IFN Treatment Research Articles

TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice.

Despite their antiviral effect, the invivo effect of interferons on HIV transmission is difficult to predict, because interferons also activate and recruit HIV-susceptible cells to sites of infection. HIV does not normally induce type I interferons in infected cells, but does if TREX1 is knocked down. Here, we investigated the effect of topical TREX1 knockdown and local interferon production on HIV transmission in human cervicovaginal explants and humanized mice. Inexplants in which TREX1 was knocked down, HIV induced interferons, which blocked infection. In humanized mice, even though TREX1 knockdown increased infiltrating immune cells, it delayed viral replication for 3-4weeks. Similarly intravaginal application of type I interferons the day before HIV infection induced interferon responsive genes, reduced inflammation, and decreased viral replication. However, intravenous interferon enhanced inflammation and infection. Thus, in models of human sexual transmission, a localized interferon response inhibits HIV transmission but systemic interferons do not.

TLR7 responsiveness of human transitional B cells

Abstract Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is known to be associated with B cell hyperactivity and production of autoantibodies (Auto-Abs) against nucleoproteins. Auto-Abs reactive to RNA-containing antigens are found in a fraction of SLE patients, particularly those with severe disease activity. Toll-like receptor 7 (TLR7), an intracellular TLR that recognizes ssRNA, has been highly implicated in the generation of pathogenic anti-RNA autoantibodies in SLE; however, the cellular origin of anti-RNA Ab–secreting cells is not well defined. We have found that increased expression of TLR7 in TLR7Tg mice results in activation of their newly-formed (NF) transitional B cells which upon stimulation produced class-switched IgGs, including anti-RNA auto-Abs. Subsequent analysis of the B cell compartment in SLE patients revealed a correlation between the levels of TLR7 expression and increases in the frequencies of circulating CD20+CD27−CD10+CD38highCD24high NF B cells. SLE patients with dysregulation of NF B cells were also more likely to have Sm/RNP auto-Abs. IFNa treatment induced more than a 10-fold increase in the mRNA expression of TLR7, UNC93B and IRF7 in NF B cells isolated from umbilical cord blood. Furthermore, IFNa-primed NF B cells became highly responsive to in vitro stimulation with TLR7 ligand-R848 and differentiated into CD27+CD38hiCD24lo plasmablasts in the absence of any other stimuli. Human NF B cells are enriched in polyreactive specificities and increases of NF B cells have been described in SLE patients. Our studies suggest that human NF B cells might be an important source of auto-Ab-secreting cells and provide a new link between innate IFNa and TLR7 signaling and B cell activation in SLE.

Oxethazaine inhibits hepatitis B virus capsid assembly by blocking the cytosolic calcium-signalling pathway.

Chronic hepatitis B virus (HBV) infection is a serious public health problem and may progress to liver fibrosis, cirrhosis and hepatocellular carcinoma. It is currently treated with PEGylated IFN-α2a and nucleoside/nucleotide analogues (NAs). However, PEGylated IFN treatment has problems of high cost, low efficiency and side effects. Long-term administration of NAs is necessary to avoid virus relapse, which can cause drug resistance and side effects. New efforts are now being directed to develop novel anti-HBV drugs targeting either additional viral targets other than viral DNA polymerase or host targets to improve the treatment of chronic hepatitis B. In this study, we discovered that oxethazaine, approved for clinic use in a few countries such as Japan, India, South Africa and Brazil, can dose-dependently reduce the levels of HBV envelope antigen, extracellular HBV DNA in supernatants and intracellular HBV total DNA. However, the levels of HBV cccDNA and HBV RNAs were not affected by oxethazaine treatment. Further study confirmed that oxethazaine acts on the virus assembly stage of the HBV life cycle. A study of the mechanisms of oxethazaine suggested that this drug inhibits HBV replication and capsid assembly by blocking the cytosolic calcium-signalling pathway. Moreover, oxethazaine could inhibit the replication of lamivudine/entecavir-dual-resistant and adefovir-resistant HBV mutants. In conclusion, our study suggests that oxethazaine may serve as a promising drug, or could be used as a starting point for anti-HBV drug discovery.

Transcriptomics in Interferon-α-Treated Patients Identifies Inflammation-, Neuroplasticity- and Oxidative Stress-Related Signatures as Predictors and Correlates of Depression.

Owing to the unique opportunity to assess individuals before and after they develop depression within a short timeframe, interferon-α (IFN-α) treatment for chronic hepatitis C virus (HCV) infection is an ideal model to identify molecular mechanisms relevant to major depression, especially in the context of enhanced inflammation. Fifty-eight patients were assessed prospectively, at baseline and monthly over 24 weeks of IFN-α treatment. New-onset cases of depression were determined using the Mini International Neuropsychiatric Interview (MINI). Whole-blood transcriptomic analyses were conducted to investigate the following: (1) baseline gene expression differences associated with future development of IFN-α-induced depression, before IFN-α, and (2) longitudinal gene expression changes from baseline to weeks 4 or 24 of IFN-α treatment, separately in those who did and did not develop depression. Transcriptomics data were analyzed using Partek Genomics Suite (1.4-fold, FDR adjusted p⩽0.05) and Ingenuity Pathway Analysis Software. Twenty patients (34%) developed IFN-α-induced depression. At baseline, 73 genes were differentially expressed in patients who later developed depression compared with those who did not. After 4 weeks of IFN-α treatment, 592 genes were modulated in the whole sample, representing primarily IFN-α-responsive genes. Substantially more genes were modulated only in patients who developed depression (n=506, compared with n=70 in patients who did not), with enrichment in inflammation-, neuroplasticity- and oxidative stress-related pathways. A similar picture was observed at week 24. Our data indicate that patients who develop IFN-α-induced depression have an increased biological sensitivity to IFN-α, as shown by larger gene expression changes, and specific signatures both as predictors and as correlates.

SCHLAFEN 5 expression correlates with intestinal metaplasia that progresses to gastric cancer.

Intestinal metaplasia (IM) is a gastric cancer precursor lesion (GCPL) and an extremely high risk factor for progression to gastric cancer (GC). Clinical guidelines recommend that patients with extensive IM undergo a gastroscopy every 3years. However, protein biomarkers that indicate a transition from IM to GC are lacking. Our group recently identified an interferon-alpha (IFNα)-responsive gene, Schlafen 4 (Slfn4), in immune cells that correlates with metaplastic changes in Helicobacter-infected mice. We therefore tested the hypothesis that a human homolog of Slfn4, namely, Schlafen 5 (SLFN5), correlates with progression of GCPL to GC. Jurkat T-lymphoid and HL-60 myeloid cell lines were treated with IFNα, and SLFN5 mRNA was quantified by quantitative PCR. SLFN5 protein expression in the inflamed gastric mucosa was co-localized to specific immune cell types by immunohistochemistry using CD20, CD2, and MAC2 antibodies. SLFN5 expression was also determined by immunohistochemistry in formalin-fixed paraffin-embedded samples from individuals with non-atrophic gastritis, atrophic gastritis, complete IM, incomplete IM, and GC, respectively. The IFNα treatment of Jurkat and HL-60 cells induced SLFN5 mRNA. SLFN5 protein was expressed mainly by T lymphocytes in inflamed gastric mucosa. The highest level of SLFN5 expression was observed in patients with IM that progressed to GC. Receiver operating characteristic curves demonstrated that correlating SLFN5 expression with the histologic diagnosis of IM significantly increased the probability of identifying patients who may progress to GC. In this study population, elevated SLFN5 protein expression in patients with IM correlated with progression to GC.

Type I interferon inhibits varicella-zoster virus replication by interfering with the dynamic interaction between mediator and IE62 within replication compartments.

BackgroundVaricella-zoster virus (VZV) is the causative agent of varicella and zoster. The immediate-early protein, IE62 is the predominant VZ virion tegument protein, transactivating the expression of all kinetic classes of VZV genes. IE62 is localized to punctae that form DNA replication compartments in the nuclei of VZV infected cells. The morphological changes and the increase in the size of replication compartments that express IE62 are correlated with production of VZ virions. Mammalian Mediator serves as a coactivator of IE62 and functions by bridging DNA-binding transcription factors¸ RNA polymerase II (RNAP II) and their target DNAs for VZV replication. While VZV is highly sensitive to type I interferons (IFNs), how IFN-α inhibits early events during VZV replication is poorly understood.ResultsIn this study, we performed in situ analysis to investigate the effects of IFN-α on the dynamic interactions of IE62 with the Mediator MED25 subunit and the RNAP II negative regulator cycle-dependent kinase 8 (CDK8) in VZV infected cells by confocal immunofluorescence. We found that in addition to dose-dependent inhibition of the yields of infectious virus by IFN treatment, IFN-α prominently impeded the development of large IE62+ nuclear compartments and significantly decreased transcription of VZV genes. Both the expression level and stable recruitment of MED25 to IE62+ replication compartments were inhibited by IFN-α. While IFN-α treatment upregulated CDK8 expression, redistribution and recruitment of CDK8 to IE62+ replication compartments in infected cells was not affected by VZV.ConclusionIFN-α exerts multiple inhibitory activities against virus infections. In this study, we provide visionary demonstration that continuous translocation of MED25 into VZV replication compartments ensures production of virions. IFN-α greatly impedes the formation of a stable complex between IE62 and the Mediator complex thereby suppresses VZV gene transcription. Our demonstration that IFN-α-induced antiviral effect against VZV infection is through inhibiting the reorganization of nuclear components uncovers a novel function of IFN-α. Targeting the interaction between IE62 and MED25 may offer a novel approach to the development of antiviral agents against VZV infection.

The anti-proliferative effects of type I IFN involve STAT6-mediated regulation of SP1 and BCL6

Type I IFN-induced STAT6 has been shown to have anti-proliferative effects in Daudi and B cells. IFN-sensitive (DS) and IFN-resistant (DR) subclones of Daudi cells were used to study the role of STAT6 in the anti-proliferative activities. Type I IFN significantly increased STAT6 mRNA and protein expression in DS but not DR cells. STAT6 knockdown significantly reduced the sensitivity to IFN in both cell lines. The molecular targets and functional importance of IFN-activated STAT6 were performed by chromatin immunoprecipitation-on-chip (ChIP-on-chip) experiments in type I IFN-treated Daudi cells. Two target genes (Sp1 and BCL6) were selected from the ChIP-on-chip data. IFN-induced STAT6 activation led to Sp1 upregulation and BCL6 downregulation in DS cells, with only minimal effects in DR cells. siRNA inhibition of STAT6 expression resulted in decreased Sp1 and BCL6 mRNA and protein levels in both DS and DR cells. IFN treatment did not increase Sp1 and BCL6 expression in a STAT2-deficient RST2 cell line, and this effect was mitigated by plasmid overexpression of STAT2, indicating that STAT2 is important for STAT6 activation. These results suggest that STAT6 plays an important role in regulating Sp1 and BCL6 through STAT2 to exert the anti-proliferative effects of type I IFN.

Circulating human cytomegalovirus-encoded HCMV-miR-US4-1 as an indicator for predicting the efficacy of IFNα treatment in chronic hepatitis B patients.

The efficacy of interferon α (IFNα) therapy for chronic hepatitis B (CHB) patients is about 40% and often associates with adverse side-effects, thus identification of an easy accessible biomarker that can predict the outcome of IFNα treatment for individual CHB patients would be greatly helpful. Recent reports by us and others show that microRNAs encoded by human cytomegalovirus (HCMV) were readily detected in human serum and can interfere with lymphocyte responses required by IFNα therapeutic effect. We thus postulate that differential expression profile of serum HCMV miRNAs in CHB patients may serve as indicator to predict the efficacy of IFNα treatment for CHB patients. Blood was drawn from 56 individual CHB patients prior to IFNα treatment. By quantifying 13 HCMV miRNAs in serum samples, we found that the levels of HCMV-miR-US4-1 and HCMV-miR-UL-148D were significantly higher in IFNα-responsive group than in IFNα-non-responsive group. In a prospective study of 96 new CHB patients, serum level of HCMV-miR-US4-1 alone classified those who were and were not responsive to IFN-α treatment with correct rate of 84.00% and 71.74%, respectively. In conclusion, our results demonstrate that serum HCMV-miR-US4-1 can serve as a novel biomarker for predicting the outcome of IFNα treatment in CHB patients.

Nitric oxide level and CD3-ζ expression in response to Interferon- Ribavirin Therapy in chronic Hepatitis C Egyptian patients.

Hepatitis C virus (HCV) infection is one of the major causes of liver diseases all over the world; it is considered one of the leading causes of cirrhosis, hepatic failure and hepatocellular carcinoma (HCC) in developed countries including Egypt. Since, the discovery of the virus, the main drug used in all antiviral protocols was interferon- α (IFN- α) but, which is not effective in 60% of these patients. The goal of this study was to measure nitric oxide synthase (NOS), and CD3-ζ in chronic HCV patients which could explain the failure from therapy. 5ml of peripheral blood were collected from 30 patients with chronic HCV infection and 10 healthy control volunteers. Patients were categorized in to responders and non-responders according to viral titre upon IFN-α treatment. CD3-ζ expression was measured in the peripheral blood by using flow_cytomery and NOS levels were assessed in the sera. Significant decreases (P˂0.001) in the expression of CD3-ζ in IFN-α non- responder was recorded when compared to responder patients and with healthy volunteers. In contrast, there were significant increases (P˂0.001) in the expression of NOS in IFN-α responder as compared to non-responder patients and healthy volunteers. Conclusion: these findings can be suggestedNitric oxide level and CD3-ζ expressionthat have immune suppressive function can be reversed and enhance responsive of HCV patients to interferon- α and ribavirin.

Genetic variation in STAT4 predicts response to interferon-α therapy for hepatitis B e antigen-positive chronic hepatitis B.

Interferon (IFN)-α is a first-line therapy for chronic hepatitis B (CHB) patients but only initiates a response in a minority of patients. A genetic variant, rs7574865 in STAT4, was recently reported to be associated with risk of developing CHB and hepatitis B virus-related hepatocellular carcinoma. We aimed to determine whether this variant is associated with the response to IFNα treatment for hepatitis B e antigen (HBeAg)-positive CHB patients. We studied 466 HBeAg-positive CHB patients who received either IFNα-2b (n = 224) or pegylated IFNα-2a (n = 242) therapy for 48 weeks and were followed for an additional 24 weeks. The rate of sustained virologic response (SVR), defined as HBeAg seroconversion along with hepatitis B virus DNA level <2000 copies/mL at week 72, was compared among patients with different genotypes of rs7574865. After 48 weeks of treatment and 24 weeks off treatment, the SVR rates in the IFNα-2b and pegylated IFNα-2a therapy groups were 30.4% and 28.9%, respectively. Compared to the rs7574865 GT/TT genotype, the GG genotype (a risk factor of CHB and hepatitis B virus-related hepatocellular carcinoma) was significantly associated with a reduced SVR rate in both patients who received IFNα-2b therapy (21.1% versus 37.2%, P = 0.01) and those who received pegylated IFNα-2a therapy (18.0% versus 41.2%, P = 9.74 × 10(-5) ). In joint analysis of the 466 patients, the GG genotype was associated with an approximately half SVR rate compared to the GT/TT genotype (19.3% versus 39.1%, P = 4.15 × 10(-6) ). A multivariate logistic regression model including rs7574865 and clinical variables showed that rs7574865 was the most significant factor for the prediction of SVR. STAT4 rs7574865 is a reliable predictor of response to IFNα therapy for HBeAg-positive CHB patients and may be used for optimizing the treatment of CHB.

Some biochemical markers and expression of indoleamine-2, 3-dioxygenase in Egyptian patients with chronic Hepatitis C.

Hepatitis C virus (HCV) is a main cause of chronic hepatitis and it may leads to cirrhosis, hepatic failure and hepatocellular carcinoma. Chronic HCV patients are subjected to treatment with ribavirin and interferon-α (IFN-α) but,which have achieved only limited success. The main aim of this study was to measure indoleamine-2, 3-dioxygenase (IDO) in chronic HCV patients which could explain the failure from therapy. Five ml of peripheral blood were collected from 30 patients with chronic HCV infection and 10 healthy control volunteers. Patients were categorized in to responders and non-responders according to viral titre upon IFN-α treatment. The levels of IDO were measured in the sera of the recruited subjects. Significant increases (P˂0.001) in the concentration of IDO were observed in IFN-α non-responder patient when compared with responders and healthy control. Conclusion: Non-responsiveness of chronic HCV patients to IFNs based therapy associated with increases in suppressive mechanisms, opening a new avenue for targeting these molecules in HCV therapy.

Probable involvement of p11 with interferon alpha induced depression.

Depression is one of the major side effects of interferon alpha (IFN-α) treatment, but the molecular mechanism underlying IFN-α-induced depression remains unclear. Several studies have shown that the serotonin receptors 5-HTR1b and 5-HTR4 play key roles in the anti-depression effects associated with p11 (S100A10). We investigated the effects of IFN-α on the regulation of p11, 5-HTR1b and 5-HTR4 in mice and human neuroblastoma cells (SH-sy5y). We found that intraperitoneal injection with IFN-α in Balb/c mice resulted in an increased immobility in FST and TST, and potently lowered the protein levels of p11, 5-HTR1b and 5-HTR4 in the hippocampus or cingulate gyrus. IFN-α significantly down-regulated the protein levels of p11, 5-HTR1b and 5-HTR4 in SH-sy5y cells, in a time- and dose-dependent manner. Our study revealed that over-expression of p11 could prevent the IFN-α-induced down-regulation of 5-HTR1b and 5-HTR4. The results indicated that IFN-α treatment resulted in p11 down-regulation, which subsequently decreased 5-HTR1b and 5-HTR4 in vitro or in vivo. Our findings suggested that p11 might be a potential regulator on 5-HTR1b and 5-HTR4 as well as a predictor of or a therapeutic target for IFN-α-induced depression.

Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma.

IntroductionInterferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling.ResultsWe could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment.ConclusionWe suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in ‘silent’ metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.

Expression of Interferon Effector Gene SART1 Correlates with Interferon Treatment Response against Hepatitis B Infection.

Interferon-α (IFN-α) has limited response rate in the treatment of chronic hepatitis B (CHB). The underlying mechanism of differential responsiveness to IFN remains elusive. It has been recently reported that SART1 mediates antiviral effects of IFN-α in the hepatitis C virus (HCV) cell culture model. In this study, we investigated the role of SART1 in antiviral activity of IFN-α against hepatitis B virus (HBV) using blood and liver biopsy samples from chronic hepatitis B patients treated with pegylated IFN-α and HepG2 cells transfected with cloned HBV DNA. We observed that the basal SART1 expression in liver and PBMCs before IFN treatment was significantly higher in responders than in nonresponders. Furthermore, baseline SART1 expression level positively correlated with the degree of HBV DNA and HBeAg decline after IFN treatment. Mechanistically, silencing SART1 abrogated the antiviral activity of IFN-α, reduced the expression of IFN-stimulated genes (ISGs) Mx, OAS, and PKR, and attenuated JAK-STAT signaling in HepG2 cells, suggesting that SART1 regulates IFN-mediated antiviral activity through JAK-STAT signaling and ISG expression. Our study elucidates the important role of SART1 in IFN-mediated anti-HBV response and provides new insights into understanding variation of IFN treatment response in CHB patients.

IFIT1 polymorphisms predict interferon-α treatment efficiency for hepatitis B virus infection.

AIMTo investigate the association between interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) polymorphisms and interferon-α (IFNα) treatment efficiency among Chinese hepatitis B virus (HBV) infection patients.METHODSTwo hundred and twenty five newly diagnosed chronic hepatitis B (CHB) patients were enrolled in the study. All of these patients received IFNα treatment for a course of 48 wk, and were followed up for 24 wk after the treatment was end. Clinical information about virological response, hepatitis B e antigen (HBeAg) seroconversion rate and combined response at the end of the treatment, as well as the sustained response by the time of following up 24 wk after the treatment, was collected. Four tag-single nucleotide polymorphisms (SNPs) of IFIT1 were selected and assessed for their association with these clinical outcomes.RESULTSAt the end of the treatment, HBeAg seroconversion was observed in 27.1% patients. Thirty-six point nine percent patients achieved virological response, and 15.6% patients exhibited combined response. Sustained response was obtained in 26.2% patients. The main HBV genotype of the study was genotype B. Patients who infected with HBV genotype B or C showed better treatment efficiency, no matter which clinical outcome was considered. Among the four SNPs assessed, rs303218 (A > G) was found to be significantly associated with the end point virological response when assuming additive model [OR = 0.64 (95%CI: 0.42-0.96), P = 0.032]. Patients who carried rs303218 GG genotype had a rather higher rate of achieving virological response (response rate: 52%, OR = 0.40, 95%CI: 0.18-0.91; P = 0.028) when compared to those had AA genotype (response rate: 27%). The most significant interaction was observed in patients who had relative lower baseline aspartate transaminase. No association between SNPs and HBeAg seroconversion, combined response or sustained response was observed.CONCLUSIONIFIT1 involves in the regulation of IFNα treatment for CHB and its polymorphism rs303218 can predict the end point virological response. The finding requires further validation.

Interferon-α reduces the gefitinib sensitivity of human non-small cell lung cancer.

Aim of the studyMany studies have shown that interferon-α (IFN-α) enhances the antiproliferative effect of gefitinib in some solid tumours. We aimed to determine the effect of combining IFN-α with gefitinib in human non-small cell lung cancer (NSCLC) cell lines (A549, H1299, HCC827) with different EGFR and K-Ras gene statuses.Material and methodsAn MTT assay was used to assess cell proliferation. Apoptosis was detected by an Annexin V/propidium iodide assay using flow cytometry, and western blotting was used to determine the expression of epidermal growth factor receptor/phosphorylated epidermal growth factor receptor (EGFR/p-EGFR) and signal transducers and activators of transcription 3/phosphorylated signal transducers and activators of transcription 3 (STAT3/p-STAT3).ResultsThere was an additive interaction when gefitinib was combined with IFN-α in all cell lines; however, there was antagonism when gefitinib followed IFN-α pretreatment in three cell lines. Notably, IFN-α pretreatment significantly reduced the gefitinib sensitivity of HCC827 cells. Surprisingly, while IFN-α inhibited STAT3 phosphorylation in cell lines, gefitinib could do so.ConclusionsThe results might confirm the hypothesis that IFN-α induces gefitinib sensitivity of NSCLC, and IFN-α inhibits phosphorylation of STAT3, which may be dependent on EGFR signal activation playing a role in the reduction of gefitinib sensitivity after IFN-α treatment in NSCLC cell lines.

Estimate the Levels of Copper in Serum Samples of Hepatitis C Patients at Different Stages after Interferon-α Therapy

In this study, we evaluated the copper (Cu) levels in serum samples of patients infected by hepatitis C virus (HCV) at different stages and examined the changes in its content before and after interferon α (IFN-α) therapy with related to healthy subjects. The 199 HCV patients of both genders at different stages including (acute, fibrosis and cirrhosis) were enrolled in this study, who have completed IFN-α treatment and were followed for at least 1 year after the end the therapy. We have also evaluated the changes in biochemical parameters before and after treatment. Blood samples were collected from patients before and after treatment of IFN-α therapy. For comparative purposes healthy subjects of same age group (30-50) were also selected. The trace level of Cu in serum samples was determined by flame atomic absorption spectrometry after preconcentration by dual-cloud point extraction method. It was observed that after INF-α treatment the level of Cu in serum samples of HCV patients were reduced in decreasing order such as acute> fibrosis> cirrhosis.

Inhibition of STAT Pathway Impairs Anti-Hepatitis C Virus Effect of Interferon Alpha

Background/Aims: Signal transducer and activator of transcription (STAT) pathway plays an important role in antiviral efficacy of interferon alpha (IFN-α). IFN-α is the main therapeutic against hepatitis C virus (HCV) infection. We explored effects of IFN-α on HCV replication and antiviral gene expression by targeting STAT. Methods: In response to IFN-α, STAT status, HCV replication, and antiviral gene expression were analyzed in human hepatoma Huh7.5.1 cells before and after cell culture-derived HCV infection. Results: IFN-α treatment induced expression and phosphorylation of STAT1 and STAT2 in Huh7.5.1 cells. Pretreatment of Huh7.5.1 cells with a mAb to IFN alpha receptor (IFNAR) 2 decreased IFN-α-dependent phosphorylation of STAT1 and STAT2, whereas pretreatment with an IFNAR1 mAb increased such phosphorylation, suggesting that IFNAR mediates IFN-α-triggered STAT signaling. During HCV infection, STAT1 and STAT2 phosphorylation could be rescued by IFN-α and IFN-α-induced phosphorylation of STAT1 and STAT2 was impaired. Inhibition of STAT pathway by Jak inhibitor I significantly enhanced HCV RNA replication and viral protein expression. Antiviral genes coding for IFN regulatory factor 9 and IFN-stimulated gene 15 were up-regulated by IFN-α during HCV infection but such up-regulation was abrogated by Jak inhibitor I. Conclusion: These results establish that activation of STAT pathway is essential for anti-HCV efficacy of IFN-α. Impairment of IFN-α-triggered STAT signaling by HCV may account for evading IFN-α response.

Impact of IL28B and OAS gene family polymorphisms on interferon treatment response in Caucasian children chronically infected with hepatitis B virus.

AIMTo investigate the impact of IL28B and OAS gene polymorphisms on interferon treatment responses in children with chronic hepatitis B.METHODSWe enrolled 52 children (between the ages of 4 and 18) with hepatitis B e antigen-negative chronic hepatitis B (CHB), who were treated with pegylated interferon alfa for 48 wk. Single nucleotide polymorphisms in the OAS1 (rs1131476), OAS2 (rs1293747), OAS3 (rs2072136), OASL (rs10849829) and IL28B (rs12979860, rs12980275 and rs8099917) genes were studied to examine their associations with responses to IFN treatment in paediatric patients. We adopted two criteria for the therapeutic response, achieving an hepatitis B virus (HBV) DNA level < 2000 IU/mL and normalization of ALT activity (< 40 IU/L). To perform the analyses, we compared the patients in terms of achieving a partial response (PR) and a complete response (CR) upon measurement at the 24-wk post-treatment follow-up.RESULTSThe PR and CR rates were 80.8% and 42.3%, respectively. Factors such as age, gender and liver histology had no impact on the type of response (partial or complete). A statistically significant relationship between higher baseline HBV DNA and ALT activity levels and lower rates of PR and CR was shown (P < 0.05). The allele association analysis revealed that only the IL-28B rs12979860 (C vs T) and IL28B rs12980275 (A vs G) markers significantly affected the achievement of PR (P = 0.021, OR = 3.3, 95%CI: 1.2-9.2 and P = 0.014, OR = 3.7, 95%CI: 1.3-10.1, respectively). However, in the genotype analysis, only IL-28B rs12980275 was significantly associated with PR (AA vs AG-GG, P = 0.014, OR = 10.9, 95%CI: 1.3-93.9). The association analysis for CR showed that the TT genotype of IL28B rs12979860 was present only in the no-CR group (P = 0.033) and the AA genotype of OASL rs10849829 was significantly more frequent in the no-CR group (P = 0.044, OR = 0.26, 95%CI: 0.07-0.88). The haplotype analysis revealed significant associations between PR and CR and OAS haplotype (P = 0.0002 and P = 0.001, respectively), but no association with IL28B haplotype was observed.CONCLUSIONIL28B and OAS polymorphisms are associated with different clinical outcomes in CHB children treated with interferon.

S-adenosyl-L-methionine modifies antioxidant-enzymes, glutathione-biosynthesis and methionine adenosyltransferases-1/2 in hepatitis C virus-expressing cells.

To elucidate the mechanism(s) by which S-adenosyl-L-methionine (SAM) decreases hepatitis C virus (HCV) expression. We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times (24-72 h), then total RNA and proteins were isolated. cDNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2 (SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A (MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1A, MAT2A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman's recycling method (0-24 h). Reactive oxidative species (ROS) levels were quantified by the dichlorofluorescein assay (0-48 h); Pyrrolidin dithiocarbamate (PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent. SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression (SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2A, since MAT1A expression was increased (2.5 fold-times at 48 h) and MAT2A was diminished (from 24 h) upon SAM treatment at both the transcriptional and translational level. A likely mechanism(s) by which SAM diminish HCV expression could involve modulating antioxidant enzymes, restoring biosynthesis of glutathione and switching MAT1/MAT2 turnover in HCV expressing cells.