IFN-beta Treatment Research Articles

Regulation of Lysosomal Associated Membrane Protein 3 (LAMP3) in Lung Epithelial Cells by Coronaviruses (SARS-CoV-1/2) and Type I Interferon Signaling

Abstract Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) infection is a major risk factor for mortality and morbidity in critical care hospitals around the world. Lung epithelial type II cells play a major role in the recognition and clearance of respiratory viruses as well as repair of lung injury in response to environmental toxicants. Gene expression profiling studies revealed that mouse lung epithelial type II cells express several cell-specific markers including surfactant proteins and Lysosomal associated membrane protein 3 (LAMP3) located in lysosomes, endosomes and lamellar bodies. These intracellular organelles are involved in vesicular transport and facilitate viral entry and release of the viral genome into the host cell cytoplasm. In this study, regulation of LAMP3 expression in human lung epithelial cells by several respiratory viruses and type I interferon signaling was investigated. Respiratory viruses including SARS-CoV-1 and SARS-CoV-2 significantly induced LAMP3 expression in lung epithelial cells within 24 hours after infection that required the presence of ACE2 viral entry receptors. Time course experiments revealed that the induced expression of LAMP3 was correlated with the induced expression of Interferon–beta (IFNB1) and STAT1 at mRNA levels. LAMP3 was also induced by direct IFN-beta treatment in multiple lung epithelial cell lines or by infection with influenza virus lacking the non-structural protein1(NS1) in NHBE bronchial epithelial cells. LAMP3 expression was also induced by several respiratory viruses in human lung epithelial cells including RSV and HPIV3. Location in lysosomes and endosomes aswell as induction by respiratory viruses and type I Interferon suggests that LAMP3 may have an important role in inter-organellar regulation of innate immunity and a potential target for therapeutic modulation in health and disease. Furthermore, bioinformatics revealed that a subset of lung type II genes were differentially regulated in the lungs of COVID-19 patients.

Soluble Receptor Isoform of IFN-Beta (sIFNAR2) in Multiple Sclerosis Patients and Their Association With the Clinical Response to IFN-Beta Treatment.

PurposeInterferon beta receptor 2 subunit (IFNAR2) can be produced as a transmembrane protein, but also as a soluble form (sIFNAR2) generated by alternative splicing or proteolytic cleavage, which has both agonist and antagonist activities for IFN-β. However, its role regarding the clinical response to IFN-β for relapsing-remitting multiple sclerosis (RRMS) is unknown. We aim to evaluate the in vitro short-term effects and after 6 and 12 months of IFN-β therapy on sIFNAR2 production and their association with the clinical response in MS patients.MethodsNinety-four RRMS patients were included and evaluated at baseline, 6 and 12 months from treatment onset. A subset of 41 patients were classified as responders and non-responders to IFN-β therapy. sIFNAR2 serum levels were measured by ELISA. mRNA expression for IFNAR1, IFNAR2 splice variants, MxA and proteases were assessed by RT-PCR. The short-term effect was evaluated in PBMC from RRMS patients after IFN-β stimulation in vitro.ResultsProtein and mRNA levels of sIFNAR2 increased after IFN-β treatment. According to the clinical response, only non-responders increased sIFNAR2 significantly at both protein and mRNA levels. sIFNAR2 gene expression correlated with the transmembrane isoform expression and was 2.3-fold higher. While MxA gene expression increased significantly after treatment, IFNAR1 and IFNAR2 only slightly increased. After short-term IFN-β in vitro induction of PBMC, 6/7 patients increased the sIFNAR2 expression.ConclusionsIFN-β administration induces the production of sIFNAR2 in RRMS and higher levels might be associated to the reduction of therapeutic response. Thus, levels of sIFNAR2 could be monitored to optimize an effective response to IFN-β therapy.

Combined Therapy of Vitamin D3-Tolerogenic Dendritic Cells and Interferon-β in a Preclinical Model of Multiple Sclerosis.

Autologous antigen-specific therapies based on tolerogenic dendritic cells (tolDC) offer the possibility to treat autoimmune diseases by restoring homeostasis and targeting specifically autoreactive responses. Here, we explore the hypothesis that systemic inflammation occurring in autoimmune diseases, such as multiple sclerosis (MS), can generate a disease-specific environment able to alter the functionality of tolDC. In this context in fact, a combined therapy of tolDC with an immunomodulatory treatment could potentiate the beneficial effect of this antigen-specific cell therapy. For this purpose, we analyzed the efficacy of a combined therapy based on the use of vitamin D3 (VitD3)-tolDC plus interferon beta (IFN-beta) in MS. VitD3-tolDC were generated from healthy donors and MS patients and co-cultured with allogeneic peripheral blood mononuclear cells, in the presence or absence of IFN-beta. In vitro, VitD3-tolDC treatment reduced the percentage of activated T cells and allogeneic proliferation, whereas VitD3-tolDC+IFN-beta treatment enhanced the suppressive ability of VitD3-tolDC and, additionally, induced a shift towards a Th2 profile. To determine the clinical benefit of the combined therapy, C57BL/6-experimental autoimmune encephalomyelitis (EAE)-induced mice were treated with antigen-specific VitD3-tolDC and/or IFN-beta. Treatment of EAE mice with combined therapy ameliorated the disease course compared to each monotherapy. These results suggest that a combined therapy based on antigen-specific VitD3-tolDC and IFN-beta may represent a promising strategy for MS patients.

Neutralizing Autoantibodies to Type I IFNs in >10% of Patients with Severe COVID-19 Pneumonia Hospitalized in Madrid, Spain.

BackgroundIn a recent study, autoantibodies neutralizing type I interferons (IFNs) were present in at least 10% of cases of critical COVID-19 pneumonia. These autoantibodies neutralized most type I IFNs but rarely IFN-beta.ObjectivesWe aimed to define the prevalence of autoantibodies neutralizing type I IFN in a cohort of patients with severe COVID-19 pneumonia treated with IFN-beta-1b during hospitalization and to analyze their impact on various clinical variables and outcomes.MethodsWe analyzed stored serum/plasma samples and clinical data of COVID-19 patients treated subcutaneously with IFN-beta-1b from March to May 2020, at the Infanta Leonor University Hospital in Madrid, Spain.ResultsThe cohort comprised 47 COVID-19 patients with severe pneumonia, 16 of whom (34%) had a critical progression requiring ICU admission. The median age was 71 years, with 28 men (58.6%). Type I IFN-alpha- and omega-neutralizing autoantibodies were found in 5 of 47 patients with severe pneumonia or critical disease (10.6%), while they were not found in any of the 118 asymptomatic controls (p = 0.0016). The autoantibodies did not neutralize IFN-beta. No demographic, comorbidity, or clinical differences were seen between individuals with or without autoantibodies. We found a significant correlation between the presence of neutralizing autoantibodies and higher C-reactive protein levels (p = 5.10e−03) and lower lymphocyte counts (p = 1.80e−02). No significant association with response to IFN-beta-1b therapy (p = 0.34) was found. Survival analysis suggested that neutralizing autoantibodies may increase the risk of death (4/5, 80% vs 12/42, 28.5%).ConclusionAutoantibodies neutralizing type I IFN underlie severe/critical COVID-19 stages in at least 10% of cases, correlate with increased C-RP and lower lymphocyte counts, and confer a trend towards increased risk of death. Subcutaneous IFN-beta treatment of hospitalized patients did not seem to improve clinical outcome. Studies of earlier, ambulatory IFN-beta treatment are warranted.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-021-01036-0.

Long-term disability outcomes in relapsing-remitting multiple sclerosis: a 10-year follow-up study.

The aim of this study is to assess the impact of interferon (IFN) beta treatment on the development of worsening disability in relapsing-remitting (RR) multiple sclerosis (MS) patients in the single-center observation cohort. This is a prospective study of 236 IFN-beta-treated and 183 untreated RRMS patients recruited consecutively at the Clinic of Neurology in Belgrade (Serbia). Out of this original cohort, 10-year follow-up data were available for 233 IFN-beta-treated and 131 untreated subjects. The median time since recruitment was 9.7years. IFN-beta treatment significantly delayed (p < 0.001) the time to reach each of the clinical outcomes (secondary progression-SP, EDSS scores 4 and 6) since recruitment. Time from the first visit to SP was reached after 9.7years for IFN-beta-treated vs. 7.8years for untreated patients. The delay for the development of EDSS score ≥ 4 from the first visit was 1.6years (8.7years for IFN-beta-treated vs. 7.1years for untreated patients). Time from the first visit to EDSS score of 6 was reached after 9.8years for IFN-beta-treated vs. 8.8years for untreated patients. The IFN-beta-treated group showed significant reduction (p < 0.001) in the risk of conversion to SP when compared with untreated patients (HR = 0.22). There was also a significant difference in reaching EDSS scores 4 and 6 (p < 0.001), in favor of the IFN-beta-treated group (HR = 0.40 and HR = 0.27, respectively). Comparison of outcomes in our IFN-beta-treated vs. untreated RRMS patients suggests that this treatment may delay development of long-term disability in MS.

Regional neuronal activity in patients with relapsing remitting multiple sclerosis.

Although interferon-beta is an established drug for relapsing remitting multiple sclerosis (RRMS), its impact on neuronal activity is not well understood. We investigated 15 patients with RRMS by [18 F]fluorodeoxyglucose positron emission tomography (FDG-PET) to assess cerebral metabolic rate of glucose (CMRglc ) before interferon-beta therapy. Further, we performed clinical and neuropsychological investigations. In nine patients, these investigations were repeated after 6months of therapy. Ten healthy controls were also studied. We found no significant differences in absolute CMRglc between patients and controls, or in patients before and during treatment. However, during treatment, relative regional glucose metabolism (rCMRlglc ) was decreased in cerebellum and increased in parts of left inferior parietal, temporo-occipital, frontal cortical areas, left striatum and right insula. In untreated patients, higher fatigue was associated with lower rCMRlglc in portions of left posterior cingulate cortex, and higher depression was associated with lower rCMRlglc within the left superior temporal sulcus. In the pooled sample, higher depression was associated with higher rCMRlglc in parts of the right precuneus. Our results indicate effects of IFN-beta treatment on cerebellar, cortical and subcortical neuronal function. Moreover, more severe fatigue and depression in untreated patients seem to be associated with reduced neuronal activity in left posterior cingulate cortex and left superior temporal cortex, respectively.

Blood lymphocyte subsets identify optimal responders to IFN-beta in MS.

Response to interferon-beta (IFN-beta) treatment is heterogeneous in multiple sclerosis (MS). We aimed to search for biomarkers predicting no evidence of disease activity (NEDA) status upon IFN-beta treatment in MS. 119 patients with relapsing-remitting MS (RRMS) initiating IFN-beta treatment were included in the study, and followed prospectively for 2years. Neutralizing antibodies (NAb) were explored in serum samples obtained after 6 and 12months of IFN-beta treatment. Soluble cytokines and blood lymphocytes were studied in basal samples by ELISA and flow cytometry, respectively. 9% of patients developed NAb. These antibodies were more frequent in patients receiving IFN-beta 1b than in those treated subcutaneous (p=0.008) or intramuscular (p<0.0001) IFN-beta 1a. No patient showing NAb remained NEDA during follow-up. Basal immunological variables are also associated with patient response. Percentages below 3% of CD19+CD5+cells (AUC 0.74, CI 0.63-0.84; OR 10.68, CI 3.55-32.15, p<0.0001; Likelihood ratio 4.28) or above 2.6% of CD8+perforin+T cells (AUC 0.79, CI 0.63-0.96; OR 6.11, CI 2.0-18.6, p=0.0009; Likelihood ratio 5.47) increased the probability of achieving NEDA status during treatment. Basal blood immune cell subsets contribute to identify MS patients with a high probability of showing an optimal response to IFN-beta.

Transcriptional dysregulation of Interferome in experimental and human Multiple Sclerosis

Recent evidence indicates that single multiple sclerosis (MS) susceptibility genes involved in interferon (IFN) signaling display altered transcript levels in peripheral blood of untreated MS subjects, suggesting that responsiveness to endogenous IFN is dysregulated during neuroinflammation. To prove this hypothesis we exploited the systematic collection of IFN regulated genes (IRG) provided by the Interferome database and mapped Interferome changes in experimental and human MS. Indeed, central nervous system tissue and encephalitogenic CD4 T cells during experimental autoimmune encephalomyelitis were characterized by massive changes in Interferome transcription. Further, the analysis of almost 500 human blood transcriptomes showed that (i) several IRG changed expression at distinct MS stages with a core of 21 transcripts concordantly dysregulated in all MS forms compared with healthy subjects; (ii) 100 differentially expressed IRG were validated in independent case-control cohorts; and (iii) 53 out of 100 dysregulated IRG were targeted by IFN-beta treatment in vivo. Finally, ex vivo and in vitro experiments established that IFN-beta administration modulated expression of two IRG, ARRB1 and CHP1, in immune cells. Our study confirms the impairment of Interferome in experimental and human MS, and describes IRG signatures at distinct disease stages which can represent novel therapeutic targets in MS.

Global methylation correlates with clinical status in multiple sclerosis patients in the first year of IFNbeta treatment

The alteration of DNA methylation patterns are a key component of disease onset and/or progression. Our objective was to evaluate the differences in Long Interspersed Nuclear Element-1 (LINE-1) methylation levels, as a surrogate marker of global DNA methylation, between multiple sclerosis (MS) patients and healthy controls. In addition, we assessed the association of LINE-1 methylation with clinical disease activity in patients treated with IFNbeta (IFNβ). We found that individuals with high levels of LINE-1 methylation showed 6-fold increased risk of suffering MS. Additionally, treated MS patients who bear high LINE-1 methylation levels had an 11-fold increased risk of clinical activity. Moreover, a negative correlation between treatment duration and percentage of LINE-1 methylation, that was statistically significant exclusively in the group of patients without clinical activity, was observed. Our data suggest that in MS patients, a slight global DNA hypermethylation occurs that may be related to the pathophysiology of the disease. In addition, global DNA methylation levels could play a role as a biomarker for the differential clinical response to IFNβ.

Increased expression ratio of matrix metalloproteinase-9 (MMP9) and tissue inhibitor of matrix metalloproteinase (TIMP-1) RNA levels in Iranian multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune disease involving the central nervous system (CNS) with unknown immunopathogenic mechanisms. Matrix metalloproteinase-9 (MMP-9) facilitates T-cell migration into the CNS while the tissue inhibitor matrix metalloproteinase-1 (TIMP-1) inhibits MMP-9 actions. The aim of this study was to evaluate the expression of TIMP-1 RNA and MMP-9/TIMP-1 RNA ratio in blood cells of Iranian patients with relapsing-remitting multiple sclerosis (RRMS) treated with IFNb. The study compared the expression level of TIMP-1 gene in RRMS samples with normal individuals in Iran and the results were compared using a ratio of MMP-9 to TIMP-1. All patients were HLA-DRB1*15 negative and were responders to interferon-beta with a normal vitamin D level. The RRMS patients manifested a lower expression level of TIMP-1 RNA than their normal counterparts although the result was not significant (P= 0.06). Also, the ratio of MMP-9 to TIMP-1 RNA increased significantly (P= 0.009). There was no linear correlation between TIMP-1 expression level and risk of Expanded Disability Status Scale of Kurtzke (EDSS); nor was there any significant correlation between expression status of TIMP-1 and duration of the disease. Although there was no significant decrease in TIMP-1 expression level, the MMP-9/TIMP-1 RNA ratio in RRMS was significantly higher than normal subjects. Further studies are recommended to compare MMP-9/TIMP-1 RNA ratio in patients before and after taking IFN-beta in order to find out if MMP-9/TIMP-1 RNA ratio can function as a proper marker of the bio efficacy of IFN-beta treatment of MS.

Systematic Review: Impact of Interferon-based Therapy on HCV-related Hepatocellular Carcinoma

Hepatitis C virus (HCV) is the leading cause of hepatocellular carcinoma (HCC), and several antiviral agents are available for the treatment of chronic HCV infection. However, the impact of antiviral therapy on the long-term outcomes of HCV-related HCC patients remains inconclusive. We aimed to examine the impact of antiviral therapy on the long-term outcomes of HCV-related HCC patients. We conducted a systematic review using PRISMA guidelines to identify trials and English-language literature from PubMed, Ovid MEDLINE, Scopus and the Cochrane Library database till August 2014. Randomized trials of antiviral treatments examining the effects of antiviral therapy on CHC patients and HCV-related HCC patients were screened and selected. We identified 6 trials evaluated the effectiveness of interferon (IFN)-alfa treatment, 3 studies examined pegylated interferon-alfa treatment, and 2 studies examined IFN-beta treatment. IFN-based therapy may decrease HCC incidence in HCV cirrhotic patients after a >5-year follow-up, improve liver reserve, decrease HCC recurrence rate, and increase survival rate in HCV-related HCC patients after curative HCC therapy. In conclusion, IFN-based therapy is beneficial and may be recommended in the management of HCV-related HCC patients who are IFN eligible.

IL-17A promotes myelin loss and inflammatory response during Cuprizone-induced demyelination

resident microglia (MG) and peripheral immune cells infiltrating into the region surrounding the infarct core and leading to secondary neurodegeneration. Current clinical therapies using thrombolytic and anti-platelet agents are limited by temporal restrictions and do not prevent the secondary, inflammation-related CNS damage. Therefore, the development of new anti-inflammatory therapies in ischemic stroke is necessary and urgent. Interferon beta (IFNbeta), a cytokine with immunomodulatory properties, was approved by the FDA for the treatment of relapsing–remitting multiple sclerosis for more than a decade. Its anti-inflammatory properties and well characterized safety profile suggest that IFNbeta has therapeutic potential for the treatment of ischemic stroke. We evaluated the therapeutic effect of IFNbeta treatment in the ischemic stroke animal model, murine transient middle cerebral artery occlusion/reperfusion (MCAO/R), as well as the regulatory effect of IFNbeta treatment on the infiltration of inflammatory peripheral immune cells into CNS infarcted region. Our results showed that IFNbeta treatment dramatically decreased infarct size in ischemic brains and that is correlated with significantly reduced neurological scores. In addition, the infiltration of inflammatory peripheral immune cells, including neutrophils and dendritic cells/macrophages, was also largely decreased in ischemic brains of IFNbeta-treated stroke mice, compared to those of control stroke mice. As MG activation and subsequent release of inflammatory cytokines in the ischemic brains exacerbate the disease of stroke, we compare MG activation and inflammatory cytokine expression in the ischemic brains of IFNbeta-treated and control stroke mice. We observed that IFNbeta treatment not only suppressed MG activation, but also inhibited inflammatory cytokine IL-1beta and MMP-9 expression in the ischemic brains. Taken altogether, our results demonstrate that IFNbeta confers a protective effect against ischemic stroke through its anti-inflammatory properties. (The study is supported by AHA grant 12SDG8170005 to JY.)

Superior antiviral and antiproliferative activity of IFN-beta vs. IFN-alpha in primary ATL cells occurs downstream of STAT1 signaling

Adult T-cell leukemia (ATL) is an aggressive CD4+CD25+ leukemia with poor prognosis, which usually develops several decades after HTLV-1 infection. In contrast to HIV-infection, the treatment of HTLV-1-associated diseases rely on a limited number of drugs. For ATL, combination therapy with IFN-alpha+AZT has shown clinical benefit in the non-lymphoma subtypes. Type I IFNs (IFN-alpha/beta) are essential cytokines with proved anti-cancer and antiviral action in vitro and in vivo. Nonetheless, their mechanisms of action in HTLV-1 infection remain unclear and a side-by-side comparison of both type I IFNs has not been performed in ATL. We show, in short-term culture of primary mononuclear cells from ATL patients, that both IFNs cause increased apoptosis, exert an anti-proliferative and antiviral effect, and decrease pro-inflammatory cytokine levels. However, IFN-beta treatment was significantly more effective in inhibiting viral p19 protein levels and lymphoproliferation, as compared to IFN-alpha. This pronounced effect of IFN-beta was explained by an induction of a higher number of known IFN-stimulated genes and antiviral genes by microarray analysis (76 vs. 26 genes were selected with p 2-fold difference vs. control). In PBMCs from healthy donors, ATL patients as well as in HTLV-1-infected cell lines, both IFNs have comparable activity in phosphorylating STATs 1 through 5 (PhosFlow), although phospho-STAT1 levels were up to tenfold higher than phospho-STAT2 through 5. This predominant STAT1-mediated antiviral gene signature was confirmed by Ingenuity Pathway analysis. In conclusion, our data suggest the superior antiviral and antiproliferative activity of IFN-beta vs. IFN-alpha occurs downstream of STAT1 signaling.

Brain atrophy as a non-response predictor to interferon-beta in relapsing–remitting multiple sclerosis

Background:Several predictors for treatment failure to interferon-beta (IFN-beta) have been proposed; however, brain atrophy has not been well studied.Methods:In this prospective and longitudinal study, all consecutive relapsing–remitting multiple sclerosis (RRMS) patients treated with sc IFN-beta-1a were included. Confirmed disability progression or a new relapse between weeks 48 and 144 after beginning with IFN-beta was considered as treatment non-response. EDSS progression, relapses, number of active lesions at 1 year (new or enlarging T2-weighted plus gadolinium-enhancing lesions, categorized in > 2 or ≤ 2), and brain parenchymal fraction (%BVC) volume change within the initial year of treatment were used as predictive factors. Cox regression model was adjusted for age, gender, and disease duration.Results:Seventy-one patients were included (71·8% female) with a follow-up of 144 weeks. Thirty-four (48%) fulfilled criteria of non-response to IFN-beta treatment. The model showed: (1) relapses+disability progression: HR = 4·6, 95% IC: 3·1–6·7 (P < 0·001); (2) relapses+BVC decrease: HR = 4·1, 95% IC: 3·2–7·3 (P = 0·001); (3) relapses+disability progression+new active lesions: HR = 10·1, 95% IC: 7·1–15·2 (P < 0·001); and (4) relapses+disability progression+new active lesions+BVC decrease: HR = 14·4, 95% IC: 11·4–21·2 (P < 0·001).Conclusions:Adding BVC measures to previously described predictive failure factors may increase sensitivity to early identify non-responder patients to IFN-beta-1a in the second and third years of therapy.

Candidate Gene Study of TRAIL and TRAIL Receptors: Association with Response to Interferon Beta Therapy in Multiple Sclerosis Patients

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10−4, pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

Anti-phospholipid antibodies are associated with response to interferon-beta1a treatment in MS: results from a 3-year longitudinal study

Objectives: To prospectively investigate the associations of baseline serum anti-phospholipid antibody (APLA) status on evolution of clinical and MRI measures in a multiple sclerosis (MS) patient cohort treated with interferon-beta (IFN-beta).Methods: Forty-seven relapsing–remitting (RR) MS patients, [26 APLA-positive (APLA+) and 21 APLA-negative (APLA−)] matched for age, sex, disease duration, MRI characteristics, disability, and time on IFN-beta treatment, were enrolled. All patients were on intramuscular (IM) IFN-beta1a for at least 3 years and remained on the same treatment over the 3-year duration of the study.Results: The APLA+ group accumulated significantly higher T2-LV over the 3-year follow-up than the APLA− group (+31% versus −1·1%, P = 0·043). The MTR of T1-LV (−3·3% versus +4·7%, P = 0·04) in the APLA+ group was lower compared to the APLA− group. At 3-year follow-up, the APLA+ group had increased tissue damage as measured by diffusion entropy (+4% versus −2·5%, P = 0·019) and whole brain volume loss (−0·69% versus −0·37%, P = 0·041), compared to the APLA− group. There were more clinical relapses in the MS APLA+ group compared to APLA− patients (18 versus 10) and a higher frequency of sustained disability progression (7/26 or 27% versus 2/21 or 9·5%).Conclusions: This study suggests that APLA+ RRMS patients treated with IFN-beta1a develop more severe MRI and clinical deterioration. Future studies are required to evaluate the role of APLA as potential biomarkers for disease prognosis versus predictors for therapeutic response to IFN-beta therapy.

MHC2TA mRNA levels and human herpesvirus 6 in multiple sclerosis patients treated with interferon beta along two-year follow-up

BackgroundIn previous studies we found that MHC2TA +1614 genotype frequency was very different when MS patients with and without human herpesvirus 6 (HHV-6) in serum samples were compared; a different clinical behavior was also described. The purpose of the study was: 1. To evaluate if MHC2TA expression in MS patients was influenced by interferon beta (IFN-beta) treatment. 2. To study MHC2TA expression in MS patients with and without minor allele C. 3. To analyze the relation between MHC2TA mRNA levels and HHV-6 active infection in MS patients.MethodsBlood and serum samples of 154 MS patients were collected in five programmed visits: basal (prior to beginning IFN-beta treatment), six, twelve, eighteen and twenty-four months later. HHV-6 in serum and MHC2TA mRNA levels were evaluated by PCR and RT-PCR, respectively. Neutralizing antibodies (NAbs) against IFN-beta were analyzed by the cytopathic effect assay.ResultsWe found that MHC2TA mRNA levels were significantly lower among MS patients with HHV-6 active infection at the basal visit (without treatment) than in those MS patients without HHV-6 active infection at the basal visit (p = 0.012); in all the positive samples we only found variant A. Furthermore, 58/99 (58.6%) MS patients without HHV-6 along the five programmed visits and an increase of MHC2TA expression after two-years of IFN-beta treatment were clinical responders vs. 5/21 (23.8%) among those MS patients with HHV-6 and a decrease of MHC2TA mRNA levels along the two-years with IFN-beta treatment (p = 0.004); no differences were found between patients with and without NAbs.ConclusionsMHC2TA mRNA levels could be decreased by the active replication of HHV-6; the absence of HHV-6 in serum and the increase of MHC2TA expression could be further studied as markers of good clinical response to IFN-beta treatment.

Elevated type I interferon-like activity in a subset of multiple sclerosis patients: molecular basis and clinical relevance

BackgroundA subset of patients with multiple sclerosis (MS) shows an increased endogenous IFN-like activity before initiation of IFN-beta treatment. The molecular basis of this phenomenon and its relevance to predict individual therapy outcomes are not yet fully understood. We studied the expression patterns of these patients, the prognostic value of an elevated IFN-like activity, and the gene regulatory effects of exogenously administered IFN-beta.MethodsMicroarray gene expression profiling was performed for 61 MS patients using peripheral blood mononuclear cells obtained before and after 1 month of IFN-beta therapy. Expression levels of genes involved in pathways either inducing or being activated by IFN-beta were compared between patients with high (MX1high cohort) and low (MX1low cohort) endogenous IFN-like activity. Patients were followed for 5 years and relapses as well as progression on the expanded disability status scale (EDSS) were documented.ResultsBefore the start of therapy, 11 patients presented elevated mRNA levels of IFN-stimulated genes indicative of a relatively high endogenous IFN-like activity (MX1high). In these patients, pathogen receptors (for example, TLR7, RIG-I and IFIH1) and transcription factors were also expressed more strongly, which could be attributed to an overactivity of IFN-stimulated gene factor 3 (ISGF3, a complex formed by STAT1, STAT2 and IFN regulatory factor 9). After 1 month of IFN-beta therapy, the expression of many pathway genes was significantly induced in MX1low patients, but remained unaltered in MX1high patients. During follow-up, relapse rate and changes in EDSS were comparable between both patient groups, with differences seen between different types of IFN-beta drug application.ConclusionsTherapeutic IFN-beta induces the transcription of several genes involved in IFN-related pathways. In a subgroup of MS patients, the expression of these genes is already increased before therapy initiation, possibly driven by an overexpression of ISGF3. Patients with high and low endogenous IFN-like activity showed similar clinical long-term courses of disease. Different results were obtained for different IFN-beta drug preparations, and this merits further investigation.

Investigation of the Effect of Human Leukocyte Antigen on the Development of Neutralizing Antibodies in Interferon-beta Treated Patients with Multiple Sclerosis (*The First Three Authors Contributed Equally to This Work) (P05.121)

Objective: Investigate if certain human leukocyte antigen (HLA) class I and II allele groups are associated with the susceptibility to develop neutralizing antibodies (NAbs) in a Swedish cohort of patients with multiple sclerosis (MS) treated with interferon-beta (IFN-beta) and whether or not an association with clinically relevant NAb-titers exists. Background A proportion of MS patients receiving IFN-beta therapy develop NAbs that interfere with treatment efficacy. However, NAbs only occur in a subset of patients, which indicates that factors like the carrying of different HLA allele groups could be involved in determining the risk for developing NAbs. Design/Methods: 1066 patients with NAb-status, from the Swedish NAb-registry, and HLA genotype data were included. Patients were regarded as NAb-positive when having at least one positive sample. Negativity was defined as never having a positive titer, with either one negative sample taken 24±3 months after the start of IFN-beta treatment or two negative samples taken between 6 to 21 and 27 to 36 months after treatment start. 419 patients matched these criteria of which 230 were NAb-positive and 189 NAb-negative. Cut-off for clinical relevant titers was a NAb-titer higher than 150 TRU/ml. The frequency distribution for each allele was analyzed by chi-square and odds ratio was assessed with 95% confidence interval, p Results: No significant association could be found between allele group frequency distribution and the development of NAbs, neither with nor without correcting for treatment. However, HLA-DRB1*15 was associated with clinically relevant titers (OR 1.45, p-value 0.036). Conclusions: NAb development being associated with specific allele groups, as described in earlier studies, could not be replicated but our results indicate that the development of clinically relevant titers is associated with HLA-DRB1*15. Knowing this could help when choosing newer substitute medications for IFN-beta. Disclosure: Dr. Fink has received research support from Biogen Idec. Dr. Lundkvist has received research support from Biogen Idec. Ms. Link has received research support from Karolinska Institutet. Dr. Kockum has nothing to disclose. Dr. Hermanrud has nothing to disclose. Dr. Brynedal has received research support from the Swedish Research Council. Dr. Hillert has received personal compensation for activities with Biogen Idec, Merck-Serono, Novartis and Teva Neuroscience as participant on advisory boards, consulting and/or speaking engagements.Dr. Hillert has received research support from Sanofi-Aventis Pharmaceuticals, Inc., Bayer Pharmaceuticals Corporation, Biogen Idec and Merck-Serono. Dr. Fogdell has received research support from Biogen Idec, Merck Serono, and Sanofi-Aventis Pharmaceuticals, Inc.

Interferon beta modulates major histocompatibility complex class I (MHC I) and CD3-zeta expression in PC12 cells

It has been demonstrated that the major histocompatibility complex of class I (MHC I) up regulation by exogenous treatment with interferon beta (IFNbeta) influences the glial reaction and synaptic elimination process. Therefore, the present study aimed to investigate the effects of IFNbeta treatment on the expression of MHC I, CD3-zeta (a subunit of MHC I receptor) and synaptic formation in PC12 cells, an in vitro model for studying the synaptic formation/elimination process. For this purpose, established cultures were subjected to IFNbeta (500 and 1000IU/ml) treatment for 5, 10 and 15 days. The cells were then fixed and processed for immunocytochemistry with antisera against MHC I (OX18), CD3-zeta and synaptophysin. The results were compared with control cultures only treated with basal medium. IFNbeta (500IU/ml) modulated the MHC I expression in PC12 cells, especially after 10 days of treatment. In this sense, IFNbeta induced MHC I as well as CD3-zeta up regulation. It was observed that the highest dose caused culture degeneration. Interestingly, differential regulation of MHC I was paralleled by enhancement in synaptic network remodeling. Altogether, the present data indicate that PC12 cells may be used as an in vitro model for studying MHC I modulation and synaptic plasticity. It also reinforced the role of IFNbeta on the synaptic elimination process.