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Interferon alpha regulated gene expression in patients initiating interferon treatment for chronic Hepatitis C
Abstract
Interferon alfa (IFN-alpha) is an approved therapeutic agent for chronic hepatitis C. To directly characterize the effects of IFN-alpha in humans, we used microarrays to profile gene expression in peripheral blood mononuclear cells (PBMCs) from hepatitis C patients treated with IFN-alpha. Seven patients were studied using two strategies: (1) in vivo: PBMCs were collected immediately before the first dose of IFN-alpha, and 3 and 6 hours after the dose; (2) ex vivo: PBMCs that were collected before the first IFN-alpha dose were incubated with IFN-alpha for 3 and 6 hours. The microarray datasets were analyzed with significance analysis of microarrays (SAM) to identify genes regulated by IFN-alpha. We identified 516 named genes up-regulated at least 2-fold, at a false discovery rate (FDR) of less than 1%. In vivo and ex vivo studies generated similar results. No genes were identified as regulated differently between these 2 experimental conditions. The up-regulated genes belonged to a broad range of functional pathways and included multiple genes thought to be involved in the direct antiviral effect of IFN-alpha. Of particular interest, 88 genes directly relating to functions of immune cells were up-regulated, including genes involved in antigen processing and presentation, T-cell activation, lymphocyte trafficking, and effector functions, suggesting that IFN-alpha up-regulates multiple genes involving different aspects of immune responses to enhance immunity against hepatitis C virus. In conclusion, IFN-alpha-inducible genes can be identified in human PBMCs in vivo as well as ex vivo. Signature changes associated with different treatment outcomes may be found among these genes.
... Among the best studied ISGs are the ones with well-defined antiviral properties, such as protein kinase R (PKR), GTPases MxA and MxB, oligoadenylate synthetase (OAS) enzymes, RNAspecific adenosine deaminase (ADAR1), and the small ubiquitin-like molecule, IFI16. Interestingly, the sets of ISGs reported as induced in studies of many cell types, conditions, and even organisms, remain remarkably consistent (Brassard et al., 2004;Der et al., 1998;Ji et al., 2003;Moschella et al., 2003;Schultz et al., 2004;Taylor et al., 2004), which not only supports the view of IFN response as an ancient cellular response (Schultz et al., 2004) but also makes it possible to predict the genes expected to be induced whenever IFN response is present. ...
... Inspection of the genes upregulated in the A and C groups revealed many known type I interferon stimulated genes (ISGs). We therefore compared our observed gene profiles obtained from SAM to published IFNα-stimulation profiles (Brassard et al., 2004;Ji et al., 2003;Moschella et al., 2003;Taylor et al., 2004) as well as profiles generated in-house and found a significant overlap ranging 15%-51% in CD4 + samples, and 24%-51% in CD8 + samples. Significantly, almost all the ISGs were found in the group of genes upregulated in A and C samples and not in the downregulated cluster observed in CD8 + cells. ...
... In fact, ever since it has been characterized by Der et.al. (Der et al., 1998), multiple studies have reported observing IFN responses with highly overlapping gene sets in a variety of in vitro and in vivo studies (Brassard et al., 2004;Ji et al., 2003;Moschella et al., 2003;Taylor et al., 2004 (Baig and Fish, 2008). In other words, mice tailor the IFN response to infections by activating specific IFN genes in specific tissues, depending on the infectious agent (Lopez and Navarro, 1998;Mesplede et al., 2003). ...
... The IFNB1 signature was taken from the differential gene expression analysis of WB from 58 multiple sclerosis patients treated with recombinant IFNB1 for one to two years and compared to 62 multiple sclerosis patients not treated with IFNB1 (MS-IFNB1) 41 . The IFNA2 signature was taken from the differential gene expression analysis of PBMC from four hepatitis C patients treated with IFNA2 for 6 h and compared to the same patients before IFNA2 administration (HepC-IFNA2) 42 To determine whether the IGS detected in SLE patients was related to a specific type of hematopoietic cell or process, linear regression analysis was carried out between the GSVA enrichment scores for cell signatures and processes and the IGS in each SLE patient from ten SLE WB and PBMC datasets. The strongest relationship to the IGS was to monocyte surface transcripts with a significant non-zero slope (p < .0001) ...
... The second reference dataset used was the IFNB1 (MS-IFNB1) signature-induced in vivo in the whole blood of 58 Multiple Sclerosis patients treated with IFNB1 (Avonex, Betaseron or Rebif) for one to two years compared to 62 MS Patients not treated with IFNB1 from Table S1 41 . The third reference dataset used was the IFNA signature-induced in 4 HepC patients treated with recombinant IFNA for 6 h compared to their PBMC before the injection of recombinant IFNA from Table 2 42 for the HepC-IFNA2 signature. Published transcripts of PBMC from patients with sepsis DE to controls (Table S2) 43 and skin biopsies from patients with dermatomyositis DE to controls (Table S2) 44 were used as comparators for Z score calculations. ...
A role for interferon (IFN) in systemic lupus erythematosus (SLE) pathogenesis is inferred from the prominent IFN gene signature (IGS), but the major IFN species and its relationship to disease activity are unknown. A bioinformatic approach employing individual IFN species gene signatures to interrogate SLE microarray datasets demonstrates a putative role for numerous IFN species, with prominent expression of IFNB1 and IFNW signatures. In contrast with other SLE-affected organs, the IGS is less prominent in lupus nephritis. SLE patients with active and inactive disease have readily detectable IGS and the IGS changes synchronously with a monocyte signature but not disease activity, and is significantly related to monocyte transcripts. Monocyte over-expression of three times as many IGS transcripts as T and B cells and IGS retention in monocytes, but not T and B cells from inactive SLE patients contribute to the lack of correlation between the IGS and SLE disease activity.
... The IFNB1 signature was taken from the differential gene expression analysis of WB from 58 multiple sclerosis patients treated with recombinant IFNB1 for one to two years and compared to 62 multiple sclerosis patients not treated with IFNB1 (MS-IFNB1) 41 . The IFNA2 signature was taken from the differential gene expression analysis of PBMC from four hepatitis C patients treated with IFNA2 for 6 h and compared to the same patients before IFNA2 administration (HepC-IFNA2) 42 To determine whether the IGS detected in SLE patients was related to a specific type of hematopoietic cell or process, linear regression analysis was carried out between the GSVA enrichment scores for cell signatures and processes and the IGS in each SLE patient from ten SLE WB and PBMC datasets. The strongest relationship to the IGS was to monocyte surface transcripts with a significant non-zero slope (p < .0001) ...
... The second reference dataset used was the IFNB1 (MS-IFNB1) signature-induced in vivo in the whole blood of 58 Multiple Sclerosis patients treated with IFNB1 (Avonex, Betaseron or Rebif) for one to two years compared to 62 MS Patients not treated with IFNB1 from Table S1 41 . The third reference dataset used was the IFNA signature-induced in 4 HepC patients treated with recombinant IFNA for 6 h compared to their PBMC before the injection of recombinant IFNA from Table 2 42 for the HepC-IFNA2 signature. Published transcripts of PBMC from patients with sepsis DE to controls (Table S2) 43 and skin biopsies from patients with dermatomyositis DE to controls (Table S2) 44 were used as comparators for Z score calculations. ...
A role for interferon (IFN) in systemic lupus erythematosus (SLE) pathogenesis is inferredfrom the prominent IFN gene signature (IGS), but the major IFN species and its relationshipto disease activity are unknown. A bioinformatic approach employing individual IFN speciesgene signatures to interrogate SLE microarray datasets demonstrates a putative role fornumerous IFN species, with prominent expression of IFNB1 and IFNW signatures. In contrastwith other SLE-affected organs, the IGS is less prominent in lupus nephritis. SLE patients withactive and inactive disease have readily detectable IGS and the IGS changes synchronouslywith a monocyte signature but not disease activity, and is significantly related to monocytetranscripts. Monocyte over-expression of three times as many IGS transcripts as T and B cellsand IGS retention in monocytes, but not T and B cells from inactive SLE patients contribute tothe lack of correlation between the IGS and SLE disease activity
... The set of differentially expressed genes included genes for the infl ammation system, the innate and adaptive immune systems, apoptosis, signal transmission, transcription, translation, degradation, and others. Among the genes whose expression showed signifi cant increases after β-IFN administration, we found "classical" genes for responses to β-IFN [5,20] -MX1 (myxovirus [infl uenza virus] resistance 1), OAS1 and OAS3 (2',5'-oligoadenylate synthetases 1 and 3), whose products have roles in the immune response to viral infections. Increases in the expression of other genes was also found -ISG20, ISG15, TNFSF10, IFIT1, STAT1, STAT2, and EIF2AK2, which have previously been ob-served in most studies [21][22][23][24][25][26] after administration of various β-IFN formulations. ...
... Activation of the transcription of these genes detected here in response to Infi beta is evidence for transmission of a signal from the IFNAR receptor activated by binding of β-IFN and is an important indicator of activation of the type I IFN signal pathway. Suffi ciently long-lasting activation of the transcription of the genes for these transcription factors on exposure to β-IFN has been seen in a number of studies [20,25,29,30]. Figure 4 (see color plates) shows a signal intensity map for the transcripts of 56 genes among the β-IFN-induced group [28] before and after administration of the fi rst Infi beta dose to MS patients. All of these other than IL7 showed increases in expression levels, the increase in more than 50% of cases reaching signifi cance. ...
The introduction of Russian analogs of β-interferon (β-IFN) produced by recombinant strains of E. coli (β-IFN-1b) into medical practice in Russia for the pathogenetic treatment of multiple sclerosis (MS) requires, in accordance with contemporary standards, confirmation by transcriptome analysis that they activate the main signal pathways involved in the mechanism of action of β-IFN. In the present report we present such an analysis for Infibeta (produced by Generium). Whole-genome transcription profiling using an Illumina HT-12 microarray was performed to identify genes showing differential expression in peripheral blood mononuclear cells from MS patients in response to exposure to this agent. Comparison of the levels of gene expression in MS patients not previously treated with any kind of immunomodulatory therapy before and 10 h after the first dose of agent identified 490 genes whose expression showed statistically significant changes, of which 191 genes showed increases in expression (up-regulated genes) and 299 genes showed decreases in expression (down-regulated genes). These genes included genes from the inflammation system, the innate and adaptive immune systems, apoptosis, signal transmission, transcription, translation, and degradation. Analysis of the overall expression of sets (groups) of genes (gene set analysis) yielded data which may provide evidence that IFN type I signal pathway genes, as well as IFN-induced genes, are involved in patients’ responses to the agent. Thus, analysis by transcriptional profiling led to the conclusion that the mechanism of the immunomodulatory action of Infibeta is identical to that described for original β-IFN formulations.
... In one relevant study, expression of ISGs was investigated in PBMC from patients with CHC initiating IFN-α therapy, including one sustained responder, one relapse and 4 non-responders to this therapy. This work included both in vivo as well as ex vivo time course experiments and the gene signature changes were found in association with the outcomes of IFN-α treatment [19]. PBMC have also been employed to assess the relation between expression of ISGs and the outcome of PegIFN/RBV treatment in HIV/HCV-co-infected patients [20]. ...
... These findings may suggest that long-term exposure to IFN caused by chronic HCV infection leads to a relative decrease in later responsiveness to this cytokine. However, it is important to note when considering these data, that in vitro stimulation with IFN-α may not exactly mimic that observed with in vivo IFN therapy, although two other reported studies comparing the in vivo and in vitro impact of IFN-α2b stimulation found that both conditions generated similar results [19,29]. In addition, it should be considered that the finding that exposure to HCV can impact the ISG expression profile in PBMC could be an outcome of direct infection of PBMC with HCV, as it has been reported previously [11,30]. ...
... Среди дифференциально экспрессирующихся генов наряду с другими присутствуют гены систем воспаления, врожденного и адаптивного иммунитета, апоптоза, передачи сигнала, транскрипции, трансляции, деградации и др. Среди генов, экспрессия которых значимо увеличилась после введения β-ИФН, мы обнаружили «классические» гены ответа на β-ИФН [5,20] -MX1 (myxovirus [influenza virus] resistance 1), OAS1 и OAS3 (2',5'-oligoadenylate synthetase 1 and 3), продукты которых участвуют в иммунном ответе на вирусную инфекцию. Выявлено также повышение экспрессии генов ISG20, ISG15, TNFSF10, IFIT1, STAT1, STAT2, EIF2AK2, ранее наблюдавшееся после введения различных препаратов β-ИФН в большинстве работ [21][22][23][24][25][26]. ...
... Сами гены STAT1 и STAT2 тоже принадлежат к ИФН-индуцируемым, имея в своей регуляторной области ISRE-мотив [28]. Активация транскрипции этих генов, выявленная нами под воздействием препарата инфибета, свидетельствует о передаче сигнала от рецептора IFNAR, активированного при связывании β-ИФН, и является важным показателем активации сигнального пути ИФН типа I. Достаточно длительная активация транскрипции генов этих транскрипционных факторов при действии β-ИФН отмечалась в ряде исследований [20,25,29,30]. ...
Implementation of the analogues of Β-interferon (Β-IFN), produced by recombinant strains of E. coli (Β-IFN-1b), for internal use in the Russian Federation for the treatment of multiple sclerosis (MS), implies a verification of their action at the transcriptome level in order to confirm the activation of the main signaling pathways involved in IFNb mechanism of action. In this work, the analysis is carried out for Infibeta (Generium, Russia). Using genome-wide transcriptional profiling with ILLUMINA HT-12 microarray, the differentially expressed genes in peripheral blood mononuclear cells of MS patients upon administration of Infibeta were studied. Comparison of gene expression levels in treatment-naive MS patients prior to first Β-IFN administration and 10 hours after it, identified 490 genes with significantly changed expression level, where 191 genes were up-regulated and 299 genes were down-regulated. Among the involved genes are those coding the components of the inflammatory system, innate and adaptive immunity, apoptosis, signal transduction, transcription, translation, degradation. Using gene set analysis, we confirmed the involvement of type I interferon signaling pathway genes and interferon-inducible genes in a patient's individual response to drug. Thus, the transcriptome profiling analysis allows concluding that the mechanism of action of Infibeta immunomodulatory drug is equal to that described for the original Β-IFN drugs.
... The activated STAT migrates to the nucleus and increases the transcription of ISGs. In peripheral blood mononuclear cells (PBMCs), interferon-α modifies the transcription of 516 genes belonging to a wide range of functional pathways, reflecting the remarkable diversity of cellular processes potentially affected by interferon-α [21]. Human type II interferon is interferon-γ. ...
In humans, insulin resistance is a physiological response to infections developed to supply sufficient energy to the activated immune system. This metabolic adaptation facilitates the immune response but usually persists after the recovery period of the infection and predisposes the hosts to type 2 diabetes and vascular injury. In patients with diabetes, superimposed insulin resistance worsens metabolic control and promotes diabetic ketoacidosis. Pathogenic mechanisms underlying insulin resistance during microbial invasions remain to be fully defined. However, interferons cause insulin resistance in healthy subjects and other population groups, and their production is increased during infections, suggesting that this group of molecules may contribute to reduced insulin sensitivity. In agreement with this notion, gene expression profiles [transcriptomes] from patients with insulin resistance show a robust overexpression of interferon-stimulated genes [interferon signature]. In addition, serum levels of interferon and surrogates for interferon activity are elevated in patients with insulin resistance. Circulating levels of interferon-γ-inducible protein-10, neopterin, and apolipoprotein L1 correlate with insulin resistance manifestations, such as hypertriglyceridemia, reduced HDL-c, visceral fat, and homeostasis model assessment-insulin resistance. Furthermore, interferon downregulation improves insulin resistance. Antimalarials such as hydroxychloroquine reduce interferon production and improve insulin resistance, reducing the risk for type 2 diabetes and cardiovascular disease. In addition, diverse clinical conditions that feature interferon upregulation are associated with insulin resistance, suggesting that interferon may be a common factor promoting this adaptive response. Among these conditions are systemic lupus erythematosus, sarcoidosis, and infections with severe acute respiratory syndrome-coronavirus-2, human immunodeficiency virus, hepatitis C virus, and Mycobacterium tuberculosis.
... No earlier data were available for discussion on the expression pattern of CTSs mRNA in PBLs, although their presence in unspecified ovary mRNA samples has been reported previously (Petanceska & Devi 1992, Bromme & Okamoto 1995, Kirschke et al. 1998, Soderstrom et al. 1999. In humans, microarray analysis has revealed that many genes, including CTSL and LAMP3, were significantly upregulated in peripheral blood mononuclear cells (PBMCs) from chronic hepatitis C patients treated with IFNα (Ji et al. 2003). These results suggest that type 1 IFN regulates the activity and expression of lysosomal CTSs (K) in the leukocytes, consistent with the present study. ...
In brief
Interferon tau (IFNT) stimulates lysosomal activation via the Janus-activated kinase in peripheral blood leukocytes during pregnancy recognition. IFNT-mediated lysosomal activation could serve as a novel marker for early pregnancy in cattle.
Abstract
IFNT is important in establishing pregnancy in ruminants. Secreted IFNT in the uterus induces the expression of an interferon-stimulated gene (ISG) in uterine tissues and peripheral blood leukocytes (PBLs). In our previous study, increased lysosome and lysosomal cathepsin (CTS) activity and mRNA expression were observed in PBLs of pregnant cows on day 18 of pregnancy. However, the mechanism of IFNT stimulation in PBLs is unclear. Here, we explored the IFNT-mediated lysosomal activation mechanisms in PBLs during early pregnancy in dairy cows. PBLs collected from the peripheral blood of Holstein cows on day 18 post artificial insemination, after confirmation of their pregnancy status, were used to detect the expression of lysosomal-associated membrane protein ( LAMP) 1, 2 , CTSB and CTSK . Expression of all genes was significantly higher in PBLs of pregnant cows than in nonpregnant cows. In vitro IFN-mediated stimulation of PBLs collected from cows that did not undergo AI significantly increased lysosomal acidification and expression of LAMP1 and 2, as well as the activities of CTSB and CTSK. Immunodetection analysis showed an increase in LAMP1 and CTSK levels in the PBLs of day 18 pregnant cows. JAK inhibitor significantly decreased lysosomal acidification, CTSK activity, LAMP1, 2, and CTSK expression in the presence of IFNT. These results suggest that IFNT regulates lysosomal function via a type 1IFN-mediated pathway in PBLs during pregnancy recognition.
... Although in this investigation our focus was on identifying protein changes that were most consistent between different donors, it may nevertheless be valuable to investigate proteins that exhibit more variable inter-donor changes. Side effects and efficacy of IFN-based therapies for viral hepatitis and cancers are known to vary between patients, which may partly be explained by differences in cellular responses to treatment (85)(86)(87). A larger study would be required to comprehensively characterize the true extent of donor-to-donor variability for such molecules. ...
The cellular response to interferon (IFN) is essential for antiviral immunity, IFN-based therapy and IFN-related disease. The plasma membrane (PM) provides a critical interface between the cell and its environment, and is the initial portal of entry for viruses. Nonetheless, the effect of IFN on PM proteins is surprisingly poorly understood, and has not been systematically investigated in primary immune cells. Here, we use multiplexed proteomics to quantify IFNα2a-stimulated PM protein changes in primary human CD14+ monocytes and CD4+ T cells from five donors, quantifying 606 and 482 PM proteins respectively. Comparison of cell surface proteomes revealed a remarkable invariance between donors in the overall composition of the cell surface from each cell type, but a marked donor-to-donor variability in the effects of IFNα2a. Furthermore, whereas only 2.7% of quantified proteins were consistently upregulated by IFNα2a at the surface of CD4+ T cells, 6.8% of proteins were consistently upregulated in primary monocytes, suggesting that the magnitude of the IFNα2a response varies according to cell type. Among these differentially regulated proteins, we found the viral target Endothelin-converting enzyme 1 (ECE1) to be an IFNα2a-stimulated protein exclusively upregulated at the surface of CD4+ T cells. We therefore provide a comprehensive map of the cell surface of IFNα2a-stimulated primary human immune cells, including previously uncharacterized interferon stimulated genes (ISGs) and candidate antiviral factors.
... . Of these, CXCL8, TLR2 and FPR1 are also upregulated ISGs [39]. ...
Background:
Disease progression in the absence of therapy varies significantly in mono-HIV and HCV infected individuals. Virus-specific CD8+ T cells play an important role in restricting lentiviral replication and determining the rate of disease progression during HIV and HCV mono- and co-infection. Thus, understanding the similarities in the characteristics of CD8+ T cells in mono-HIV and HCV infection at the transcriptomic level contributes to the development of antiviral therapy. In this study, a meta-analysis of CD8+ T cell gene expression profiles derived from mono-HIV and HCV infected individuals at different stages of disease progression, was conducted to understand the common changes experienced by CD8+ T cells.
Methods:
Five microarray datasets, reporting CD8+ T cell mRNA expression of the mono-HIV and HCV infected patients, were retrieved from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were identified via integrative meta-analysis of expression data (INMEX) program. Network analysis methods were used to assess protein-protein interaction (PPI) networks, Gene Ontology (GO) terms and pathway enrichment for DEGs. MirDIP and miRDB online prediction tools were used to predict potential microRNAs (miRNAs) targeting hub genes.
Results:
First, we identified 625 and 154 DEGs in the CD8+ T cells originating from mono-HIV and HCV chronic progressor patients, respectively, compared to healthy individuals. Among them, interferon-stimulated genes (ISGs) including ISG15, IFIT3, ILI44L, CXCL8, FPR1 and TLR2, were upregulated after mono-HIV and HCV infection. Pathway enrichment analysis of DEGs showed that the "cytokine-cytokine receptor interaction" and "NF-kappa B" signaling pathways were upregulated after mono-HIV and HCV infection. In addition, we identified 92 and 50 DEGs in the CD8+ T cells of HIV non-progressor and HCV resolver patients, respectively, compared with corresponding chronic progressors. We observed attenuated mitosis and reduced ISG expression in HIV non-progressors and HCV resolvers compared with the corresponding chronic progressors. Finally, we identified miRNA-143-3p, predicted to target both IFIT3 in HIV and STAT5A in HCV infection.
Conclusions:
We identified DEGs and transcriptional patterns in mono-HIV and HCV infected individuals at different stages of disease progression and identified miRNA-143-3p with potential to intervene disease progression, which provides a new strategy for developing targeted therapies.
... A very large number of genes are modified by the treatment in vitro of PBMC with interferon and ribavirin [7,19]. Similar results have been found recently following the treatment of A549 cells and Huh 7 cells with IFN-acon1 [10]. ...
Contains 166 gene-days of 37 viral titer linked genes (probe sets). Not less than 3 gene-days of the gene have component PC2 values more than 5 (these gene-days are pink dots of the Fig. 4). The component PC2 is PCA second component after multidimensional scaling of 8 variables: correlation coefficients in the space of (52*51)/2 dimensions between Gcov_MVcov, GDcov_MVcov, Gcov_VTcov, GDcov_VTcov, Geu_MVeu, GDeu_MVeu, Geu_VTeu, GDeu_VTeu for all gene-days. Columns of the Table S1 are as follows: SID - Affymetrix probe set ID; DAY - day of the treatment; Static_CA_1013 - log-transformed and day0 normalized gene expression values [(Ln(di)- Ln(d0))] for a patient 1013. The response based classification of the patient and her/his race are identified in the patient's name; Slow_CA_1016; Rapid_AA_1018; .................. - 52 patients of the study; VTeu_Geu - the following 8 columns are correlations between corresponding vectors in the space of (52*51)/2 dimensions. These correlations are 8 initial variables for the multidimensional scaling procedure; VTeu_GDeu; MVeu_Geu; MVeu_GDeu; VTcov_Gcov; VTcov_GDcov; MVcov_Gcov; MVcov_GDcov; GENE_SYMBOL - the following are gene names and descriptions; LOCUSLINK; UNIGENE; GENBANK; GENE; SOURCE_ID; DESCRIPTION; PC1 - the multidimensional scaling first component; PC2 - the multidimensional scaling second component.
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... 3 Chen et al 4 first described the differences in ISG activation in the liver in subjects prone to HCV elimination in comparison with nonresponders to therapy. HCV infection modifies the baseline expression profiles of ISG compared with healthy subjects both in hepatocytes and peripheral blood mononuclear cells (PBMC), [4][5][6][7] and the course of the ISG expression profiles is most likely unfavorable with persistence of the chronic HCV infection. Clinical data are in accordance with this hypothesis; the capacity to eliminate the virus with interferon alpha and RBV is reduced in the course of the disease. ...
Background and aims
Patients with advanced liver fibrosis owing to chronic hepatitis C virus genotype 1 represent a difficult-to-treat group even if a protease inhibitor is added to pegylated interferon alpha and ribavirin. Therefore, only patients with a high chance of cure should be treated with interferon-based treatment.
Patients and methods
Expression of IFNG, IFNLR1, and interferon-sensitive genes CXCL9, IFI16, IFI27, ISG15, and USP18 in peripheral blood mononuclear cells was assessed before and during the initial 12 weeks of treatment. The studied group consisted of 26 treatment-experienced patients of average age of 50 years with advanced liver fibrosis compared to seven healthy volunteers. Fourteen patients were treated with pegylated interferon alpha 2b, ribavirin, and boceprevir and 12 patients with telaprevir. The overall sustained virological response (SVR) rate was 69% (18/26).
Results
A significant difference in the initial expression (median, interquartile range [IQR]) of CXCL9 2.9×, IQR: 1.7–12.4 vs 1.2×, IQR: 0.5–1.8; (P=0.01) IFNG 7.3×, IQR: 1.7–32.6 vs 0.7×, IQR: 0.4–1.3; P=0.002 and USP18 3.7×, IQR: 2.1–7.7 vs 1.4×, IQR: 0.9–1.6; (P=0.03) was found between the SVR and non-SVR groups. Expression of all analyzed genes was progressively increasing during the first 12 weeks of therapy, but a significant difference between SVR and non-SVR group was found only in USP18 expression at week 12 (P=0.001). Initial expression of four genes predicted SVR in univariate analysis (CXCL9 [OR: 12.00, 95% CI: 1.21–118.89], IFI27 [OR: 12.00, 95% CI: 1.21–118.89], IFNG [OR: 10.50, 95% CI: 1.50–73.67], USP18 [OR: 21.00, 95% CI: 2.05–215.18]). In multivariate analysis, only the initial expression of USP18 was identified as a predictor of SVR (P=0.047).
Conclusion
Initial expression of USP18 and the course of its activation could be a reliable predictor of SVR achievement.
... Los objetivos del tratamiento del VHC son erradicar la infección y mejorar el grado de fibrosis e inflamación hepática (Pestka 2000;Castet et al. 2002;Radaeva et al. 2002;Ji et al. 2003) (Figura 7). Algunas de las proteínas con actividad antiviral derivadas de la transcripción de los ISGs son la proteína kinasa dependiente de RNA (PKR), la proteína 1 de resistencia a myxovirus (MxA) y la 2',5'oligoadenilato sintetasa (OAS). ...
... The signature of the Influenza virus was not associated with T2DM (Influenza-induced genes, FDR = 0.43 (or 43 %), Influenza-repressed genes, FDR = 0.14, data not shown). Because type I interferon is typically induced by viral infections we also included an ex vivo type I interferon-induced geneset in PBMC from IFNα-treated patients [29]. The type I IFN-induced geneset was also significantly increased in T2DM (Table 3 and Fig. 2). ...
Metabolic syndrome (MetS) is characterized by central obesity, insulin resistance, dysglycemia, and a pro-atherogenic plasma lipid profile. MetS creates a high risk for development of type 2 diabetes (T2DM) and cardiovascular disease (CVD), presumably by altering inflammatory responses. Presently, it is unknown how the chronic metabolic disturbances in acute hyperglycemia, MetS and T2DM affect the immune activity of peripheral blood cells.
We performed genome-wide expression analysis of peripheral blood cells obtained from patients with T2DM (n = 6) and age-, sex- , BMI- and blood pressure-matched obese individuals with MetS (n = 4) and lean healthy normoglycemic controls (n = 3), both under fasting conditions and after controlled induction of acute hyperglycemia during a 70 min hyperglycemic clamp. Differential gene expression during fasting conditions was confirmed by real-time PCR, for which we included additional age-, sex-, BMI-, and blood pressure-matched obese individuals with (n = 4) or without (n = 4) MetS.
Pathway and Gene ontology analysis applied to baseline expression profiles of peripheral blood cells from MetS and T2DM patients revealed metabolic changes, highly similar to a reoviral infection gene signature in T2DM patients. Transcription factor binding site analysis indicated that increased HIF-1α activity, a transcription factor induced by either hypoxia or oxidative stress, is responsible for this aberrant metabolic profile in peripheral blood cells from T2DM patients. Acute hyperglycemia in healthy controls resulted in reduced expression of cytotoxicity-related genes, representing NK- and CD8+ cells. In obese controls, MetS and especially T2DM patients, baseline expression of genes involved in cytotoxicity was already low, compared to healthy controls and did not further decrease upon acute hyperglycemia.
The reduced activity of cytotoxic genes in T2DM is explained by chronic hyperglycemia, but its acute effects are restricted to healthy controls. Genome expression of circulating leukocytes from T2DM patients differs from MetS individuals by a specific reovirus signature. Our data thus suggest a role for suppressed anti-viral capacity in the etiology of diabetes.
... El genotipo es el que más importancia tiene, tanta que, aunque la dosis de IFN permanece fija, la duración del tratamiento y la dosis de RBV variarán en función del genotipo. Ya en los primeros estudios con IFNestándar y RBV se demostró que los pacientes con genotipo 1 eran más resistentes al tratamiento 31 Por tanto, estos pacientes han de ser considerados "difíciles de tratar". ...
... 26 The IFN-α-based therapy induces expression of various genes, which include genes involved in antigen presentation and T-cell activation. 27,28 The IFN-stimulated genes related to T-cell activation also are induced during treatment of HCV with IFN-α. 29 In the present study, we hypothesized that PRA change can be used as a surrogate marker of SVR indicating that there was no correlation between HCV-related autoimmunity and PRA levels. ...
Chronic hepatitis C virus infection compromises hemodialysis patients and increases liver-related mortality. Interferon treatment is associated with improved sustained virological response rates and increased risk of graft loss after kidney transplant. This may be related to the development of antihuman leukocyte antigen antibodies, which may be a surrogate marker of potent immune response. We evaluated panel reactive antibody 1 and 2 levels for prediction of sustained viral response in patients with kidney transplant.
In this retrospective cohort study, we reviewed data from hepatitis C virusinfected hemodialysis patients who received interferon treatment before kidney transplant. Panel reactive antibody > 20% was considered positive. Sustained viral response rates for interferon treatment were obtained and compared with panel reactive antibody 1 and 2 values.
There were 40 patients (16 female and 24 male patients; mean age, 41.5 y; range, 18-65 y). Sustained viral response rate was 18/40 (45%). Panel reactive antibody 1 was negative in 31 patients and positive in 9 patients. Sustained viral response ratio was not correlated with panel reactive antibody 1 positivity. Panel reactive antibody 2 was negative in 31 patients (sustained viral response: present, 11 patients; absent, 20 patients) and positive in 9 patients (sustained viral response: present, 7 patients; absent, 2 patients). Sustained viral response ratio was significantly correlated with panel reactive antibody 2 positivity.
We showed a correlation between panel reactive antibody 2 positivity and sustained viral response rates that may be a predictive tool for hepatitis C virus treatment response. In patients with other complications that compromise hepatitis C virus treatment, panel reactive antibody 2 may be a surrogate marker for sustained viral response prediction. The induction of cellular immunity may cause clearance of hepatitis C virus infection and formation of high panel reactive antibody 2 levels.
... Labels indicate in any given year the subjects matters, ordered alphabetically along the x-axis, for which the number of publication reaches a count of 4, with the number of publications being plotted along the y-axis. Early adoption was observed in the field of autoimmunity, with Lupus, Rheumatoid Arthritis and Multiple Sclerosis reaching this threshold in 2006 [9,20,21], and with both cancer and hepatitis research field also contributing pioneering studies [22,23]. In 2010 study counts neared or surpassed 20 in all these research areas. ...
The immune system plays a key role in health maintenance and pathogenesis of a wide range of diseases. Leukocytes that are present in the blood convey valuable information about the status of the immune system. Blood transcriptomics, which consists in profiling blood transcript abundance on genome-wide scales, has gained in popularity over the past several years. Indeed, practicality and simplicity largely makes up for what this approach may lack in terms of cell population-level resolution. An extensive survey of the literature reveals increasingly widespread use across virtually all fields of medicine as well as across a number of different animal species, including model organisms but also animals of economical importance. Dissemination across such a wide range of disciplines holds the promise of adding a new perspective, breadth or context, to the considerable depth afforded by whole genome profiling of blood transcript abundance. Indeed, it is only through such contextualization that a truly global perspective will be gained from the use of systems approaches. Also discussed are opportunities that may arise for the fields of immunology and medicine from using blood transcriptomics as a common denominator for developing interactions and cooperation across fields of research that have traditionally been and largely remain compartmentalized. Finally, an argument is made for building immunology research capacity using blood transcriptomics platforms in low-resource and high-disease burden settings.
Copyright © 2015. Published by Elsevier Ltd.
... Together with the discovery of the above SNPs, it became clear that the concerted action of multiple ISGs must also be considered to fully understand the limits of IFN alpha treatment. Administration of IFN induces an up-regulation of ISGs in PBMC and the liver of HCV-infected individuals; the type, and especially the magnitude, of the IFN induced response differed between responder and non-responder HCV positive individuals [53,[78][79][80]. HCV infected patients who were less likely to respond to the IFN alpha therapy exhibited paradoxically a higher constitutive ISG expression compared to those that achieved a good response [53,78,[80][81][82][83]. Notably, the presence of high basal levels of ISGs was associated with a lack of increased expression after IFN administration [83,84]. ...
... In line with this idea, Chen et al. have demonstrated that a preexisting high level of ISG expression in liver biopsies is strongly indicative of a poor response to peg-IFN-a/ribavirin therapy in CHC patients [65]. Of note, a wide array of ISG is induced in vivo by IFN therapy also in PBMC, even though no relationship between preexisting IFN signature in PBMC and response to therapy has been reported [66,67]. Therefore, the extent of the previous exposure to endogenous IFNs should be taken into account in evaluating Treg modulation in response to therapy. ...
... These include the identification of genes differentially ex-pressed between breast cancer individuals with mutation in the BRCA1 versus BRCA2 gene (Hendenfalk et al., 2001), acute myeloid and lymphoblastic leukemia (Golub et al., 1999), cells in cancer and normal tissues (Alon et al., 1999). Other studies of differential gene expression include studies of the effects of interferon-alphas in humans (Ji et al., 2003), the proliferation of Pre B cells in vitro , and biliary tract carcinoma (Hansel et al., 2003), among others. Numerous experimental designs and analysis methods for detecting DE have been proposed, including , , , Tusher et al. (2001), andSmyth (2004) among others. ...
High-throughput laboratory measurement technologies, including microarrays for genomics and proteomics, are now typically used in biomedical studies ranging from animal models to human clinical trials. These methods, such as gene expression mi-croarrays, aim to capture the global expression patterns of thousands of genes or pro-teins simultaneously. A common feature is the high-dimensionality of the resulting data. Post-study analytical challenges involve methods to extract the meaningful in-formation from the millions of data points. However, there is a need to develop sys-tematic approaches to planning such studies. In this work, we provide a synthesis of the available and current trends in sample size and power analysis for genomics stud-ies. Our emphasis will be on clarifying assumptions of the available methods as well as their applicability in practice, including the assumption of independent gene expres-sion. We also emphasize emerging sample size design methods that focus on the false discovery rate (FDR) over the traditional family-wise error rate as a criterion.
... Leukocytes are sensitive responders to IFN-α and provide a more accessible alternative, requiring just a peripheral blood sample. In fact, in vivo upregulation of ISGs in PBMCs following therapeutic IFN-α is similar to ISG upregulation in vitro following IFN-α stimulation [13], suggesting in vitro PBMC responsiveness may indeed be an accurate reflection of in vivo clinical response. ...
Background
Treatment with interferon-alpha (IFN-α) and ribavirin successfully clears hepatitis C virus (HCV) infection in 50% of patients infected with genotype 1. Addition of NS3-4A protease inhibitors (PIs) increases response rates but results in additional side effects and significant economic costs. Here, we hypothesised that in vitro responsiveness of peripheral blood mononuclear cells (PBMCs) to IFN-α stimulation would identify patients who achieved sustained virological response (SVR) on dual therapy alone and thus not require addition of PIs.
Methods
PBMCs were isolated from HCV infected patients (n = 42), infected with either HCV genotype 1 or genotype 3, before commencing therapy and stimulated in vitro with IFN-α. Expression of the IFN stimulated genes (ISGs) PKR, OAS and MxA was measured and correlated with subsequent treatment response and IL28B genotype.
Results
Genotype 1 infected patients who achieved SVR had significantly higher pre-treatment expression of PKR (p = 0.0148), OAS (p = 0.0019) and MxA (p = 0.0019) in IFN-α stimulated PBMCs, compared to genotype 1 infected patients who did not achieve SVR or patients infected with genotype 3, whose in vitro ISG expression did not correlate with clinical responsiveness. IL28B genotype (rs12979860) did not correlate with endogenous or IFN-α stimulated ISG responsiveness.
Conclusions
In vitro responsiveness of PBMCs to IFN-α from genotype 1 infected patients predicts clinical responsiveness to dual therapy, independently of IL28B genotype. These results indicate that this sub-group of HCV infected patients could be identified pre-treatment and successfully treated without PIs, thus reducing adverse side effects and emergence of PI resistant virus while making significant economic savings.
... Using microarrays to identify transcriptional changes indicative of clinical responsiveness to IFN-a may one day assist in identifying patients that are strong candidates for IFN-a therapy and=or to monitor cytotoxicity. [76] Even though only a few ISGs have been fully characterized and the majority remain poorly understood, several studies demonstrated that ISGs have inhibitory effect on the viral replication of many DNA and RNA viruses. [77,78] All known human IFN-a subtypes show the same antiviral and antiproliferative activities in bioassays. ...
In 2007, the world solemnized the golden jubilee of the discovery of interferon (IFN). Interferon is a small protein messenger called pluripotent cytokine produced by several cells of the host in response to various biological as well as synthetic stimuli. There are three major classes of interferons in humans: IFN-alpha, -beta and -gamma. As a treatment option, interferon alpha (IFN-α) is the most effective one. IFN-α has proved to be effective as anti-viral therapy and tumors fighting drug in the past two decades. Meanwhile, great progress has been achieved in establishing IFN-α as the first choice of anti-viral therapy for chronic hepatitis C virus (HCV) patients. Recently, novel pegylated IFN-α 2 products with extended in vivo half-lives and consensus interferon; an artificially engineered type I interferon have been developed to substantially improve treatment regimes for HCV patients. Undesirable acute and chronic side-effects in addition to immunogenicity of therapeutic IFN products remain constraints to conquer for further improvements in clinical applications of IFN. It is certainly expected that more research will be conducted in the future, not only to face these challenges but also to extend the range of IFN products and their clinical targets. The objective herein is to review the current therapeutic alpha interferons production, formulation technologies and prospective future for original entity and its biogeneric version.
... The more rapid the stimulation is, the greater are the chances of success. The study of mononuclear cells ex vivo and in vivo demonstrated that, 3-6 h after the administration of conventional IFN-α, 516 genes were upregulated, of which 88 with actions directly linked to immune functions [17]; the same phenomenon was observed for PEG-IFN, also differentiating responders from nonresponders using the intensity of expression in certain IFN-inducible genes (2'5'OAS, MX1, IRF-7 and TLR-7), greater in the responders and lesser in the Afro-Americans [18,19]. ...
... Previous studies suggested that PDCD5 could inhibit the growth of some tumor cells (cervical cancer, ovarian cancer, hepatocellular cancer, renal clear cell carcinoma, etc.) by accelerating apoptosis and cooperating with radiotherapy and chemotherapy [3][4][5][6][7] . Also, the decreased expression of PDCD5 has been reported in various human tumors, such as breast cancer [8] , hepatocellular carcinoma [9] , cervical cancer [10] , gastric cancer [11] , lung cancer [12] , acute and chronic myelogenous leukemia [13][14][15] , astrocytic gliomas [16] and prostate cancer [17] . ...
Programmed cell death 5 (PDCD5) is an apoptosis related gene and plays an important role in the pathogenesis and development of cancer. Whether PDCD5 is present in peripheral blood serum has not been reported. The aim of this study is to determine the contents of PDCD5 protein in peripheral blood serum of cancer patients, as well as normal subjects.
ELISA was used to detect the serum PDCD5 concentrations in 100 normal persons, 83 patients with breast cancer, 74 patients with gastrointestinal tract cancer and 41 patients with lung cancer. The results were statistically analyzed and discussed.
PDCD5 could be detected in peripheral blood serum in both normal subjects and cancer patients. The serum PDCD5 contents in normal persons ranged from 3.8 to 6.1 ng/ml with a median of 4.70±0.68 ng/ml. For cancer patients the PDCD5 levels were 4.59±0.90, 4.79±1.14 and 10.43±22.34 ng/ml for breast cancer, gastrointestinal cancer and lung cancer patients respectively. There was no statistically significant difference between the serum PDCD5 concentrations of normal persons and cancer patients.
PDCD5 is present in peripheral blood. The PDCD5 levels in cancer patients are not statistically different from that of normal persons, though decreased expression of PDCD5 in malignant tissues has been found.
... In lieu of performing serial liver biopsies, we studied the effect of RBV over time on the mRNA expression of 5 selected ISGs in PBMCs [25,26] and on the serum protein levels of IP-10, a known ISG. We did not find any effect of RBV on the magnitude and temporal pattern of IFN-induced ISG expression in the PBMCs. ...
Objective:
Ribavirin improves treatment response to pegylated-interferon (PEG-IFN) in chronic hepatitis C but the mechanism remains controversial. We studied correlates of response and mechanism of action of ribavirin in treatment of hepatitis C.
Design:
70 treatment-naive patients were randomised to 4 weeks of ribavirin (1000-1200 mg/d) or none, followed by PEG-IFNα-2a and ribavirin at standard doses and durations. Patients were also randomised to a liver biopsy 24 h before or 6 h after starting PEG-IFN. Hepatic gene expression was assessed by microarray and interferon-stimulated gene (ISG) expression quantified by nCounter platform. Temporal changes in ISG expression were assessed by qPCR in peripheral-blood mononuclear cells (PBMC) and by serum levels of IP-10.
Results:
After 4 weeks of ribavirin monotherapy, hepatitis C virus (HCV) levels decreased by 0.5±0.5 log10 (p=0.009 vs controls) and ALT by 33% (p<0.001). Ribavirin pretreatment, while modestly augmenting ISG induction by PEG-IFN, did not modify the virological response to subsequent PEG-IFN and ribavirin treatment. However, biochemical, but not virological, response to ribavirin monotherapy predicted response to subsequent combination treatment (rapid virological response, 71% in biochemical responders vs 22% non-responders, p=0.01; early virological response, 100% vs 68%, p=0.03; sustained virological response 83% vs 41%, p=0.053). Ribavirin monotherapy lowered serum IP-10 levels but had no effect on ISG expression in PBMC.
Conclusions:
Ribavirin is a weak antiviral but its clinical effect seems to be mediated by a separate, indirect mechanism, which may act to reset IFN-responsiveness in HCV-infected liver.
... ding to Tang et al., IFN-α attachment to interferon alpha receptors (IFNAR) [39] and the very rapid activation of Interferon Stimulated Genes (ISG) after interferon alpha administration [40] could explain the effect of IFN-α as a potent immunostimulant of an innate response in the first few hours and an adaptive response after the first four weeks. [41]. The degree of the ISG-induced innate response may result in a rapid decline of HCV replication (as measured by a decrease of HCV-RNA levels), which, if significant enough, causes very distinct CD4 and CD8 responses (shifting the immune response from innate to adaptive). Clinically, this phenomenon defines a group of patients known as r ...
Hepatitis C (HCV), a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma, is the most common indication for liver transplantation in the United States. Although annual incidence of infection has declined since the 1980s, aging of the currently infected population is expected to result in an increase in HCV burden. HCV is prone to develop resistance to antiviral drugs, and despite considerable efforts to understand the virus for effective treatments, our knowledge remains incomplete. This paper reviews HCV resistance mechanisms, the traditional treatment with and the new standard of care for hepatitis C treatment. Although these new treatments remain PEG-IFN-α- and ribavirin-based, they add one of the newly FDA approved direct antiviral agents, telaprevir or boceprevir. This new "triple therapy" has resulted in greater viral cure rates, although treatment failure remains a possibility. The future may belong to nucleoside/nucleotide analogues, non-nucleoside RNA-dependent RNA polymerase inhibitors, or cyclophilin inhibitors, and the treatment of HCV may ultimately parallel that of HIV. However, research should focus not only on effective treatments, but also on the development of a HCV vaccine, as this may prove to be the most cost-effective method of eradicating this disease.
... Interestingly, even after 24 h of treatment, IFNdependent gene expression can help predict the probability of achieving an SVR [54] . Many of the genes, found to be upregulated in non-responders and responders, encode molecules secreted in the serum (cytokines) [55,56]. Thus, they are a logical approach to the development of serum markers to predict treatment response. ...
HCV infection is a global health problem that affects 170 million people worldwide. The severity of the disease varies from asymptomatic chronic infection to cirrhosis and hepatocellular carcinoma (HCC). Recently, the standard of care for genotype 1 patients has greatly improved with the addition of protease inhibitors (telaprevir or boceprevir) to pegylated interferon (PegIFN) and ribavirin (RBV). The prediction of fibrosis progression and the response to antiviral treatment are two major issues in the management of patients with chronic hepatitis C. Differential expression of mRNAs was first analyzed for both progression of fibrosis and treatment response. Specific polymorphisms, associated with either fibrosis or viral response, were identified thanks to major improvements in genome scanning technologies. Since 2009, several independent genome wide association studies (GWAS) have reported an association between genetic polymorphisms within the IL-28B promoter and both natural and treatment-induced clearance in genotype 1 infected patients. These different studies showed the strong association and the importance of IL-28B polymorphisms in the treatment response. Combining the different genetic factors could improve their predictive value and help identify patients at a high risk of progression of fibrosis as well as those with a lower chance of responding to treatment. The aim of this review was to discuss the genomic factors (mRNAs, miRNAs, and SNPs) and HCV infection with clinical implications for either progression of fibrosis or treatment response. Recent findings on the IL-28B polymorphism and its application in clinical practice will also be discussed.
The Mre11 complex (comprising Mre11, Rad50, Nbs1) is integral to the maintenance of genome stability. We previously showed that a hypomorphic Mre11 mutant mouse strain ( Mre11 ATLD1/ATLD1 ) was highly susceptible to oncogene induced breast cancer. Here we used a mammary organoid system to examine which Mre11 dependent responses are tumor suppressive. We found that Mre11 ATLD1/ATLD1 organoids exhibited an elevated interferon stimulated gene (ISG) signature and sustained changes in chromatin accessibility. This Mre11 ATLD1/ATLD1 phenotype depended on DNA binding of a nuclear innate immune sensor, IFI205. Ablation of Ifi205 in Mre11 ATLD1/ATLD1 organoids restored baseline and oncogene-induced chromatin accessibility patterns to those observed in WT . Implantation of Mre11 ATLD1/ATLD1 organoids and activation of oncogene led to aggressive metastatic breast cancer. This outcome was reversed in implanted Ifi205 -/- Mre11 ATLD1/ATLD1 organoids. These data reveal a connection between innate immune signaling and tumor suppression in mammary epithelium. Given the abundance of aberrant DNA structures that arise in the context of genome instability syndromes, the data further suggest that cancer predisposition in those contexts may be partially attributable to tonic innate immune transcriptional programs.
Interferons type I (IFN-I), activated following a bacterial or viral infection, play a major role in the induction and regulation of the immune system. The immune response results in viral RNA and binds to receptors such as RIG-I-like receptors (RLRs) or Toll-like receptors, leading to the IFN-I signaling cascade. Thanks to its cellular function, IFN-I is widely used in therapies for such diseases as multiple sclerosis (MS) and hepatitis C disease (HCD).
MS is a neurological, autoimmune, chronic inflammatory disease of the central nervous system (CNS). During MS, nerve cell demyelination is observed due to the myelin heaths and oligodendrocyte damage. As a result, neuronal signal and neuron communication are attenuated. The mechanism of MS is still unknown. MS therapy applies interferon-β (IFN-β). IFN-β therapy has been used since the last century, but the therapeutic mechanism of IFN-β has not been completely understood. MS can lead to four syndromes: clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS).
HCD occurs as a result of infection with the hepatitis C virus (HCV), belonging to the Flaviviridae family. HCV is a blood-borne virus with a positive single-stranded RNA. A vaccine for HCV is not available yet. HCD can lead to liver damage or cancer. In HCD interferon-α therapy (IFN-α) is applied. As with MS, the mechanism of IFN-α therapy is not completely known.
The aim of this review - to analyze the possibilities of traditional therapy and opening new perspectives and approaches to personalized theraру of patients with chronic viral hepatitis C. There are considered the viral and patien tfactors that have an mpact on the efficacy of theтру with interferon-a2 and ribavirin. There are presented data on the molecular mechanism of action of these drugs preparations. There are considered new antiviral drugs (protease inhibitors) and pharmacological agents being under development and targeted to viral polymerase andprotein NS5A.
Viral infections are a global public health problem and can contribute significantly to patient morbidity and mortality. It is estimated that there are 350 million people worldwide who are infected with hepatitis B virus and 17,000 new cases per year of hepatitis C are identified (Wasley et al., MMWR Morb Mortal Wkly Rep 57(SS-2):1–24, 2008). Herpes viruses are common, with an estimated seroprevalence of 50% for herpes simplex virus type-1 and 20% herpes simplex virus type-2 seroprevalence among adults in the United States (Piret and Boivin, Antimicrob Agents Chemother 55(2):459–472, 2011). Influenza infections worldwide lead to serious morbidity and have been associated with thousands of deaths each year; 18,500 deaths were attributed to 2009 influenza A H1N1 pandemic alone (MMWR Morb Mortal Wkly Rep 59(33):1057–1089, 2010; Dawood et al., Lancet Infect Dis 12(9):687–695, 2012). It is imperative that clinicians have an intimate knowledge of any medication used in their practice and a working knowledgebase of pharmacokinetics and pharmacodynamics is especially true in the discipline of infectious diseases. This chapter provides a detail review of pharmacokinetic and pharmacodynamic parameters of antiviral agents utilized to treat select viral infections in patients undergoing transplantation.
Ubiquitin‐specific protease 18 (USP18) is an important inhibitor of interferon (IFN) antiviral activity, and the aim of this study was to investigate the association between the USP18 mRNA level change in peripheral blood mononuclear cells (PBMCs) when stimulated with IFN in vitro before initiating treatment and the treatment outcomes in HBeAg‐positive chronic hepatitis B (CHB) patients treated with IFN. A total of 44 patients who received standard IFN‐based anti‐HBV therapy and follow‐up were enrolled in the study. The in vitro IFN‐induced USP18 mRNA change (USP18IFN‐N) was measured via comparison of quantitative PCR‐determined USP18 transcription levels of BPMCs cultured with and without IFN stimulation. Either for virological (VR) or serological response (SR), the baseline USP18IFN‐N were significantly higher (p = 0.018 for VR, p =0.008 for SR) among non‐responders (n = 23 for VR, n = 33 for SR) than that of responders (n = 21 for VR, n=11 for SR). Multivariate analyses revealed baseline USP18IFN‐N was a novel independent predictor for either VR (OR = 0.292, 95% CI = 0.102‐0.835, p = 0.022) or SR (OR = 0.173, 95% CI = 0.035‐0.849, p = 0.031) in our cohort. In addition, baseline USP18IFN‐N in combination with HBV DNA loads or HBeAg levels showed improved accuracy of pretreatment prediction for VR or SR responders, respectively. Baseline USP18IFN‐N levels are associated with both virological and serological response, and have the potential to become a clinical predictor for treatment outcomes in HBeAg‐positive CHB patients before initiating IFN‐α therapy.
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Hepatitis C virus is an important risk factor of liver cirrhosis and hepatocellular carcinoma. The commonly used treatment strategies, based on pegylated interferon alfa and ribavirin, do not provide a fully satisfactory clinical effect. The reasons for treatment failure are multifactorial and are attributed to both host and viral factors. Some of them are already employed in clinical practice to adjust the treatment strategy (i. e. pretreatment viral load, viral load kinetics, HCV genotype), but the knowledge in this field is still unsatisfactory. The present review summarizes the causes of therapy failure, with emphasis on the high viral genetic variability and assesses the potential of this unique viral feature to be used as a treatment prognostic marker.
The viral and patient factors affecting the efficacy of therapy were discussed. The modern data on the molecular mechanism of action of main drugs for hepatitis C therapy were presented. New antiviral drugs (protease inhibitors) and pharmacological substances under development targeted against viral polymerase and protein NS5A were considered.
Interferon-alpha (IFN-α) is a key mediator of antiviral immune responses used clinically for hepatitis C treatment. Though effective, IFN-α induces marked behavioral changes that, when severe, can appear indistinguishable from major depression. Curiously, fatigue and motivational impairment evolve rapidly, suggesting acute engagement of immune-brain communicatory pathways, yet mood impairments typically emerge later, after weeks of treatment. Whether this reflects prolonged modulation of motivational processes underpinning fatigue or separate neurobiological mechanisms is currently unclear.
Here, we used quantitative magnetization transfer (qMT) imaging, an advanced microstructural neuroimaging technique sensitive to effects of inflammation, in a prospective study design to measure acute brain changes to IFN-α and relate these to later development of discrete behavioral changes. Twenty-three patients initiating IFN-α treatment for hepatitis C underwent qMT imaging and blood sampling at baseline and 4 hours after their first IFN-α injection. Comprehensive behavioral and psychological assessments were completed at both scanning sessions and at treatment weeks 4, 8, 12, and 24.
IFN-α injection stimulated an acute inflammatory cytokine response and evoked fatigue that peaked between 4 and 12 weeks, preceding mood change by 4 weeks. In the brain, IFN-α induced an acute change in striatal microstructure that additionally predicted development of fatigue but not mood symptoms.
Our findings highlight qMT as an in vivo biomarker of central effects of peripheral inflammation. We demonstrate exquisite sensitivity of the striatum to IFN-α, implicate striatal perturbation in IFN-α-induced fatigue, and dissociate this from mechanisms underlying IFN-α-induced mood symptoms, providing empirical support for distinct neural substrates mediating actions on motivation and mood.
Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.
To evaluate the frequency of single-nucleotide polymorphism at the -88 myxovirus resistance (MxA) gene promoter region in relation to the status of hepatitis C virus (HCV) progression and response to combined interferon (IFN) in chronic HCV Egyptian patients. One hundred ten subjects were enrolled in the study; 60 HCV genotype 4-infected patients who underwent combined IFN therapy and 50 healthy individuals. All subjects were genotyped for -88 MxA polymorphism by the restriction fragment length polymorphism technique. There was an increasing trend of response to combined IFN treatment as 34.9% of GG, 64.3% of GT, and 66.7% of TT genotypes were sustained responders (P=0.05). The T allele was significantly affecting the response rate more than G allele (P=0.032). Moreover, the hepatic fibrosis score and hepatitis activity were higher in GG genotypes compared with the GT and TT genotypes. The multivariate analysis showed that the MxA GG genotype was an independent factor increasing the no response to IFN therapy (P=0.04, odds ratio [OR] 3.822, 95% confidence interval [CI] 1.056-11.092), also MxA G allele (P=0.0372, OR 2.905, 95% CI 1.066-7.919). MxA -88 polymorphism might be a potential biomarker to predict response to IFN and disease progression in chronic HCV-infected patients.
To evaluate the frequency of single-nucleotide polymorphism at the-88 myxovirus resistance (MxA) gene promoter region in relation to the status of hepatitis C virus (HCV) progression and response to combined interferon (IFN) in chronic HCV Egyptian patients. One hundred ten subjects were enrolled in the study; 60 HCV genotype 4-infected patients who underwent combined IFN therapy and 50 healthy individuals. All subjects were genotyped for-88 MxA polymorphism by the restriction fragment length polymorphism technique. There was an increasing trend of response to combined IFN treatment as 34.9% of GG, 64.3% of GT, and 66.7% of TT genotypes were sustained responders (P = 0.05). The T allele was significantly affecting the response rate more than G allele (P = 0.032). Moreover, the hepatic fibrosis score and hepatitis activity were higher in GG genotypes compared with the GT and TT genotypes. The multivariate analysis showed that the MxA GG genotype was an independent factor increasing the no response to IFN therapy (P = 0.04, odds ratio [OR] 3.822, 95% confidence interval [CI] 1.056– 11.092), also MxA G allele (P = 0.0372, OR 2.905, 95% CI 1.066–7.919). MxA-88 polymorphism might be a potential biomarker to predict response to IFN and disease progression in chronic HCV-infected patients.
Although in vitro studies have been performed to dissect the mechanism of action of IFNα, detailed in vivo studies on the long-term effects of IFNα on monocytes have not been performed. Here we examined peripheral blood from 14 chronic HCV patients at baseline and 12 weeks after start of IFNα-based therapy. Monocytes were phenotyped by flow-cytometry and their function evaluated upon TLR stimulation and assessed by multiplex cytokine assays.
During therapy of HCV patients, monocytes displayed a hyperactive state as evidenced by increased TLR-induced pro-inflammatory cytokine levels, as well as enhanced CD69 and CD83 mRNA and protein expression. Moreover, monocytes from 8 patients at baseline and 12 weeks after start of IFNα-based therapy were transcriptomically profiled by high throughput RNA-sequencing. Detailed RNA-seq analysis of monocytes showed significant ISG mRNA induction during therapy. Importantly, IFNα-based therapy activated TLR7 signaling pathways, as demonstrated by up-regulated expression of TLR7, MyD88, and IRF7 mRNA, whereas other TLR family members as well as CD1c, CLEC4C, and CLEC9A were not induced. The induction of TLR7 responsiveness of monocytes by IFNα in vivo in HCV patients is relevant for the development of TLR7 agonists that are currently under development as a promising immunotherapeutic compounds to treat chronic viral hepatitis.
Therapeutic vaccine candidates against hepatitis C virus infection have demonstrated immunogenicity, but none of them have been able to induce complete viral elimination. Reviewed evidence shows hepatitis C virus-specific T cell exhaustion as a major obstacle for the immune containment of the virus. The possibility that hepatitis C virus-specific T cells may be rescued from exhaustion by interferon-based treatment makes this therapy a promising candidate to combine with hepatitis C virus-specific therapeutic vaccine interventions. This review summarizes the main effects of interferon-based therapy on hepatitis C virus-specific cellular immune response in chronic patients and, focuses on its potential to favorably impact the performance of therapeutic vaccine approaches for sustained viral clarification.
DNAJ/HSP40 is an evolutionarily conserved family of proteins bearing various functions. Historically, it has been emphasized that HSP40/DNAJ family proteins play a positive role in infection of various viruses. We identified DNAJ/HSP40B6 as a potential negative regulator of HIV-1 replication in our genetic screens. In this study, we investigated the functional interactions between HIV-1 and HSP40 family members.
We took genetic and comparative virology approaches to expand the primary observation.
Multiple HSP40/DNAJ proteins were tested for their ability to inhibit replication of adenovirus, herpes simplex virus type 1, HIV-1, and vaccinia virus. The mechanism of inhibition was investigated by using HSP40/DNAJ mutants and measuring the efficiencies of each viral replication steps.
HSP40A1, B1, B6, and C5, but not C3, were found to be able to limit HIV-1 production. This effect was specific to HIV-1 for such effects were not detected in adenovirus, herpes simplex virus type 1, and vaccinia virus. Genetic analyses suggested that the conserved DNAJ domain was responsible for the inhibition of HIV-1 production through which HSP40 regulates HSP70 ATPase activity. Interestingly, HSP40s lowered the levels of steady-state viral messenger RNA. This was not attributed to the inhibition of Tat/long terminal repeat-driven transcription but the downregulation of Rev expression.
This is the first report providing evidence that HSP70-HSP40 complex confers an innate resistance specific to HIV-1. For their interferon-inducible nature, HSP40 family members should account for the anti-HIV-1 function of interferon.
Chronic hepatitis C virus infection occurs worldwide and affects over 2.7 million adults in North America. Current standard of care is the combination of pegylated (peg) interferon and ribavirin for 24 weeks in hepatitis C virus genotypes 2 or 3 and at least 48 weeks in chronic hepatitis C virus infection genotypes 1 or 4. Peginterferon-alpha 2(alpha) is a 40-kDa linear pegylated molecule that alters the pharmacokinetic properties of unmodified interferon-alpha. In clinical trials evaluating the combination of peginterferon-alpha 2(alpha) and ribavirin in chronic chronic hepatitis C virus infection, sustained virologic response rates have been achieved in 46-52% of patients with genotype 1 and 76-80% of patients with genotypes 2 or 3. Studies on the role of longer treatment duration and retreatment in prior nonresponders are in progress. The main toxicities of peginterferon-alpha 2(alpha) are flu-like symptoms and neuropsychiatric disorders, especially depression and cytopenias, and are found in similar rates to those observed with regular inteferon. Future areas of study include the role of peginterferon-alpha 2(alpha) in combination with the newer oral chronic hepatitis C virus infection active agents in development.
Twenty years ago, Emeritus Professor Sir Charles Stuart-Harris prefaced a collection of papers from a colloquium on “Problems
of Antiviral Therapy” with the following comments.
Key concepts: • Interferon-alpha has a broad spectrum of immunomodulatory activities on both innate and adaptive immune responses. • Ribavirin has immunomodulatory properties and both in vitro and in vivo was shown to modulate the cytokine profile. • The greater efficacy of interferon-alpha plus ribavirin therapy is a result of multiple synergistic effects including antiviral activity, favourable cytokine profile, increased antiviral T-cell reactivity in fast-responders, upregulation of antiviral gene expression. • In hepatitis C treatment, interferon-alpha and ribavirin appear to work mainly by direct interference with HCV life cycle, rather than by enhancement of cellular immunity to HCV. Interferon-alpha (IFN-α) has been the mainstay of treatment for hepatitis C even before the identification of the hepatitis C virus (HCV). The initial results with recombinant IFN-α or with natural, lymphoblastoid IFN-α alone were disappointing with only a small proportion of patients achieving a sustained virological response. The empirical addition of ribavirin to standard interferon significantly improved the long-term virological response. The development of pegylated interferons led to further improvement in the treatment outcome, and currently the combination of pegylated IFN-α plus ribavirin is the standard of care for antiviral therapy in patients with chronic hepatitis C. Both IFN-α and ribavirin possess broad spectrum antiviral and immunomodulatory activities, however the mechanisms of action of these two drugs in hepatitis C treatment, and the hierarchy of their effects in achieving HCV clearance are not fully understood.
Programmed cell death 5 (PDCD5) is a novel apoptotic regulatory gene that promotes apoptosis in various tumor cells. Studies have shown that PDCD5 accelerates the apoptosis of synoviocytes in vitro, implying a potential role in rheumatoid arthritis (RA) pathogenesis. This study examined the expression of PDCD5 in serum and synovial fluid of RA patients, its effect on the expression of inflammatory cytokine, interleukin-17 (IL-17), and the assessment of disease activity in RA.
PDCD5 and IL-17 levels in serum and synovial fluid from 18 patients with RA and 22 patients with osteoarthritis (OA) were detected using enzyme-linked immunosorbent assay (ELISA). Concentrations of serum PDCD5 in 40 healthy people were also detected as controls. As disease activity indices, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and X-ray grading scale were also evaluated.
Serum and synovial fluid PDCD5 levels in RA patients were significantly higher than those in OA and healthy controls. Serum PDCD5 level was inversely correlated to CRP and ESR, and was significantly higher in the RF negative group than in the positive group. PDCD5 level was also negatively correlated with IL-17 levels both in serum and synovial fluid of RA patients. However, differences in synovial fluid PDCD5 level from RA patients at different Larsen stages were not detectable.
PDCD5 affects RA pathogenesis. Insufficient apoptosis of fibroblast-like synoviocytes and inflammatory cells in RA could increase the expression of PDCD5 protein. As PDCD5 levels correlated negatively with disease activity indices and IL-17 level, PDCD5 could become a target in the diagnosis and treatment of RA.
Chronic infection with hepatitis C virus (HCV) is a major public health problem, with perhaps 180 million people infected worldwide. A significant proportion of these will eventually develop clinical complications, such as cirrhosis, liver decompensation and hepatocellular carcinoma. Sustained virological response (SVR) to antiviral therapy is associated with improvement in liver histology and survival free of liver-related complications. Great effort has been made to improve SVR rate by adapting the duration of therapy according to HCV genotype and to on-treatment response. Rapid virological response (RVR, undetectable HCV RNA at week 4) usually has a high positive predictive value for achieving SVR and early virological response (EVR, ≥ 2 log reduction or undetectable HCV RNA at week 12) exhibits a high negative predictive value for non-response. Individualized approach can improve cost-effectiveness of HCV antiviral therapy by reducing side effects and the costs of therapy associated with unnecessary exposure to treatment and through extending therapy for those with unfavorable features. This article summarizes recent data on strategies of individualized treatment in naïve patients with mono-infection by the different HCV genotypes. The management of common side effects, the impact of HCV infection on health-related quality of life and the potential applications of host genomics in HCV therapy are also briefly discussed.
Background:
The interaction between interferon-stimulated genes (ISGs) expression, IL28B genotypes and hepatitis C treatment outcomes has been mainly evaluated in the liver tissue from hepatitis C virus (HCV)-monoinfected patients but with controversial results. Herein, we examined whether more easily accessible peripheral blood mononuclear cells (PBMCs) could be used for this purpose in HIV-HCV coinfected patients, a population in whom HCV-induced liver disease progression occurs more rapidly and treatment response is lower.
Methods:
Gene expression profiles were examined using the human whole genome Agilent microarray platform in PBMCs collected from HIV/HCV-coinfected patients who had completed a course of peginterferon/ribavirin therapy with validated outcomes. Patients were split out according to the achievement of sustained virological response (SVR) and IL28B rs12979860 genotypes. The GeneSpringGX software was used to select genes differentially expressed in the different groups.
Results:
Nineteen HIV/HCV-coinfected individuals receiving antiretroviral therapy and having undetectable plasma HIV-RNA were examined. Global gene expression profiles showed 42 genes differentially expressed according to treatment outcome and 56 according to IL28B genotype. Common genes were not found and functions differed for genes belonging to either group. Whereas at least 26 out of 37 repressed genes (70.3%) in SVR patients were ISGs, none of the 56 differentially expressed genes in carriers of distinct IL28B variants were ISGs (P < 0.0001).
Conclusion:
Baseline expression of ISGs in PBMCs from HCV/HIV-coinfected patients influence the response to peginterferon/ribavirin therapy, regardless of IL28B genotypes. PBMC specimens can reliably be used for evaluating ISGs expression in clinical practice.
Interferon-α and ribavirin combination therapy has been the current choice for treating chronic hepatitis C (CHC) patients. The treatment takes 6 to 12 months but the overall sustained response rate is only around 50% and often brings significant adverse effects to some patients. The treatment outcome has been shown to be associated with various viral factors, such as viral loads before and during treatment and, most importantly, viral genotypes and quasispecies. Host factors that may affect drug response include age, gender, ethnicity, HLA alleles and stage of liver fibrosis. Recent studies have further indicated an association between patients' genotypes and their treatment efficacy. In this review article, we evaluate and summarize factors that may contribute to the treatment outcome of CHC. The content is divided into four categories: 1. viral factors: viral load, viral genotype and early viral response; 2. general host factors: gender, age, treatment period/dosage and kinds of IFN; 3. host genetic polymorphisms, particularly single nucleotide polymorphism (SNP) association studies; and 4. gene expression profiles in hepatitis C liver tissues. In summary, recent advances in pharmacogenomics have demonstrated the potential applications of genetic polymorphisms and expression patterns in determining treatment responsiveness in chronic hepatitis C. By combining both human and viral genotypes and their expression, it becomes plausible to satisfactorily assess the clinical outcomes prior to interferon combination treatment for CHC patients.
Interferon-α (IFN-α) are cytokines endowed with multiple biologic effects, including activities on cells of the immune system, which are important for inducing protective antiviral and antitumor responses. Studies in mouse models have been instrumental for understanding the immune adjuvant activity of these cytokines and some of their mechanisms of action. In particular, recent studies conducted on both mouse and human models suggest that IFN-α act as effective immune adjuvants for inducing antiviral and antitumor immunity and that the effects of IFN on the differentiation and activation of dendritic cells (DC) play an important role in the induction of protective responses. In spite of the long record of IFN-α clinical use, a few clinical trials have attempted to evaluate the efficacy of these cytokines used as vaccine adjuvants. Recently, studies on the IFN-α signature in cells from patients treated with IFN-α under different modalities and various clinical settings have provided important insights for understanding the in vivo mechanisms of the IFN immune adjuvant activity in humans and may contribute to the identification of molecular markers with a clinical response. These studies further support the interest of evaluating the clinical efficacy of IFN-α when used as a vaccine adjuvant and also suggest that the DC generated in vitro from monocytes in the presence of this cytokine can exhibit a special advantage for the development of effective therapeutic vaccination strategies in cancer patients.
We investigated the global changes in mRNA abundance in Escherichia coli elicited by various perturbations of tryptophan metabolism. To do so we printed DNA microarrays containing 95% of all annotated E. coli ORFs. We determined the expression profile that is predominantly dictated by the activity of the tryptophan repressor. Only three operons, trp, mtr, and aroH, exhibited appreciable expression changes consistent with this profile. The quantitative changes we observed in mRNA levels for the five genes of the trp operon were consistent within a factor of 2, with expectations based on established Trp protein levels. Several operons known to be regulated by the TyrR protein, aroF-tyrA, aroL, aroP, and aroG, were down-regulated on addition of tryptophan. TyrR can be activated by any one of the three aromatic amino acids. Only one operon, tnaAB, was significantly activated by the presence of tryptophan in the medium. We uncovered a plethora of likely indirect effects of changes in tryptophan metabolism on intracellular mRNA pools, most prominent of which was the sensitivity of arginine biosynthetic operons to tryptophan starvation.
A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed
robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes.
Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled
detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression
measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.
If viruses, by their variety, distribution, potential virulence and sheer inexhaustibility, present a permanent threat to the well-being of organisms, one might wonder why good health still exists. Yet healthy organisms do exist, though not because they simply evade the tireless viral intruders, but because they constantly mobilize potent defense forces. In vertebrates, T and B cell-mediated immunity is usually credited with being of prime importance in antiviral defense, but often, the first hurdles viruses have to clear are not antibodies and T cells but the flow of mucus, the acidity of the stomach, the paucity of appropriate cellular virus receptors, or the inadequacy of a synthetic machinery that viruses need to usurp for their replication. These hurdles are host defense mechanisms no less important for the homeostasis of health than the specific immune system; they are placed at various levels and make the interplay between virus and host a notoriously complex affair. The method of choice to elucidate such defense mechanisms at the molecular level is the analysis of genetic variants in which these defense forces are altered. Although such variants abound from plants (FRASER 1990) to mammals (for mice, see GREEN 1989), only a few of the associated genes have been isolated or characterized (for example, DÉzÉlÉe et al. 1989; Dveksler et al. 1991; Yokomori and Lai 1992; NÉdellec étal. 1994; Bureau et al. 1993), most likely because many of these variants are multigenic in origin and difficult to analyze.
The pleiotropic activities of interferons (IFNs) are mediated primarily through the transcriptional regulation of many downstream effector genes. The mRNA profiles from IFN-alpha, -beta, or -gamma treatments of the human fibrosarcoma cell line, HT1080, were determined by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes. Among these were transcripts for known IFN-stimulated genes (ISGs), the expression of which were consistent with previous studies in which the particular ISG was characterized as responsive to either Type I (alpha, beta) or Type II (gamma) IFNs, or both. Importantly, many novel IFN-stimulated genes were identified that were diverse in their known biological functions. For instance, several novel ISGs were identified that are implicated in apoptosis (including RAP46/Bag-1, phospholipid scramblase, and hypoxia inducible factor-1alpha). Furthermore, several IFN-repressed genes also were identified. These results demonstrate the usefulness of oligonucleotide arrays in monitoring mammalian gene expression on a broad and unprecedented scale. In particular, these findings provide insights into the basic mechanisms of IFN actions and ultimately may contribute to better therapeutic uses for IFNs.
Antigen injection into animals causes antigen-specific T cells to become activated and, rapidly thereafter, die. This antigen-induced death is inhibited by inflammation. To find out how inflammation has this effect, various cytokines were tested for their ability to interfere with the rapid death of activated T cells. T cells were activated in vivo, isolated, and cultured with the test reagents. Two groups of cytokines were active, members of the interleukin 2 family and the interferons (IFNs) alpha and beta. This activity of IFN-alpha/beta has not been described previously. It was due to direct effects of the IFNs on the T cells and was not mediated by induction of a second cytokine such as interleukin 15. IFN-gamma did not slow the death of activated T cells, and therefore the activity of IFN-alpha/beta was not mediated only by activation of Stat 1, a protein that is affected by both classes of IFN. IFN-alpha/beta did not raise the levels of Bcl-2 or Bcl-XL in T cells. Therefore, their activity was distinct from that of members of the interleukin 2 family or CD28 engagement. Since IFN-alpha/beta are very efficiently generated in response to viral and bacterial infections, these molecules may be among the signals that the immune system uses to prevent activated T cell death during infections.
Interferons (IFNs) are the most important cytokines in antiviral immune responses. “Natural IFN-producing cells” (IPCs) in
human blood express CD4 and major histocompatibility complex class II proteins, but have not been isolated and further characterized
because of their rarity, rapid apoptosis, and lack of lineage markers. Purified IPCs are here shown to be the CD4+CD11c− type 2 dendritic cell precursors (pDC2s), which produce 200 to 1000 times more IFN than other blood cells after microbial
challenge. pDC2s are thus an effector cell type of the immune system, critical for antiviral and antitumor immune responses.
The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.
IFNs play critical roles in host defense by modulating the expression of various genes via signal transducer and activator of transcription factors. We show that IFNalpha/beta activates another important transcription factor, NF-kappaB. DNA-binding activity of NF-kappaB was induced by multiple type 1 IFNs and was promoted by IFN in a diverse group of human, monkey, rat, and murine cells. Human IFN promoted NF-kappaB activation in murine cells that express the human IFNalpha/beta receptor-1 signal-transducing chain of the type 1 IFN receptor. IFN promotes inhibitor of kappa B alpha (IkappaBalpha) serine phosphorylation and degradation, and stimulates NF-kappaB DNA-binding and transcriptional activity. Importantly, IFN promotes cell survival by protecting cells against a variety of proapoptotic stimuli, such as virus infection and antibody-mediated crosslinking. Expression of superrepressor forms of IkappaBalpha, besides inhibiting IFN-mediated NF-kappaB activation and IkappaBalpha degradation, also enhanced apoptotic cell death in IFN-treated cells. We conclude that NF-kappaB activation by IFNalpha/beta is integrated into a signaling pathway through the IFNalpha/beta receptor-1 chain of the type 1 IFN receptor that promotes cell survival in apposition to various apoptotic stimuli.
The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L., Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77-80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C., Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73-76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A. (2000) Nucleic Acids Res., 28, 10-14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332-333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45-48] and can be accessed at http://genome-www.stanford.edu/microarray.
Interferons (IFNs) are a family of multifunctional cytokines that activate transcription of subsets of genes. The gene products induced by IFNs are responsible for IFN antiviral, antiproliferative, and immunomodulatory properties. To obtain a more comprehensive list and a better understanding of the genes regulated by IFNs, we compiled data from many experiments, using two different microarray formats. The combined data sets identified >300 IFN-stimulated genes (ISGs). To provide new insight into IFN-induced cellular phenotypes, we assigned these ISGs to functional categories. The data are accessible on the World Wide Web at http://www.lerner.ccf.org/labs/williams/, including functional categories and individual genes listed in a searchable database. The entries are linked to GenBank and Unigene sequence information and other resources. The goal is to eventually compile a comprehensive list of all ISGs. Recognition of the functions of the ISGs and their specific roles in the biological effects of IFNs is leading to a greater appreciation of the many facets of these intriguing and essential cytokines. This review focuses on the functions of the ISGs identified by analyzing the microarray data and focuses particularly on new insights into the protein kinase RNA-regulated (PRKR) protein, which have been made possible with the availability of PRKR-null mice.
Treatment with peginterferon alfa-2a alone produces significantly higher sustained virologic responses than treatment with interferon alfa-2a alone in patients with chronic hepatitis C virus (HCV) infection. We compared the efficacy and safety of peginterferon alfa-2a plus ribavirin, interferon alfa-2b plus ribavirin, and peginterferon alfa-2a alone in the initial treatment of chronic hepatitis C.
A total of 1121 patients were randomly assigned to treatment and received at least one dose of study medication, consisting of 180 microg of peginterferon alfa-2a once weekly plus daily ribavirin (1000 or 1200 mg, depending on body weight), weekly peginterferon alfa-2a plus daily placebo, or 3 million units of interferon alfa-2b thrice weekly plus daily ribavirin for 48 weeks.
A significantly higher proportion of patients who received peginterferon alfa-2a plus ribavirin had a sustained virologic response (defined as the absence of detectable HCV RNA 24 weeks after cessation of therapy) than of patients who received interferon alfa-2b plus ribavirin (56 percent vs. 44 percent, P<0.001) or peginterferon alfa-2a alone (56 percent vs. 29 percent, P<0.001). The proportions of patients with HCV genotype 1 who had sustained virologic responses were 46 percent, 36 percent, and 21 percent, respectively, for the three regimens. Among patients with HCV genotype 1 and high base-line levels of HCV RNA, the proportions of those with sustained virologic responses were 41 percent, 33 percent, and 13 percent, respectively. The overall safety profiles of the three treatment regimens were similar; the incidence of influenza-like symptoms and depression was lower in the groups receiving peginterferon alfa-2a than in the group receiving interferon alfa-2b plus ribavirin.
In patients with chronic hepatitis C, once-weekly peginterferon alfa-2a plus ribavirin was tolerated as well as interferon alfa-2b plus ribavirin and produced significant improvements in the rate of sustained virologic response, as compared with interferon alfa-2b plus ribavirin or peginterferon alfa-2a alone.
The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces
for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site
for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data
once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological
data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to
connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L.,
Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77–80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C.,
Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73–76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A.
(2000) Nucleic Acids Res., 28, 10–14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332–333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45–48] and can be accessed at http://genome-www.stanford.edu/microarray.
Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-α rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.
Only 15 to 20 percent of patients with chronic hepatitis C have a sustained virologic response to interferon therapy. We compared the efficacy and safety of recombinant interferon alfa-2b alone with those of a combination of interferon alfa-2b and ribavirin for the initial treatment of patients with chronic hepatitis C.
We randomly assigned 912 patients with chronic hepatitis C to receive standard-dose interferon alfa-2b alone or in combination with ribavirin (1000 or 1200 mg orally per day, depending on body weight) for 24 or 48 weeks. Efficacy was assessed by measurements of serum hepatitis C virus (HCV) RNA and serum aminotransferases and by liver biopsy.
The rate of sustained virologic response (defined as an undetectable serum HCV RNA level 24 weeks after treatment was completed) was higher among patients who received combination therapy for either 24 weeks (70 of 228 patients, 31 percent) or 48 weeks (87 of 228 patients, 38 percent) than among patients who received interferon alone for either 24 weeks (13 of 231 patients, 6 percent) or 48 weeks (29 of 225 patients, 13 percent) (P<0.001 for the comparison of interferon alone with both 24 weeks and 48 weeks of combination treatment). Among patients with HCV genotype 1 infection, the best response occurred in those who were treated for 48 weeks with interferon and ribavirin. Histologic improvement was more common in patients who were treated with combination therapy for either 24 weeks (57 percent) or 48 weeks (61 percent) than in those who were treated with interferon alone for either 24 weeks (44 percent) or 48 weeks (41 percent). The drug doses had to be reduced and treatment discontinued more often in patients who were treated with combination therapy.
In patients with chronic hepatitis C, initial therapy with interferon and ribavirin was more effective than treatment with interferon alone.
This chapter describes one of the currently used microarray technologies commonly called “spotting” or “printing” because DNAs are physically spotted on a solid substrate in which short oligonucleotides is synthesized directly on a solid support. In standard spotting applications, large collections of DNA samples are assembled in 96- or 384-well plates. DNA microarrays are used for a variety of purposes; essentially any property of a DNA sequence that can be made experimentally to result in differential recovery of that sequence can be assayed for thousands of sequences at once by DNA microarray hybridization. The chapter focuses on the application of DNA microarrays to gene expression studies and discusses general principles of whole genome expression monitoring as well as detailing the specific process of making and using spotted DNA microarrays.
The healthy liver of adult humans has little or no lymphocyte component and the histological finding of intrahepatic lymphocytes (IHL) is evidence of liver pathology. In a liver injured by chronic hepatitis C, the most common chronic liver disease, most IHL are activated/pro-inflammatory cells, which are particularly enriched for effectors of innate immunity (natural killer (NK), natural T, and other NK-like T cells). IHL do not undergo clonal expansion in the liver but migrate from extrahepatic sites to the chronically infected liver, where they display effector function and subsequently die, suggesting that maintenance of the IHL pool depends on continuous lymphocyte migration. The cytotoxic and inflammatory functions of these IHL have three potential outcomes: 1) they could be helpful in clearing the virus (a rare case in hepatitis C virus (HCV) infection); 2) they could be useless and have no effect on the infection; or 3) they could be harmful, whereby overaggressive lymphocyte responses destroy the liver in a continuous and unsuccessful attempt to clear the virus. Unfortunately, we do not know as of yet which of these possibilities is the case and, therefore, a more complete picture of the intrahepatic immune response will be relevant to the development of new therapeutic strategies against HCV. Additionally and from a more general perspective, due to the availability of biopsied material and the high prevalence (~3%) of HCV infection worldwide, studying the chronically inflamed liver of hepatitis C patients is an ideal model to investigate the poorly understood processes of lymphocyte trafficking, activation and death to non-lymphoid sites of chronic inflammation in man.
On the basis of cell surface markers, mature T cells are considered to have either a naïve or a memory phenotype. These cells exhibit distinct types of kinetic behaviour in vivo. While naive-phenotype cells persist long term in a non-dividing state. memory phenotype T cells include cycling cells and exhibit a more rapid rate of turnover; this has also been shown to be true for cells that can be definitively identified as naive or memory T cells respectively. The number of memory-phenotype (CD44in) CD8+ T cells entering cell cycle is greatly increased after In vivo exposure to viruses, bacteria or components of bacteria. Accelerated turnover of memory T cells also occurs after the injection of a variety cytokines that are induced by infectious agents, including type I interferon (IFN-I), Although naive-phenotype T cells do not divide in response to these cytokines, they do exhibit signs of activation, including upregulation of CD69 after exposure to lFN-1, These findings suggest that the dissimilar in vivo kinetics of naive- and memory-phenotype T cells might reflect their divergent responses to cytokines. Furthermore, the ability of infection- induced cytokines to stimulate non-specific proliferation of memory phenotype T cells and partial activation of naive-phenotype T cells implies that they play a complex role during primary immune responses w infectious agents.
Considerable progress has been made in the understanding of the molecular biology of the human interferon system. The genes encoding the interferons, their receptors, and the proteins that mediate many of their biological effects have been molecularly cloned and characterized. The availability of complete cDNA clones of components of the interferon systems has contributed significantly to our understanding of both the biology and the biochemistry of the antiviral actions of interferons. At the biological level, the antiviral effects of interferon may be viewed to be virus-type nonspecific. That is, treatment of cells with one type or even subspecies of interferon often leads to the generation of an antiviral state effective against a wide array of different RNA and DNA animal viruses. However, at the biochemical level, the antiviral action of interferon is often virus-type selective. That is, the apparent molecular mechanism which is primarily responsible for the inhibition of virus replication may differ considerably between virus types, and even host cells. For example, the IFN-regulated Mx protein selectively inhibits influenza virus but not other viruses when constitutively expressed in mouse cells. The IFN-regulated 2',5'-oligoadenylate synthetase selectively inhibits EMC and mengo viruses, two picornaviruses, but not viruses of other families when constitutively expressed in transfected cells. Some viruses are typically insensitive to the antiviral effects of interferon, both in cell culture and in intact animals. This lack of sensitivity to IFN may result from a virus-mediated direct antagonism of the interferon system. For example, in the case of adenovirus, the activation of the IFN-regulated RNA-dependent P1/elF-2 protein kinase is blocked by the virus-associated VA RNA. The relative sensitivity to interferon of different animal viruses varies appreciably. All three of the basic components required to measure an antiviral response may play a role in determining the relative effectiveness of the antiviral response: the species of interferon administered; the kind of cell treated; and, the type of virus used to challenge the interferon-treated host cell. Thus, the relative sensitivity to interferon observed for a particular interferon-cell-virus combination is likely the result of the equilibrium between the many agonists and antagonists which contribute to the overall response. That is, the relative sensitivity of a virus to the inhibitory action of IFN is governed by the qualitative nature and quantitative amount of the individual IFN-regulated cell proteins that may collectively contribute to the inhibition of virus replication.(ABSTRACT TRUNCATED AT 400 WORDS)
In a three-way crossover design, 12 healthy male volunteers received 5 X 10(6) IU/m2 body surface area interferon alpha-2b(IFN alpha-2b) by intravenous (IV) infusion over 30 minutes, intramuscular (IM) injections, and subcutaneous (SC) injections. Blood and urine samples were collected at specified times, and analysis of IFN alpha-2b concentrations was performed by immunoradiometric assay. "Flulike" symptoms were the most frequently reported adverse experiences and were independent of the route of administration. After a 30-minute IV infusion, IFN alpha-2b disappeared rapidly from serum, with distribution and elimination phase half-lives of 0.1 hour and 1.7 hours, respectively. Interferon alpha-2b was absorbed slowly after IM and SC administration, with similar absorption half-lives of 5.8 and 5.5 hours, respectively. The observed maximal concentrations after IM and SC administration were 42.1 IU/mL at six hours and 45.8 IU/mL at eight hours, respectively. Interferon alpha-2b was eliminated with half-lives of 2.2 hours after IM administration and 2.9 hours after SC administration. The areas under the serum concentration-time curves for the SC and IM doses were higher than those obtained for the IV infusion. Measurable amounts of IFN alpha-2b were not found in urine regardless of the route of administration.
RNA genomes evolve largely on the basis of single point mutations introduced by imprecise RNA polymerases, or by recombination. Clusters of certain transitions (biased hypermutations) were detected first in the genomes of persistent viruses, and in the past year have also been found in the genomes of lytic RNA viruses. A cellular RNA-modifying enzyme probably introduces the clustered transitions and thus contributes to the evolution of RNA viruses.
The adaptive immune response protects us from infection in a world of pathogens that is forever evolving new variants. As the system is built on the generation of an open repertoire of receptors, the recognition of self is unavoidable, and is guarded against by deletion during lymphocyte development of those cells that are specific for ubiquitous self antigens, and the silencing of those that are specific for self antigens only encountered after cells achieve functional maturity in the periphery. This silencing occurs when lymphocytes recognize antigens in the absence of suitable costimulatory molecules. By contrast, when the same cell encounters the same ligand on a cell that expresses costimulatory molecules, it will proliferate and differentiate into an effector cell. These effector cells mediate protective immunity when the antigen is carried by a pathogen, but they can mount autoimmune responses if the antigen is derived from self. The major costimulatory molecules for CD4 T cells appear to be B7 and B7.2 that bind to the CD28 and CTLA-4 receptors on the T cell. The signals from the TCR appear to be integrated with those from the costimulator receptor, and the T cell response depends on the precise nature of these signals, further conditioned by cytokines present in the environment of the responding cell. B cells can be viewed in a similar way, with the costimulatory molecule CD40 ligand and cytokines coming mainly from CD4 helper T cells determining the fate of the responding B cell. The TCR is not simply an on and off switch, since the precise way in which the TCR is ligated determines the differentiation of the T cell and can alter the effector responses of established T cell lines. Thus, the response capabilities of T cells are more flexible than originally believed, and much of this flexibility comes from the interplay of TCR signals and signs from the environment. If the biochemical nature of these differential signaling pathways were known, it might be possible to develop simple pharmacological agents capable of diverting T cell responses from harmful to innocuous by getting the T cell to reinterpret the signals it is receiving via its receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
The integration and control of systemic immune responses depends on the regulated trafficking of lymphocytes. This lymphocyte
“homing” process disperses the immunologic repertoire, directs lymphocyte subsets to the specialized microenvironments that
control their differentiation and regulate their survival, and targets immune effector cells to sites of antigenic or microbial
invasion. Recent advances reveal that the exquisite specificity of lymphocyte homing is determined by combinatorial “decision
processes” involving multistep sequential engagement of adhesion and signaling receptors. These homing-related interactions
are seamlessly integrated into the overall interaction of the lymphocyte with its environment and participate directly in
the control of lymphocyte function, life-span, and population dynamics. In this article a review of the molecular basis of
lymphocyte homing is presented, and mechanisms by which homing physiology regulates the homeostasis of immunologic resources
are proposed.
The 2-5A system is an RNA degradation pathway that can be induced by the interferons (IFNs). Treatment of cells with IFN activates genes encoding several double-stranded RNA (dsRNA)-dependent synthetases. These enzymes generate 5'-triphosphorylated, 2',5'-phosphodiester-linked oligoadenylates (2-5A) from ATP. The effects of 2-5A in cells are transient since 2-5A is unstable in cells due to the activities of phosphodiesterase and phosphatase. 2-5A activates the endoribonuclease 2-5A-dependent RNase L, causing degradation of single-stranded RNA with moderate specificity. The human 2-5A-dependent RNase is an 83.5 kDa polypeptide that has little, if any, RNase activity, unless 2-5A is present. 2-5A binding to RNase L switches the enzyme from its off-state to its on-state. At least three 2',5'-linked oligoadenylates and a single 5'-phosphoryl group are required for maximal activation of the RNase. Even though the constitutive presence of 2-5A-dependent RNase is observed in nearly all mammalian cell types, cellular amounts of 2-5A-dependent mRNA and activity can increase after IFN treatment. One well-established role of the 2-5A system is as a host defense against some types of viruses. Since virus infection of cells results in the production and secretion of IFNs, and since dsRNA is both a frequent product of virus infection and an activator of 2-5A synthesis, the replication of encephalomyocarditis virus, which produces dsRNA during its life cycle, is greatly suppressed in IFN-treated cells as a direct result of RNA decay by the activated 2-5A-dependent RNase. This review covers the organic chemistry, enzymology, and molecular biology of 2-5A and its associated enzymes. Additional possible biological roles of the 2-5A system, such as in cell growth and differentiation, human immunodeficiency virus replication, heat shock, atherosclerotic plaque, pathogenesis of Type I diabetes, and apoptosis, are presented.
Mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. These proteins have attracted attention because some display antiviral activity against pathogenic RNA viruses, for example against members of the orthomyxovirus (influenzavirus) family or the bunyavirus family. Transfected cells and transgenic mice expressing Mx proteins are highly resistant to Mx-sensitive viruses, demonstrating that Mx proteins are powerful antiviral agents. In humans, synthesis of MxA is observed during self-limiting viral infections and may thus promote recovery from disease.
To better understand the dynamics of hepatitis C virus and the antiviral effect of interferon-α-2b (IFN), viral decline in
23 patients during therapy was analyzed with a mathematical model. The analysis indicates that the major initial effect of
IFN is to block virion production or release, with blocking efficacies of 81, 95, and 96% for daily doses of 5, 10, and 15
million international units, respectively. The estimated virion half-life (t
1/2) was, on average, 2.7 hours, with pretreatment production and clearance of 1012 virions per day. The estimated infected cell death rate exhibited large interpatient variation (corresponding t
1/2 = 1.7 to 70 days), was inversely correlated with baseline viral load, and was positively correlated with alanine aminotransferase
levels. Fast death rates were predictive of virus being undetectable by polymerase chain reaction at 3 months. These findings
show that infection with hepatitis C virus is highly dynamic and that early monitoring of viral load can help guide therapy.
A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.
To investigate the type of immunity responsible for resolution of hepatitis C virus (HCV) infection, we monitored antibody and intrahepatic cytotoxic T lymphocyte (CTL) responses during acute (<20 weeks) infection in chimpanzees. Two animals who terminated infection made strong CTL but poor antibody responses. In both resolvers, CTL targeted at least six viral regions. In contrast, animals developing chronic hepatitis generated weaker acute CTL responses. Extensive analysis of the fine specificity of the CTL in one resolver revealed nine peptide epitopes and restriction by all six MHC class I allotypes. Every specificity shown during acute hepatitis persisted in normal liver tissue more than 1 yr after resolution. These results suggest that CD8+CTL are better correlated with protection against HCV infection than antibodies.
The class I major histocompatibility complex (MHC class I) presents 8-10 residue peptides to cytotoxic T lymphocytes. Most of these antigenic peptides are generated during protein degradation in the cytoplasm and are then transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Several lines of evidence have indicated that the proteasome is the major proteolytic activity responsible for generation of antigenic peptides--probably most conclusive has been the finding that specific inhibitors of the proteasome block antigen presentation. However, other proteases (e.g. the signal peptidase) may also generate some epitopes, particularly those on certain MHC class I alleles. The proteasome is responsible for generating the precise C termini of many presented peptides, and appears to be the only activity in cells that can make this cleavage. In contrast, aminopeptidases in the cytoplasm and endoplasmic reticulum can trim the N terminus of extended peptides to their proper size. Interestingly, the cellular content of proteases involved in the production and destruction of antigenic peptides is modified by interferon-gamma (IFN-gamma) treatment of cells. IFN-gamma induces the expression of three new proteasome beta subunits that are preferentially incorporated into new proteasomes and alter their pattern of peptidase activities. These changes are likely to enhance the yield of peptides with C termini appropriate for MHC binding and have been shown to enhance the presentation of at least some antigens. IFN-gamma also upregulates leucine aminopeptidase, which should promote the removal of N-terminal flanking residues of antigenic peptides. Also, this cytokine downregulates the expression of a metallo-proteinase, thimet oligopeptidase, that actively destroys many antigenic peptides. Thus, IFN-gamma appears to increase the supply of peptides by stimulating their generation and decreasing their destruction. The specificity and content of these various proteases should determine the amount of peptides available for antigen presentation. Also, the efficiency with which a peptide is presented is determined by the protein's half life (e.g. its ubiquitination rate) and the sequences flanking antigenic peptides, which influence the rates of proteolytic cleavage and destruction.
Recent advances in our understanding of cytolytic effector mechanisms include the partial characterization of caspase-independent apoptotic pathways triggered by granzymes, a realization of the vital importance of perforin and granzymes in the defence against certain virus infections in vivo and the first description of hereditary immunodeficiency due to disordered perforin expression in humans.
The healthy liver of adult humans has little or no lymphocyte component and the histological finding of intrahepatic lymphocytes (IHL) is evidence of liver pathology. In a liver injured by chronic hepatitis C, the most common chronic liver disease, most IHL are activated/pro-inflammatory cells, which are particularly enriched for effectors of innate immunity (natural killer (NK), natural T, and other NK-like T cells). IHL do not undergo clonal expansion in the liver but migrate from extrahepatic sites to the chronically infected liver, where they display effector function and subsequently die, suggesting that maintenance of the IHL pool depends on continuous lymphocyte migration. The cytotoxic and inflammatory functions of these IHL have three potential outcomes: 1) they could be helpful in clearing the virus (a rare case in hepatitis C virus (HCV) infection); 2) they could be useless and have no effect on the infection; or 3) they could be harmful, whereby overaggressive lymphocyte responses destroy the liver in a continuous and unsuccessful attempt to clear the virus. Unfortunately, we do not know as of yet which of these possibilities is the case and, therefore, a more complete picture of the intrahepatic immune response will be relevant to the development of new therapeutic strategies against HCV. Additionally and from a more general perspective, due to the availability of biopsied material and the high prevalence (approximately 3%) of HCV infection worldwide, studying the chronically inflamed liver of hepatitis C patients is an ideal model to investigate the poorly understood processes of lymphocyte trafficking, activation and death to non-lymphoid sites of chronic inflammation in man.
RNA editing, the post-transcriptional alteration of a gene-encoded sequence, is a widespread phenomenon in eukaryotes. As a consequence of RNA editing, functionally distinct proteins can be produced from a single gene. The molecular mechanisms involved include single or multiple base insertions or deletions as well as base substitutions. In mammals, one type of substitutional RNA editing, characterized by site-specific base-modification, was shown to modulate important physiological processes. The underlying reaction mechanism of substitutional RNA editing involves hydrolytic deamination of cytosine or adenosine bases to uracil or inosine, respectively. Protein factors have been characterized that are able to induce RNA editing in vitro. A supergene family of RNA-dependent deaminases has emerged with the recent addition of adenosine deaminases specific for tRNA. Here we review the developments that have substantially increased our understanding of base-modification RNA editing over the past few years, with an emphasis on mechanistic differences, evolutionary aspects and the first insights into the regulation of editing activity.
Mathematical models have been used to study the dynamics of HIV. Using these same principles, the dynamics of hepatitis C virus (HCV) are reviewed during interferon (IFN) therapy. After initiating IFN treatment, there is an IFN dose-dependent exponential decline in viral RNA levels within the first 48 hours. This rapid 1.0 to 2.0 log decline was best explained by an effect of IFN in inhibiting viral production with a varying degree of effectiveness. By applying mathematical principles, viral serum half-life was estimated to be 3.0 hours and viral production rat was calculated to be 1.0 x 10(12) virions per day. After this rapid first-phase decline there was a slower second phase decline in viral levels that was highly variable between subjects. This phase was dependent on the rate of elimination of HCV-infected liver cells. The rapidity of the second phase proved to be the best predictor of early viral clearance. The use of these models to understand the life cycle of viruses and their response to therapy is reviewed.
Major insights into events that control Th1/Th2 differentiation have been acquired recently, and highlight the role of Type I IFN in Th1 generation, by inducing up-regulation of the IL-12 receptor beta(2) subunit. IFN-alpha induces responsiveness to IL-12, and here we have investigated the source and the circumstances under which IFN-alpha is produced, in the absence of viral infections. Human dendritic cells (DC) were co-cultured with autologous T cells activated by cross-linking the CD3 complex. DC were also cultured with L cells expressing human CD40 ligand (CD40L). Our results show that large amounts (>200 IU IFN-alpha from 2.5x10(4) cells) of IFN-alpha are produced by DC following interaction with stimulated T cells. Similar effects were observed when DC were cultured with CD40L-expressing transfectants, although the amount of IFN-alpha produced was reduced, suggesting that the CD40-CD40L interaction may be important. These results show that stimulated T cells can solicit the signals from DC that allow their polarization towards a Th1 phenotype. Type I DC produce Type I IFN not only following viral infection but also during an immunological interaction and this may be the basic mechanism that assists in the development of a Th1 response.
Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-alpha rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.
The estimated prevalence of hepatitis C virus infection in the US is approximately 1.8%. Although interferon monotherapy and combination therapy of interferon with ribavirin represent mainstay for treating HCV infection, the rate of sustained virologic response remains suboptimal. The growing evidence suggested that the clinical sequence and treatment response of chronic hepatitis C are determined by a dynamic, complex tripartite relationship among HCV infection, the host immune response, and the effect of different interferon regimens. The treatment response is associated with various viral factors including the pretreatment viral level, dynamic change of viral level during treatment, viral genotype quasispecies and nucleotide mutation in nonstructural protein 5A of hepatitis C virus. Host factors that may affect treatment response include age, gender, race, HLA alleles and the host immune responses. Interferon regimens, including type, dose, frequency and duration of treatment and combination of interferon with other anti-HCV agents also alter the therapeutic response. Understanding these complicated interaction may provide better insights into the mechanism(s) of interferon response, leading to more effective clinical application of interferon therapy.
This review describes the contribution of noncytolytic mechanisms to the control of viral infections with a particular emphasis on the role of cytokines in these processes. It has long been known that most cell types in the body respond to an incoming viral infection by rapidly secreting antiviral cytokines such as interferon alpha/beta (IFN-alpha/beta). After binding to specific receptors on the surface of infected cells, IFN-alpha/beta has the potential to trigger the activation of multiple noncytolytic intracellular antiviral pathways that can target many steps in the viral life cycle, thereby limiting the amplification and spread of the virus and attenuating the infection. Clearance of established viral infections, however, requires additional functions of the immune response. The accepted dogma is that complete clearance of intracellular viruses by the immune response depends on the destruction of infected cells by the effector cells of the innate and adaptive immune system [natural killer (NK) cells and cytotoxic T cells (CTLs)]. This notion, however, has been recently challenged by experimental evidence showing that much of the antiviral potential of these cells reflects their ability to produce antiviral cytokines such as IFN-gamma and tumor necrosis factor (TNF)-alpha at the site of the infection. Indeed, these cytokines can purge viruses from infected cells noncytopathically as long as the cell is able to activate antiviral mechanisms and the virus is sensitive to them. Importantly, the same cytokines also control viral infections indirectly, by modulating the induction, amplification, recruitment, and effector functions of the immune response and by upregulating antigen processing and display of viral epitopes at the surface of infected cells. In keeping with these concepts, it is not surprising that a number of viruses encode proteins that have the potential to inhibit the antiviral activity of cytokines.
Microarrays can measure the expression of thousands of genes to identify changes in expression between different biological states. Methods are needed to determine the significance of these changes while accounting for the enormous number of genes. We describe a method, Significance Analysis of Microarrays (SAM), that assigns a score to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, the false discovery rate (FDR). When the transcriptional response of human cells to ionizing radiation was measured by microarrays, SAM identified 34 genes that changed at least 1.5-fold with an estimated FDR of 12%, compared with FDRs of 60 and 84% by using conventional methods of analysis. Of the 34 genes, 19 were involved in cell cycle regulation and 3 in apoptosis. Surprisingly, four nucleotide excision repair genes were induced, suggesting that this repair pathway for UV-damaged DNA might play a previously unrecognized role in repairing DNA damaged by ionizing radiation.
The lysosomal cysteine peptidases cathepsin B and cathepsin L are abundant and ubiquitously expressed members of the papain family, and both enzymes contribute to the terminal degradation of proteins in the lysosome. However, there is accumulating evidence for specific functions of lysosomal proteases in health and disease. The generation of 'knock out' mouse strains that are deficient in lysosomal proteases provides a valuable tool for evaluation of existing hypotheses and gaining new insights into the in vivo functions of these proteases. In this minireview, we summarise and discuss the findings obtained by analysis of mice that are devoid of cathepsin B or cathepsin L. In brief, cathepsin L appears to be critically involved in epidermal homeostasis, regulation of the hair cycle, and MHC class II-mediated antigen presentation in cortical epithelial cells of the thymus. Cathepsin B plays a major role in pathological trypsinogen activation in the early course of experimental pancreatitis and contributes significantly to TNF-alpha induced hepatocyte apoptosis.
A sustained virological response (SVR) rate of 41% has been achieved with interferon alfa-2b plus ribavirin therapy of chronic hepatitis C. In this randomised trial, peginterferon alfa-2b plus ribavirin was compared with interferon alfa-2b plus ribavirin.
1530 patients with chronic hepatitis C were assigned interferon alfa-2b (3 MU subcutaneously three times per week) plus ribavirin 1000-1200 mg/day orally, peginterferon alfa-2b 1.5 microg/kg each week plus 800 mg/day ribavirin, or peginterferon alfa-2b 1.5 microg/kg per week for 4 weeks then 0.5 microg/kg per week plus ribavirin 1000-1200 mg/day for 48 weeks. The primary endpoint was the SVR rate (undetectable hepatitis C virus [HCV] RNA in serum at 24-week follow-up). Analyses were based on patients who received at least one dose of study medication.
The SVR rate was significantly higher (p=0.01 for both comparisons) in the higher-dose peginterferon group (274/511 [54%]) than in the lower-dose peginterferon (244/514 [47%]) or interferon (235/505 [47%]) groups. Among patients with HCV genotype 1 infection, the corresponding SVR rates were 42% (145/348), 34% (118/349), and 33% (114/343). The rate for patients with genotype 2 and 3 infections was about 80% for all treatment groups. Secondary analyses identified bodyweight as an important predictor of SVR, prompting comparison of the interferon regimens after adjusting ribavirin for bodyweight (mg/kg). Side-effect profiles were similar between the treatment groups.
In patients with chronic hepatitis C, the most effective therapy is the combination of peginterferon alfa-2b 1.5 microg/kg per week plus ribavirin. The benefit is mostly achieved in patients with HCV genotype 1 infections.
Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2',5'-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2'-5'-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-alpha, IFN-beta, and IFN-gamma, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2',5'-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.
Interferon (IFN)-alpha therapy is currently the primary choice for viral hepatitis and a promising treatment for hepatocellular carcinoma (HCC). Primary mouse and rat hepatocytes respond poorly to IFN-alpha stimulation. Thus, it is very important to examine the IFN-alpha signal pathway in primary human hepatocytes.
The IFN-alpha-activated signals and genes in primary human hepatocytes and hepatoma cells were examined by Western blotting and microarray analyses.
Primary human hepatocytes respond very well to IFN-alpha stimulation as shown by activation of multiple signal transducer and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple genes. The differential response to IFN-alpha stimulation in primary human and mouse hepatocytes may be caused by expression of predominant functional IFN-alpha receptor 2c (IFNAR2c) in primary human hepatocytes vs. expression of predominant inhibitory IFNAR2a in mouse hepatocytes. Microarray analyses of primary human hepatocytes show that IFN-alpha up-regulates about 44 genes by over 2-fold and down-regulates about 9 genes by 50%. The up-regulated genes include a variety of antiviral and tumor suppressors/proapoptotic genes. The down-regulated genes include c-myc and c-Met, the hepatocyte growth factor (HGF) receptor. Down-regulation of c-Met is caused by IFN-alpha suppression of the c-Met promoter through down-regulation of Sp1 binding and results in attenuation of HGF-induced signals and cell proliferation.
IFN-alpha directly targets human hepatocytes, followed by activation of multiple STATs and regulation of a wide variety of genes, which may contribute to the antiviral and antitumor activities of IFN-alpha in human liver.
CD8+ T-cell response is thought to be important for the control of hepatitis C virus (HCV) as well as for the liver cell injury caused by HCV infection. Studies on antigen-specific CD8+ T cells had long been hampered by lack of suitable techniques. Recently developed single-cell based assays, including peptide major histocompatibility complex (MHC) tetramer staining and intracellular cytokine staining, have greatly enhanced the opportunities for directly studying HCV-specific CD8+ T cells. Thanks to these novel assays the quantitative and qualitative nature of HCV-specific CD8+ T cells, including their number, phenotype, and effector functions, are starting to be revealed. However, much important information remains missing, including the signals for differentiation and migration of HCV-specific CD8+ T cells and the precise functions of antigen-specific effector cells in the virus-infected liver. The urgent need for effective immunotherapy and vaccines can not be met without a better understanding of the CD8+ T-cell response in HCV infection, which calls for a comprehensive strategy to study such cells directly using sensitive and quantitative assays.
Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interven-tional Therapy Group
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McHutchison JG, Gordon SC, Schiff ER, Shiffman ML, Lee WM, Rustgi VK, Goodman ZD, et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interven-tional Therapy Group. N Engl J Med 1998;339:1485-1492.