Esther Larrea

A clinical trial of CTLA-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis C

BACKGROUND & AIMS Tremelimumab is a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), an inhibitory co-receptor that interferes with T cell activation and proliferation. The purpose of this pilot clinical trial was to test the antitumor and antiviral effect of tremelimumab in patients with hepatocellular carcinoma (HCC) and chronic hepatitis C virus (HCV) infection; and to study the safety of its administration to cirrhotic patients. METHODS Tremelimumab at a dose of 15 mg/kg IV every 90 days was administered until tumor progression or severe toxicity. Twenty patients were assessable for toxicity and viral response and 17 were assessable for tumor response. Most patients were in the advanced stage and 43% had an altered liver function (Child-Pugh class B). RESULTS A good safety profile was recorded and no patient needed steroids because of severe immune-mediated adverse events. Some patients had a transient albeit intense elevation of transaminases after the first dose, but not following subsequent cycles. Partial response rate was 17.6% and disease control rate was 76.4%. Time to progression was 6.48 months (95% CI 3.95-9.14). A significant drop in viral load was observed while new emerging variants of the hypervariable region 1 of HCV replaced the predominant variants present before therapy, particularly in those patients with a more prominent drop in viral load. This antiviral effect was associated with an enhanced specific anti-HCV immune response. CONCLUSIONS Tremelimumab safety profile and antitumor and antiviral activity, in patients with advanced HCC developed on HCV-induced liver cirrhosis, support further investigation.

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

Tumor necrosis factor α (TNF‐α) 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‐α gene in patients with chronic hepatitis C. Serum TNF‐α 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‐α 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‐α 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‐α mRNA were lower in long‐term responders to interferon than in cases who failed to respond to the treatment. Values of TNF‐α 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‐α transcripts in peripheral mononuclear cells but it was found that high TNF‐α values were associated with genotype 1b. In conclusion, there is an enhanced expression of TNF‐α in HCV infection. High levels of this cytokine may play a role in the resistance to interferon therapy.

Superoxide Dismutase in Patients With Chronic Hepatitis C Virus Infection

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.

Upregulation of Indoleamine 2,3-Dioxygenase in Hepatitis C Virus Infection

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.

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

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 (1) 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. (HEPATOLOGY 2007;45:602–613.)

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

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: STAT3α 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)-α and IFN-γ) were increased in HCV infected livers, particularly in those with high inflammatory activity. Conversely, phosphorylated STAT2 (a factor selectively activated by IFN-α) was undetectable in livers with HCV infection, a finding that was associated with marked downregulation of the two functional subunits of the IFN-α receptor. HCV replication in Huh7 cells caused STAT3α downregulation and blocked STAT3 phosphorylation by either IFN-α or cardiotrophin 1. HCV replication in Huh7 cells also inhibited STAT1 and STAT2 activation by IFN-α while there was no impairment of STAT1 phosphorylation by the proinflammatory cytokine IFN-γ. 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.

Hepatitis C virus induces the expression of CCL17 and CCL22 chemokines that attract regulatory T cells to the site of infection

BACKGROUND & AIMS The mechanisms by which Foxp3+ T regulatory cells (Treg) accumulate in HCV infected livers are not known. Here, we studied the role of chemokines CCL17 and CCL22 in this process. METHODS Chemokine mRNA levels were determined by qPCR in liver biopsies from 26 HCV chronically infected patients (CHC), 11 patients with treatment-induced sustained virological response (SVR), 16 patients with other liver diseases unrelated to HCV, and 24 normal livers. Double-immunofluorescence Foxp3/CD3 or CD11c/CCL22 was performed in liver sections. Chemokine production by monocyte-derived dendritic cells (MDDC) co-cultured with uninfected or HCV-JFH1 infected Huh7 cells was measured by qPCR and ELISA. Chemotactic activity of culture supernatants was also tested. RESULTS Foxp3+ Treg were increased in CHC livers as compared to controls. Patients with CHC showed elevated intrahepatic levels of CCL17 mRNA compared to normal livers or livers from subjects with SVR or other forms of liver disease. Intrahepatic CCL22 expression was also higher in CHC than in healthy subjects or SVR patients but similar to that observed in other liver diseases. Dendritic cells producing CCL22 could be found inside the hepatic lobule in CHC patients. Contact between MDDC and HCV-JFH1-infected Huh7 cells induced the expression of CCL17 and CCL22 in a process partially dependent on ICAM-1. Transwell experiments showed that upregulation of these chemokines enhanced Treg migration. CONCLUSIONS Contact of HCV-infected cells with dendritic cells induces the production of Treg-attracting chemokines, an effect which may favour liver accumulation of Treg in CHC. Our findings contribute to explain the mechanism by which HCV escapes the immune response and thus reveals novel therapeutic targets.

Nuclear factor‐κB in the liver of patients with chronic hepatitis C: Decreased RelA expression is associated with enhanced fibrosis progression

The mechanisms of liver damage in chronic hepatitis C virus (HCV) infection are poorly understood. The transcription factor, nuclear factor‐κB (NF‐κB), 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‐κB 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‐κB system by semiquantitative polymerase chain reaction (PCR). We found that NF‐κB was activated in the liver of patients with chronic hepatitis C. Neither NF‐κB activity nor the RNA levels of NF‐κB 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‐κB, 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‐κB 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.

Effects of IFN-α as a signal-3 cytokine on human naïve and antigen-experienced CD8(+) T cells.

IFN‐α/β link innate and adaptive immune responses by directly acting on naïve CD8+ T cells. This concept unveiled in mice remains unexplored in humans. To investigate that, human CD8+CD45RO− cells were stimulated with beads coated with anti‐CD3 and anti‐CD28 mAb, mimicking Ag (type‐1) and co‐stimulatory (type‐2) signals, in the presence or absence of IFN‐α and their transcriptional profiles were defined by cDNA‐microarrays. We show that IFN‐α provides a strong third signal directly to human CD8+ T cells resulting in regulation of critical genes for their overall activation. This transcriptional effect was substantiated at the protein level and verified by functional assays. Interestingly, the biological effects derived from this stimulation vary depending on the CD8+ T‐cell population. Thus, whereas IFN‐α increases the proliferative capacity of naïve CD8+ T cells, it inhibits or does not affect the proliferation of Ag‐experienced cells, such as memory and effector CTL, including CMV‐specific lymphocytes. Cytolysis and IFN‐γ‐secretion of all these populations are enhanced by IFN‐α‐derived signals, which are critical in naïve CD8+ T cells for acquisition of effector functions. Our findings in human CD8+ T cells are informative to understand and improve IFN‐α‐based therapies for viral and malignant diseases.

Prediction of sustained remission of chronic hepatitis C after a 12-month course of alfa interferon

alpha-Interferon therapy normalizes aminotransferase levels in approximately 50% of the patients with chronic hepatitis C, but post-therapy relapses are common and predictive factors of sustained response remain largely unknown. We retrospectively assessed several parameters as predictors of sustained remission after a 12-month course of lymphoblastoid alpha-interferon: the Knodell histological activity index, serum levels of procollagen type III peptide, serum HCV-RNA, anti-alpha-interferon antibodies, and anti-HCV antibodies (C-100-3), all at month 12. Thirty-seven patients were studied. Fourteen patients were non-responders (38%), 15 patients experienced a sustained response (40.5%) and eight patients responded similarly but relapsed after alpha-interferon withdrawal (21.5%). A decrease in the histological activity index above 5, normalization of procollagen type III peptide levels (< 12 ng/ml) and the absence of viremia after treatment were all significantly associated with a sustained response (p = 0.008, p = 0.007 and p = 0.037, respectively). Anti-interferon antibodies were detected in only one non-responder patient. Anti-C-100-3 antibodies became undetectable at month 12 in 5 of the 15 sustained responders. The best prediction of sustained response was obtained from the three variables independent of multivariate analysis according to the following equation: F = 0.872 + 0.067 x K (decrease of histological index) -0.052 x P (procollagen type III peptide levels at month 12) -0.28 x R (HCV-RNA at month 12; R = 2 when present and R = 1 when absent). A score higher than 0 predicted sustained remission with a 100% sensitivity and specificity in this series of patients.(ABSTRACT TRUNCATED AT 250 WORDS)