Activity Of Interferon-beta Research Articles

Stability and Activity of Interferon Beta to Treat Idiopathic Pulmonary Fibrosis with Different Nebulizer Technologies.

Background: Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by lung scarring, which results in breathing difficulty. Currently, patients with IPF exhibit a poor survival rate and have access to very limited therapeutic options. Interferon beta (IFN-β) has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsing forms of multiple sclerosis, and it has also been shown to exhibit therapeutic potential in IPF. However, clinical use of IFN-β did not lead to improved overall survival in IPF patients in existing studies. One possibility is the limited efficiency of IFN-β delivery through intravenous or subcutaneous injection. Materials and Methods: The aerosol particle size distribution was determined with a laser diffraction particle size analyzer to characterize the droplet size and fine particle fraction generated by three types of nebulizers: jet, ultrasonic, and mesh. A breathing simulator was used to assess the delivery efficiency of IFN-β, and the temperature in the medication reservoirs was monitored with a thermocouple during nebulization. To further evaluate the antifibrotic activity of IFN-β pre- and postnebulization, bleomycin (BLM)- or transforming growth factor-beta (TGF-β)-treated human lung fibroblast (HLF) cells were used. Cell viability was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Transwell migration assay and Q-PCR analysis were used to evaluate cell migration and the myofibroblast differentiation ability, respectively. IFN-β protein samples were prepared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, and the expression of IFN-β was assessed by western blotting. Results: Among the current drug delivery systems, aerosolized medication has shown increased efficacy of drug delivery for treating respiratory diseases when compared with parenteral drugs. It was found that neither the structural integrity nor the biological function of nebulized IFN-β was compromised by the nebulization process of the mesh nebulizer. In addition, in BLM dose-response or TGF-β-induced lung fibroblast proliferation assays, these effects could be reversed by both parenteral and inhaled IFN-β nebulized with the mesh nebulizer. Nebulized IFN-β with the mesh nebulizer also significantly inhibited the migration and myofibroblast differentiation ability of TGF-β-treated HLF cells. Conclusions: The investigations revealed the potential efficacy of IFN-β in the treatment of IPF with the mesh nebulizer, demonstrating the higher efficiency of IFN-β delivered through the mesh nebulizer.

Validation of a test procedure for determination of specific activity of human recombinant interferon beta (Infibeta®) using various cell/virus combinations

Interferon beta preparations have demonstrated efficacy in the treatment of multiple sclerosis. One of the most important quality attributes that support efficacy and safety of interferon beta preparations is specific antiviral activity. Interferon beta activity is determined by the biological test method which can lead to an erroneous final result due to the system uncertainties that contribute to the overall uncertainty. Accurate assessment of specific activity plays an important role in adequate determination of the product’s therapeutic dose, therefore the test method standardisation and validation are of particular relevance. The aim of the study was to validate a test procedure for assessing specific activity of human recombinant interferon beta preparations, using various cell/virus combinations, as illustrated by the example of Infibeta®. Materials and methods: WISH, Vero, A-549, and MDBK cells in combination with mouse encephalomyocarditis virus were used in the study. The testing was performed using the biological test method based on the interferon ability to suppress virus-induced cytopathic effects in cell cultures. The results were processed using methods of mathematical statistics and the GraphPadPrism, Statistical 2/0 software package. Results: the paper compares the results of specific activity determination of Infibeta® (interferon beta-1b) using various cell/virus combinations. The dose-response curves were used to compare the test results. It was demonstrated that all the tested cell lines could be used in the biological test procedure for determination of interferon beta specific activity. However, the best results were obtained with A-549, WISH cells in combination with mouse encephalomyocarditis virus. The following validation characteristics were determined in the cell/virus systems: specificity, linearity, precision, and robustness. Conclusions: the study validated the test procedure that enables measurement of interferon beta specific activity in the range of 4.8–11.2 IU/mL at a satisfactory accuracy level, which guarantees reliable test results. The study demonstrated robustness, intermediate precision, and reproducibility of the test procedure.

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins.

Interferon-β (IFN-β) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-β and S100P lowering IFN-β cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633–639). S100P is a member of large family of multifunctional Ca2+-binding proteins with cytokine-like activities. To probe selectivity of IFN-β—S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca2+-dependent binding to IFN-β with equilibrium dissociation constants, Kd, of 0.04–1.5 µM for their Ca2+-bound homodimeric forms. Calcium depletion abolishes the S100—IFN-β interactions. Monomerization of S100A1/A4/A6 decreases Kd values down to 0.11–1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-β-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-β activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-β.

Grouper PKR activation inhibits red-spotted grouper nervous necrosis virus (RGNNV) replication in infected cells

The double-stranded RNA-activated protein kinase (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, PKR inhibits or promotes viral replication. In the present study, PKR homologues of orange-spotted grouper (Epinephelus coioides) (EcPKR) were cloned and the involvement of EcPKR during Red-spotted grouper nervous necrosis virus (RGNNV) infection was investigated. EcPKR encodes a 621-amino acid polypeptide that is closely related to the equivalent protein in Larimichthys crocea. EcPKR encoded two dsRNA binding domains and a Serine/Threonine protein kinase domain. Quantitative real-time PCR (qRT-PCR) analysis indicated that EcPKR was present in all examined tissues, with higher expression in spleen, intestine and gill. When stimulated with poly(I:C), the expression of EcPKR in the grouper spleen was increased, with highest expression 12 h post stimulation. EcPKR concentration was significantly increased in RGNNV-infected cells, with highest expression at 36 h post stimulation. EcPKR is mainly present in the cytoplasm. Overexpression of EcPKR in grouper spleen (GS) cells inhibits the transcription of the RGNNV genes. Furthermore, our results show that EcPKR overexpression significantly enhances the immune response of interferon and the activation of interferon-beta (IFN-β), interferon stimulated response element (ISRE) and nuclear factor-kappa B (NF-κB). Taken together, these results are important for better understanding of the function of PKR in fish and reveal its involvement in host response to immune challenges in RGNNV.

Molecular cloning of porcine 2′,5′-oligoadenylate synthetase-like protein and its role in porcine reproductive and respiratory syndrome virus infection

Porcine 2′,5′-oligoadenylate synthetase-like protein is an essential antiviral protein induced by interferons; however, its bioinformatics, genetic characteristics and immunological characteristics related to porcine reproductive and respiratory syndrome virus are still unknown. In this study, porcine 2′,5′-oligoadenylate synthetase-like protein was cloned, and various attributes were predicted by bioinformatics analysis. Through RNAi depletion and overexpression methods, it was determined that porcine OASL not only inhibits porcine reproductive and respiratory virus replication but also activates interferon-beta production and the interferon-beta promoter, promoting the expression of interferon-beta mRNA. Through the depletion of different amino acids at the N and C termini, the antiviral activity and promoting the activity of interferon beta were evaluated. The results demonstrated that 31–60 amino acids at the N terminus were critical for virus replication. This study laid a theoretical foundation for exploring the characteristics of the porcine 2′,5′-oligoadenylate synthetase-like protein and suggested a new strategy for the prevention and control of porcine reproductive and respiratory syndrome virus and investigation of the therapeutic mechanism of this protein.

Interferon-Beta: Neutralizing Antibodies, Binding Antibodies, Pharmacokinetics and Pharmacodynamics, and Clinical Outcomes.

Antibodies to interferon-beta (IFNb) may occur during treatment with this drug and can be measured at several levels, the totality of antibodies referred to as antidrug antibodies (ADA) or binding antibodies, and in case of interference with the drug activity referred to as neutralizing antibodies (NAB). Antibodies can also interfere with the biological activity of IFNb as measured by pharmacodynamic markers. To get a complete picture of the interference between IFNb as a drug and the ADA, all the 3 above levels need to be considered. Furthermore, the interaction of these biomarkers changes over time with a shift of antibody properties with respect to immunoglobulin subtypes, affinity, and titers of antibodies. In case of persistent NAB, the clinical benefit of IFNb in the treatment of multiple sclerosis is abolished. In this report, the current knowledge on these issues will be reviewed. The data have been presented at a meeting in Coral Gables, Florida on April 18-21, 2012.

Identifying responders and nonresponders to interferon therapy in multiple sclerosis.

Interferon beta is a well established disease-modifying agent used for relapsing-remitting multiple sclerosis. Despite treatment, a relevant proportion of patients continue to experience clinical (ie, relapses, worsening of disability) and magnetic resonance imaging (MRI) activity. Early identification of responders and nonresponders to interferon beta is strongly recommended to select patients who need a prompt switch to another disease-modifying agent and to ultimately avoid accumulation of fixed disability over time. Detecting responders and nonresponders to interferon beta can be challenging, mainly because of the lack of a clear and shared clinical definition of response to treatment. Clinical features at the start of treatment should be considered as prognostic factors, but MRI parameters assessed during treatment, such as contrast-enhancing lesions or new T2-hyperintense lesions, may be sensitive markers of response to interferon beta. Quantitative scoring systems derived from a combination of relapses and MRI activity have recently been proposed as practical tools for use in the everyday clinical setting. Blood biomarkers, such as neutralizing antibodies to interferon beta and Myxovirus resistance protein A, provide further useful information for detecting responders and nonresponders to interferon beta. However, since the presence of neutralizing antibodies can only partially explain the nonresponse to interferon beta, biomarkers of interferon beta activity possibly related to the pathogenesis of the disease could represent a future step toward a tailored, long-lasting effective treatment against multiple sclerosis.

Noninvasive molecular imaging of interferon beta activation in mouse liver

Interferon beta (IFN-β) is the priming cytokine in the interferons (IFNs) response that plays essential roles in innate immune system. Only very few studies on IFN activation in animals have been reported before, therefore, we embarked to develop a novel method to dynamically examine IFN-β activation in mouse liver by noninvasive molecular imaging. Interferon beta promoter-directed firefly luciferase gene was integrated into chromosomes of hepatocytes by hydrodynamic injection. Mouse hepatitis virus type 3 (MHV-3) and polyinosinic-polycytidylic acid [poly(I:C)] were used to stimulate the activation of IFN-β. Luciferase activity was used as an indicator of the IFN-β promoter activity in vitro and in vivo. The expression level of IFN-β in the sera and firefly luciferase in the liver was assessed by ELISA and bioluminescence imaging respectively. Western blot was used for detecting proteins expression. A rapid and elevated luciferase expression in the mouse liver induced by poly (I:C) and MHV-3 was detected by bioluminescence imaging. The detectable level of IFN-β in the sera was not induced by MHV-3. Moreover, IFN-β activation was significantly inhibited by the hepatitis C virus (HCV) NS3/4A protease in mouse liver. These results were consistent with IFN-β production in the sera. Therefore, a novel visual method to monitor IFN-β promoter activity was established in the current study. This novel sensitive method can be used for not only assessing IFN-β activation or inhibition in the liver under different conditions, but also screening drug candidates of stimulating or inhibiting of IFN-β production.

Neutralizing antibodies in interferon beta treated patients with multiple sclerosis: knowing what to do now

Longterm treatment of parenterally administered interferon beta (IFNb) is one of the mainstays of multiple sclerosis (MS) therapy [1]. The first trials were conducted in the early 1980s when human fibroblast derived IFNb was administered intrathecally in an open-label fashion [2]. In ten patients treated monthly for 6 months a significant reduction of the annualized relapse rate was noted compared to the relapse rate at baseline. This effect was not observed in ten control patients with MS that, for ethical reasons, did not undergo repeat sham lumbar punctures. This prompted a larger multicentre trial in which again human IFNb purified from cultured fibroblasts was administered intrathecally [3]. After the advent of recombinant techniques to produce larger amounts of IFNb, the first pivotal trial of IFNb-1b administered subcutaneously every other day in 372 patients with relapsing-remitting MS was published in 1993 [4], and the drug was approved by the FDA for use in MS the same year. Since then two additional IFNb-1a products were evaluated in pivotal trials that yielded positive results and have been marketed subsequently [5, 6]. Treatment with IFNb was also shown to delay disease progression and halt disease activity in patients with clinically isolated syndromes [7–9]. This observation along with neuropathological and MR imaging studies emphasized the importance of early diagnosis and treatment initiation of patients [10]. Overall the effect size of the IFNb preparations is comparable and moderate [1] and the effects of these drugs have been proven to be sustained for more than 15 years [11]. Additionally the long-term safety profile of IFNb treatment is excellent [12, 13]. However, with a so called biological, i.e. a proteinaceous therapeutic compound, the issue of IFNb immunogenicity is inherent and has remained a matter of debate [14]. Antibodies against IFNb can be detected in up to 47% of patients undergoing treatment with IFNb (Table 1) [14]. These antibodies are either purely binding, and then termed binding antibodies (BAbs) or inhibit IFNb activity and then termed neutralizing antibodies (NAbs) (Fig. 1). The proportion of NAbs is variable and differs between available IFNb products (table 1) [14]. Once the bioactivity of injected IFNb is blocked by persistent and hightiter NAbs the beneficial clinically detectable treatment effects of IFNb seem to vanish, albeit with a latency of some one to four years. A diminution of the downregulatory effect on MR disease activity becomes apparent earlier, i.e. 6-18 months after development of high titer Nabs. [15]. Immediate negative effects have not been sufficiently demonstrated. It is of note that most analyses were done in a post-hoc fashion on data from trials originally designed and powered to show treatment effects of IFNb rather than detrimental effects of NAbs [16]. Thus, there is ongoing controversy regarding the clinical impact of NAbs on the management of MS patients [17]. Current European guidelines recommend switching therapy in patients with persistently high-titer NAbs with suspected clinical or MRI activity [16, 18]. Importantly, NAbs may also affect endogenous IFNb as cross-reactivity between antibodies against IFNb-1a or IFNb-1b and endogenous, human fibroblast derived IFNb was shown only recently by us [19]. Antibodies to IFNb are measured in a two-step procedure. First, BAbs are detected by standard antibody assays. In a second step the neutralizing capacity of these T. Menge H.-P. Hartung (&) B. C. Kieseier Department of Neurology, Medical Faculty, Heinrich-Heine-University, Moorenstrasse 5, 40225 Dusseldorf, Germany e-mail: Hans-Peter.Hartung@uni-duesseldorf.de

One-year evaluation of factors affecting the biological activity of interferon beta in multiple sclerosis patients

MxA is an antiviral protein induced by type I interferons (IFN) and some viruses; MxA gene expression is an appropriate marker for measuring biologic activity of exogenous IFNβ, as its induction indicates IFNAR receptor stimulation. A recent study has shown that measurement of MxA mRNA, after 1 year of treatment, predicts clinical responsiveness to IFNβ therapy. Loss of IFNβ bioactivity is mostly due to anti-IFNβ antibodies (both neutralizing and binding), non-compliance and receptor saturation. The aim of this study was to evaluate all possible causes of loss of IFNβ bioactivity after 1 year in treated patients. One hundred sixty-seven multiple sclerosis (MS) patients were included. One year after beginning IFNβ therapy, each patient underwent a blood test; MxA gene expression was measured by real time PCR, antiviral CPE assay to detect neutralizing antibodies (NAbs), and capture-ELISA (cELISA) to measure binding antibodies (BAbs). For MxA an upper normal threshold of 87 (RE) was considered, 20 TRU/mL was the threshold for NAbs, and 1 U for BAbs positivity. Thirty-seven out of 167 patients (22%) were MxA-negative; of these, 22 were both BAbs and NAbs+, whereas 12 were BAbs+ but Nabs-, and three were both BAbs and NAbs-. The following conclusions were drawn from the study: (1) MxA mRNA should be measured after 1 year of IFNβ therapy; (2) after 1 year of IFNβ treatment, absence of IFNβ bioactivity was detected in 22% of the patients; (3) different biological phenomena and reduced compliance explain this absence; (4) identification of the reason for absence of IFN bioactivity improves patients' management.

PPAR γ activation potentiates interferon-beta activity in human pancreas adenocarcinoma through post-translational regulation of P21 and P27

PPAR γ activation potentiates interferon-beta activity in human pancreas adenocarcinoma through post-translational regulation of P21 and P27

Differential Gene Expression and Translational Approaches to Identify Biomarkers of Interferon Beta Activity in Multiple Sclerosis

More than 16 years ago human interferon-β-1b (IFN-β-1β) was shown to be effective in the treatment of the relapsing-remitting form of multiple sclerosis (MS). Over time, IFN-β has been demonstrated to be both a safe and effective treatment. However, the mechanism of action of IFN-β in MS remains unknown. To better understand the mechanism of action of IFN-β, considerable effort has been made in transcriptional profiling of peripheral blood mononuclear cells collected from MS patients. IFN-β is known to induce a large number of genes that play an important role in regulating responses to viral infection, immune modulation, and cell proliferation. Identifying differentially induced genes that are linked to the beneficial effects observed during treatment is under active investigation. IFN biomarkers in MS patients have been proposed but have not been clearly confirmed in independent studies or consistently correlated with clinical measures of disease progression. Organizing single genes or gene signatures grouped according to molecular mechanisms meaningful in MS may help to link IFN activity measurements to clinical outcomes. In this review, IFN activity measurements will be discussed with a specific emphasis on what is known about differential gene expression and treatment effects in MS.

Are Corticosteroids Efficacious for Preventing or Treating Neutralizing Antibodies in Multiple Sclerosis Patients Treated With Beta-Interferons?

Persistent, high-titer neutralizing antibodies (NAbs) reduce or eliminate the biologic activity of interferon-beta (IFNB) therapies for multiple sclerosis (MS) and are associated with reduction in efficacy. Most patients who develop NAbs have preceding detectable binding antibodies. There is no consensus on prevention or management of NAb-positive patients but switching to noninterferon therapy and use of immunosuppressive strategies, especially corticosteroids, has been proposed. To evaluate the evidence supporting the efficacy of corticosteroid therapy for (a) reducing the incidence of NAbs; and (b) improving markers of interferon bioavailability, reducing NAbs titers, and improving clinical outcomes in NAb-positive patients with MS receiving IFNB therapy. The objective was addressed through the development of a structured critically appraised topic. This included a case scenario, structured question, search strategy, critical appraisal, results, evidence summary, commentary, and bottom line conclusions. Participants included consultant and resident neurologists, a medical librarian, clinical epidemiologists, and content experts in the fields of neuroimmunology and multiple sclerosis. We selected 4 papers for detailed review, all of which used pulsed methylprednisolone therapy, either alone or in combination with another immunosuppressive therapy. A randomized open-label trial showed that monthly intravenous methylprednisolone initiated at INFB therapy onset was associated with a lower risk of an ever-positive NAb result but was insufficiently sensitive to detect an effect on more clinically meaningful high-titer NAbs. A randomized controlled trial for patients with active disease despite IFNB therapy showed that baseline NAb titers were reduced by subsequent intravenous methylprednisolone treatment, but the incidence of NAbs was too small to assess clinical relevance. Small open-label observational studies suggest that pulse methylprednisolone, alone or in combination with azathioprine, does not restore the bioavailability of IFNB. Pulse methylprednisolone therapy may reduce the risk of developing NAbs (but possibly not high-titer NAbs of clinical importance) when coadministered with newly initiated IFNB therapy. However, current evidence suggests that methylprednisolone therapy does not restore IFNB biologic response in established NAB-positive MS patients.

Time course transcriptomics of IFNB1b drug therapy in multiple sclerosis

Despite its generalized use as drug therapy for multiple sclerosis (MS), the molecular mechanisms of action of interferon beta (IFNB) are still poorly understood. IFNB therapy is long-termed and clinical effects are not immediate, therefore reliable early biomarkers for IFNB activity should maintain a differential expression over time, but longitudinal studies at a transcriptional level have been rare. Microarrays were used to monitor 18 IFNB1b treated MS patients at four time points spanning a period of 1 year. Genes showing in the majority of patients the greatest and most consistent changes in their expression levels were studied. Interferon regulated genes were significantly overrepresented. Fifteen markers were differentially expressed during all three time points and followed a consistent time course pattern: EIF2AK2, IFI6, IFI44, IFI44L, IFIH1, IFIT1, IFIT2, IFIT3, ISG15, MX1, OASL, RSAD2, SN, XAF1 and the marker 238704_at. Except for the last one, these biomarkers were all formerly identified as being indicative for IFNB activity. Expression changes were both early detectable and long lasting and could thus be optimal biomarkers for IFNB activity in long-term studies. Other known biomarkers of IFNB activity were found to be differentially expressed just for certain periods after therapy onset: Interleukin-8 was a short lasting marker and changes in STAT1 were detected with delay.

Translational approaches and patient profiling to identify relevant biomarkers of interferon beta activity in multiple sclerosis

Translational approaches and patient profiling to identify relevant biomarkers of interferon beta activity in multiple sclerosis

Enhancement of Chemokine Expression by Interferon Beta Therapy in Patients With Multiple Sclerosis

Interferon beta has been approved for the treatment of multiple sclerosis (MS). It is believed that immunomodulatory rather than antiviral activity of interferon beta is responsible for disease amelioration. The impact of interferon beta on the chemoattraction of immune cells has not been fully addressed. To address the influence of interferon beta on the expression of chemokines and their receptors in a standardized setting. The expression of 14 chemokines and 14 chemokine receptor genes was determined by quantitative real-time polymerase chain reaction from fresh blood samples. Outpatient units in Germany. Patients Untreated and interferon beta-treated patients with MS who tested positive and negative for neutralizing antibodies (NABs) were recruited from August 24, 2006, through December 15, 2006, for the initial study and from March 12, 2007, through April 2, 2007, for the validation study. Gene expression and serum chemokine protein levels. CCL1, CCL2, CCL7, CXCL10, CXCL11, and CCR1 gene expression was strongly upregulated in interferon beta-treated, NAB-negative MS patients. In contrast, gene expression in interferon beta-treated, NAB-positive MS patients did not differ from untreated control donor individuals. Antibody titers inversely correlated with chemokine and chemokine receptor gene expression. Accordingly, serum chemokine protein levels of interferon beta-treated, NAB-negative MS patients were significantly higher than in untreated or interferon beta-treated, NAB-positive MS patients. We demonstrate that interferon beta strongly upregulates a set of chemokines and CCR1 in peripheral immune cells. The peripheral upregulation of these chemokines may reduce the chemoattraction of immune cells to the central nervous system and thus add to the therapeutic effects of interferon beta.

Interferon-β treatment in multiple sclerosis attenuates inflammatory gene expression through inducible activity of the phosphatase SHP-1

Interferon-beta is a current treatment for multiple sclerosis (MS). Interferon-beta is thought to exert its therapeutic effects on MS by down-modulating the immune response by multiple potential pathways. Here, we document that treatment of MS patients with interferon beta-1a (Rebif) results in a significant increase in the levels and function of the protein tyrosine phosphatase SHP-1 in PBMCs. SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and CNS demyelination as evidenced in mice deficient in SHP-1. In order to examine the functional significance of SHP-1 induction in MS PBMCs, we analyzed the activity of proinflammatory signaling molecules STAT1, STAT6, and NF-kappaB, which are known SHP-1 targets. Interferon-beta treatment in vivo resulted in decreased NF-kappaB and STAT6 activation and increased STAT1 activation. Further analysis in vitro showed that cultured PBMCs of MS patients and normal subjects had a significant SHP-1 induction following interferon-beta treatment that correlated with decreased NF-kappaB and STAT6 activation. Most importantly, experimental depletion of SHP-1 in cultured PBMCs abolished the anti-inflammatory effects of interferon-beta treatment, indicating that SHP-1 is a predominant mediator of interferon-beta activity. In conclusion, interferon-beta treatment upregulates SHP-1 expression resulting in decreased transcription factor activation and inflammatory gene expression important in MS pathogenesis.

Sendai virus defective-interfering genomes and the activation of interferon-beta

The ability of some Sendai virus stocks to strongly activate IFNβ has long been known to be associated with defective-interfering (DI) genomes. We have compared SeV stocks containing various copyback and internal deletion DI genomes (and those containing only nondefective (ND) genomes) for their ability to activate reporter genes driven by the IFNβ promoter. We found that this property was primarily due to the presence of copyback DI genomes and correlated with their ability to self-anneal and form dsRNA. The level of IFNβ activation was found to be proportional to that of DI genome replication and to the ratio of DI to ND genomes during infection. Over-expression of the viral V and C proteins was as effective in blocking the copyback DI-induced activation of the IFNβ promoter as it was in reducing poly-I/C-induced activation, providing evidence that these DI infections activate IFNβ via dsRNA. Infection with an SeV stock that is highly contaminated with copyback DI genomes is thus a very particular way of potently activating IFNβ, presumably by providing plentiful dsRNA under conditions of reduced expression of viral products which block the host antiviral response.

Biological markers of interferon-beta therapy: comparison among interferon-stimulated genes MxA, TRAIL and XAF-1

Biological activity of interferon-beta (IFNβ) can be assessed by measuring IFN-stimulated genes (ISGs). Among them, myxovirus resistance protein A (MxA) appears to have the highest specificity, but it has no role in the pathogenesis of multiple sclerosis (MS). To investigate the reliability of MxA as a biomarker, we compared its expression to that of two other ISGs: TNF-related apoptosis-inducing ligand (TRAIL) and X-linked inhibitor of apoptosis factor-1 (XAF-1). Both were shown to be involved in immunoregulatory mechanisms and might play a role in MS. Quantitative-PCR measurements were performed in peripheral blood mononuclear cells from 73 MS patients after short-term and long-term treatment with IFNβ. A time-dependent response for multiple ISGs was observed in all patients after short-term treatment. In contrast, long-term treatment induced concurrent inhibition of ISGs in 12.3% (9/73) of patients, in whom neutralizing antibodies (NAbs) were detectable. Besides, 22% (16/73) of chronically treated patients showed a non-NAbs-related abrogation of TRAIL expression. In summary, 1) MxA expression was significantly higher than both TRAIL and XAF-1, and 2) MxA was the most sensitive gene to detect decreased bioavailability due to NAbs. These findings identify MxA as an appropriate biomarker for IFNβ, although there is no evidence for a functional role of it in MS.

A Toll-like receptor–independent antiviral response induced by double-stranded B-form DNA

The innate immune system recognizes nucleic acids during infection or tissue damage; however, the mechanisms of intracellular recognition of DNA have not been fully elucidated. Here we show that intracellular administration of double-stranded B-form DNA (B-DNA) triggered antiviral responses including production of type I interferons and chemokines independently of Toll-like receptors or the helicase RIG-I. B-DNA activated transcription factor IRF3 and the promoter of the gene encoding interferon-beta through a signaling pathway that required the kinases TBK1 and IKKi, whereas there was substantial activation of transcription factor NF-kappaB independent of both TBK and IKKi. IPS-1, an adaptor molecule linking RIG-I and TBK1, was involved in B-DNA-induced activation of interferon-beta and NF-kappaB. B-DNA signaling by this pathway conferred resistance to viral infection in a way dependent on both TBK1 and IKKi. These results suggest that both TBK1 and IKKi are required for innate immune activation by B-DNA, which might be important in antiviral innate immunity and other DNA-associated immune disorders.