detected, suggesting a participation of infiltrating leukocytes in the production of IFN-alpha in livers with chronic hepatitis C. As compared with controls, patients with chronic hepatitis C showed a significant increase in IFN-beta mRNA in both the liver and PBMC, while IFN-alpha mRNA was significantly increased in PBMC but markedly reduced in liver tissue. In conclusion, IFN-alpha5 is the sole IFN-alpha subtype expressed in normal liver tissue. The hepatic levels of IFN-alpha are reduced in chronic hepatitis C, an event that may favor viral persistence.

Nuclear factor-kappa B in the liver of patients with chronic hepatitis C: decreased RelA expression is associated with enhanced fibrosis progression

2012-10-09T10:44:48Z

Title: Nuclear factor-kappa B in the liver of patients with chronic hepatitis C: decreased RelA expression is associated with enhanced fibrosis progression Author(s) : Boya, P. (Patricia); Larrea, E. (Esther); Sola, J. (Josu); Majano, P.L. (Pedro Lorenzo); Jimenez, C. (Carlos); Civeira, M.P. (María Pilar); Prieto, J. (Jesús) Abstract: The mechanisms of liver damage in chronic hepatitis C virus (HCV) infection are poorly understood. The transcription factor, nuclear factor-kappa B (NF-kappa B), regulates the expression of genes involved in apoptosis, inflammation, and antiviral response. It plays a protective role in several forms of liver damage. In this study, we analyzed NF-kappa B by gel mobility shift assay and immunohistochemistry in liver biopsies from HCV-infected patients, and we have determined the hepatic levels of the components of the NF-kappa B system by semiquantitative polymerase chain reaction (PCR). We found that NF-kappa B was activated in the liver of patients with chronic hepatitis C. Neither NF-kappa B activity nor the RNA levels of NF-kappa B subunits showed correlation with liver inflammatory activity, viral load, or HCV genotype. By contrast, hepatic mRNA values of RelA, the main element of active NF-kappa B, correlated inversely with apoptosis (r = -.68; P <.05) and with the rate of fibrosis progression (r = -.51; P <.04). In intermediate/rapid fibrosers, RelA mRNA levels were significantly decreased as compared with slow fibrosers (P <.003) and with normal livers (P <.03). In conclusion, we found that NF-kappa B is activated in chronic HCV-infected livers, and that the expression of RelA is inversely correlated with liver cell apoptosis and with the rate of fibrosis progression. Our data thus suggest that RelA expression may protect against liver fibrosis and hepatocellular damage.

Tumor necrosis factor alpha gene expression and the response to interferon in chronic hepatitis C

2012-10-06T00:06:47Z

Title: Tumor necrosis factor alpha gene expression and the response to interferon in chronic hepatitis C Author(s) : Larrea, E. (Esther); Garcia, N. (Nicolás); Qian, C. (Cheng); Civeira, M.P. (María Pilar); Prieto, J. (Jesús) Abstract: Tumor necrosis factor alpha (TNF-alpha) is a cytokine with pleiotropic properties that is induced in a variety of pathological situations including viral infections. In this work, we analyzed the expression of TNF-alpha gene in patients with chronic hepatitis C. Serum TNF-alpha levels were found to be elevated in all chronic hepatitis C patients including those cases presenting sustained biochemical remission of the disease after interferon therapy. Untreated patients with chronic hepatitis C showed increased TNF-alpha messenger RNA (mRNA) levels in the liver and mononuclear cells as compared with healthy controls. After completion of treatment with interferon, patients experiencing sustained complete response showed values of TNF-alpha mRNA, both in the liver and in peripheral mononuclear cells, within the normal range, significantly lower than patients who did not respond to interferon and than those with complete response who relapsed after interferon withdrawal. Pretreatment values of TNF-alpha mRNA were lower in long-term responders to interferon than in cases who failed to respond to the treatment. Values of TNF-alpha mRNA in the liver or in mononuclear cells were higher in specimens with positive hepatitis C virus (HCV) RNA than in those samples where the virus was undetectable. Neither the intensity of the liver damage nor the amount of HCV RNA in serum or in cells showed correlation with the levels of TNF-alpha transcripts in peripheral mononuclear cells but it was found that high TNF-alpha values were associated with genotype 1b. In conclusion, there is an enhanced expression of TNF-alpha in HCV infection. High levels of this cytokine may play a role in the resistance to interferon therapy.

Antioxidant status and glutathione metabolism in peripheral blood mononuclear cells from patients with chronic hepatitis C

2012-10-06T00:06:19Z

Title: Antioxidant status and glutathione metabolism in peripheral blood mononuclear cells from patients with chronic hepatitis C Author(s) : Boya, P. (Patricia); Peña, A. (Andrés) de la; Beloqui, O. (Óscar); Larrea, E. (Esther); Conchillo, M. (M.); Castelruiz, Y. (Yurdana); Civeira, M.P. (María Pilar); Prieto, J. (Jesús) Abstract: BACKGROUND/AIMS: Oxidative stress could play a role in the pathogenesis of hepatitis C virus infection. We investigated the oxidant/antioxidant status in peripheral blood mononuclear cells from patients with chronic hepatitis C and controls. METHODS/RESULTS: Lipid peroxidation products and superoxide dismutase activity in peripheral blood mononuclear cells were higher in chronic hepatitis C patients than in healthy subjects while glutathione S-transferase activity was reduced in patients as compared to controls. Catalase, glutathione peroxidase and glutathione reductase were similar in chronic hepatitis C and normal individuals. No statistically significant differences were found between patients and controls with regard to glutathione levels in peripheral blood mononuclear cells, but 35% of patients with chronic hepatitis C showed values of glutathione and oxidized glutathione which were below and above, respectively, the limits of normal controls. Finally, the glutathione synthetic capacity of the cytosol of peripheral blood mononuclear cells was significantly higher in patients than in controls, indicating increased glutathione turnover in lymphocytes from patients with chronic hepatitis C. CONCLUSIONS: Oxidative stress is observed in peripheral blood mononuclear cells from chronic hepatitis C patients. This process might alter lymphocyte function and facilitate the chronicity of the infection.

Superoxide dismutase in patients with chronic hepatitis C virus infection

2012-10-03T00:03:48Z

Title: Superoxide dismutase in patients with chronic hepatitis C virus infection Author(s) : Larrea, E. (Esther); Beloqui, O. (Óscar); Muñoz-Navas, M. (Miguel); Civeira, M.P. (María Pilar); Prieto, J. (Jesús) Abstract: It has been reported that hepatitis C virus (HCV) may cause oxidative stress in infected cells. Patients with chronic hepatitis C exhibit an increased production of tumor necrosis factor-alpha (TNF alpha), a cytokine that can produce oxidative stress by stimulating the generation of reactive oxygen species (ROS). Cell defense against ROS includes overexpression of Mn-superoxide dismutase (SOD), an inducible mitochondrial enzyme. To investigate cell defense against oxidative stress in HCV infection, we analyzed Mn-SOD mRNA in liver and in peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis C. Mn-SOD expression in PBMC was significantly increased in patients with HCV infection. Patients with sustained virological and biochemical response after therapy showed significantly lower Mn-SOD than patients with positive viremia. By contrast, Mn-SOD expression was not enhanced in the liver of patients with chronic hepatitis C. The values of Mn-SOD mRNA did not correlate with TNF alpha mRNA expression, viral load, or liver disease activity. Our results indicate that in HCV infection an induction of Mn-SOD was present in PBMC but absent in the liver, suggesting that this organ could be less protected against oxidative damage. Oxidative stress could participate in the pathogenesis of HCV infection.

Cardiotrophin-1 promotes a high survival rate in rabbits with lethal fulminant hepatitis of viral origin

2012-08-11T00:09:38Z

Title: Cardiotrophin-1 promotes a high survival rate in rabbits with lethal fulminant hepatitis of viral origin Author(s) : Tuñon, M.J. (María Jesús); San-Miguel, B. (Beatriz); Crespo, I. (Irene); Riezu-Boj, J.I. (José Ignacio); Larrea, E. (Esther); Alvarez, M. (Marcelino); Gonzalez, I. (Iranzu); Bustos, M. (Matilde); Gonzalez-Gallego, J. (Javier); Prieto, J. (Jesús) Abstract: Rabbit hemorrhagic disease virus (RHDV) causes lethal fulminant hepatitis closely resembling acute liver failure (ALF) in humans. In this study, we investigated whether cardiotrophin-1 (CT-1), a cytokine with hepatoprotective properties, could attenuate liver damage and prolong survival in virus-induced ALF. Twenty-four rabbits were infected with 2 × 10(4) hemagglutination units of RHDV. Twelve received five doses of CT-1 (100 μg/kg) starting at 12 h postinfection (hpi) (the first three doses every 6 h and then two additional doses at 48 and 72 hpi), while the rest received saline. The animals were analyzed for survival, serum biochemistry, and viral load. Another cohort (n = 22) was infected and treated similarly, but animals were sacrificed at 30 and 36 hpi to analyze liver histology, viral load, and the expression of factors implicated in liver damage and repair. All infected rabbits that received saline died by 60 hpi, while 67% of the CT-1-treated animals survived until the end of the study. Treated animals showed improved liver function and histology, while the viral loads were similar. In the livers of CT-1-treated rabbits we observed reduction of oxidative stress, diminished PARP1/2 and JNK activation, and decreased inflammatory reaction, as reflected by reduced expression of tumor necrosis factor alpha, interleukin-1β, Toll-like receptor 4, VCAM-1, and MMP-9. In addition, CT-1-treated rabbits exhibited marked upregulation of TIMP-1 and increased expression of cytoprotective and proregenerative growth factors, including platelet-derived growth factor B, epidermal growth factor, platelet-derived growth factor receptor β, and c-Met. In conclusion, in a lethal form of acute viral hepatitis, CT-1 increases animal survival by attenuating inflammation and activating cytoprotective mechanisms, thus representing a promising therapy for ALF of viral origin.

IFN-alpha5 mediates stronger Tyk2-stat-dependent activation and higher expression of 2',5'-oligoadenylate synthetase than IFN-alpha2 in liver cells

2012-08-07T00:10:02Z

Title: IFN-alpha5 mediates stronger Tyk2-stat-dependent activation and higher expression of 2',5'-oligoadenylate synthetase than IFN-alpha2 in liver cells Author(s) : Larrea, E. (Esther); Aldabe, R. (Rafael); Riezu-Boj, J.I. (José Ignacio); Guitart, A. (Anunciata); Civeira, M.P. (María Pilar); Prieto, J. (Jesús); Baixeras, E. (Elena) Abstract: 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.

Vaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity

2012-04-23T07:44:12Z

Title: Vaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity Author(s) : Rollier, C.S. (Christine S.); Paranhos-Baccala, G. (Glaucia); Verschoor, E.J. (Ernst J.); Verstrepen, B.E. (Babs E.); Drexhage, J.A.R. (Joost A.R.); Fagrouch, Z. (Zahra); Berland, J.L. (Jean-Luc); Komurian-Pradel, F. (Florence); Duverger, B. (Blandine); Himoudi, N. (Nourredine); Staib, C. (Caroline); Meyr, M. (Marcus); Whelan, M. (Myke); Whelan, J.A. (Joseph A.); Adams, V.A. (Victoria A.); Larrea, E. (Esther); Riezu-Boj, J.I. (José Ignacio); Lasarte, J.J. (Juan José); Bartosch, B. (Birke); Cosset, F. L. (Francoise-L.); Spaan, W.J.M. (Willy J.M.); Diepolder, H.M. (Helmut M.); Pape, G.R. (Gerd R.); Sutter, G. (Gerd); Inchauspe, G. (Geneviese); Heeney, J.L. (Jonathan L.) Abstract: Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P=0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 [corrected] and PD-1 levels in liver correlated with clearance or chronicity. CONCLUSION: Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history.

Induction of immunosuppressive molecules and regulatory T cells counteracts the antitumor effect of interleukin-12-based gene therapy in a transgenic mouse model of liver cancer

2012-04-23T07:41:09Z

Title: Induction of immunosuppressive molecules and regulatory T cells counteracts the antitumor effect of interleukin-12-based gene therapy in a transgenic mouse model of liver cancer Author(s) : Zabala, M. (Maider); Lasarte, J.J. (Juan José); Perret, C. (Christine); Sola, J. (Josu); Berraondo, P. (Pedro); Alfaro, M. (Maite); Larrea, E. (Esther); Prieto, J. (Jesús); Kramer, M.G. (María Grabiela) Abstract: BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) often lacks curative treatment; therefore new efficient therapies are needed. In this work we aimed at evaluating the antitumor effect of interleukin-12 (IL-12)-based gene therapy on HCC occurring spontaneously in mice. METHODS: A plasmid-vector expressing IL-12 in a liver-specific and doxycycline (Dox)-inducible manner was transferred by hydrodynamic injection to the liver of L-PK/c-myc mice with HCC. IL-12 expression was induced by administering Dox (3 cycles of 1 month duration separated by 1 month rest). RESULTS: Dox administration increased serum IL-12 and IFN-gamma and induced tumor lymphocytic infiltration in all treated mice which was accompanied by tumor stabilization or regression in 40% of animals. The antitumor effect did not correlate with levels of IL-12 or IFN-gamma nor with the intensity of tumor mononuclear infiltration. However, tumors from non-responder mice showed more abundance of Foxp3+ regulatory T cells and higher expression of the immunosuppressive molecules PD-1, PD-L1, VEGF, CTLA-4, IDO, and IL-10 than those that responded to therapy. CONCLUSIONS: Although long-term induction of IL-12 expression in the liver can inhibit HCC growth, the efficacy of the treatment appears to be limited by the activation of immunosuppressive mechanisms.

Enhancement of peptide immunogenicity by insertion of a cathepsin B cleavage site between determinants recognized by B and T cells

2012-04-18T00:10:36Z

Title: Enhancement of peptide immunogenicity by insertion of a cathepsin B cleavage site between determinants recognized by B and T cells Author(s) : Sarobe, P. (Pablo); Lasarte, J.J. (Juan José); Larrea, E. (Esther); Golvano, J.J. (José Javier); Prieto, I. (Inés); Gullon, A. (Arturo); Prieto, J. (Jesús); Borras-Cuesta, F. (Francisco) Abstract: The insertion of two lysine residues (cleavage sites of cathepsin B) at the boundary of a peptide recognized by B cells (BD) and a class-II- presentable sequence (TDh) enhanced the anti-BD antibody induction capacity of this type of peptide construct, as well as production of IL2. It is postulated that these lysines generate a neoprocessable site which helps in release of the TDh moiety from the construct, enabling its presentation to class II molecules, an essential step in clonal expansion of the antibody-producing B cell after internalization of the construct via the BD moiety.