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

Purpose: Interferon 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-ß. However, its role regarding the clinical response to IFN-ß 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-ß therapy on sIFNAR2 production and their association with the clinical response in MS patients. Methods: Ninety-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-ß 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-ß stimulation in vitro. Results: Protein and mRNA levels of sIFNAR2 increased after IFN-ß 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-ß in vitro induction of PBMC, 6/7 patients increased the sIFNAR2 expression. Conclusions: IFN-ß 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-ß therapy.

Skin Phototype Could Be a Risk Factor for Multiple Sclerosis.

Environmental and genetic factors are assumed to be necessary for the development of multiple sclerosis (MS), however its interactions are still unclear. For this reason here, we have not only analyzed the impact on increased risk of MS of the best known factors (HLA-DRB1*15:01 allele, sun exposure, vitamin D levels, smoking habit), but we have included another factor (skin phototype) that has not been analyzed in depth until now. This study included 149 MS patients and 147 controls. A multivariate logistic regression (LR) model was carried out to determine the impact of each of the factors on the increased risk of MS. Receiver Operating Characteristics (ROC) analysis was performed to evaluate predictive value of the models. Our multifactorial LR model of susceptibility showed that females with light brown skin (LBS), smokers and who had HLA-DRB1*15:01 allele had a higher MS risk (LBS: OR = 5.90, IC95% = 2.39-15.45; smoker: OR = 4.52, IC95% = 2.69-7.72; presence of HLA-DRB1*15:01: OR = 2.39, IC95% = 1.30-4.50; female: OR = 1.88, IC95% = 1.08-3.30). This model had an acceptable discriminant value with an Area Under a Curve AUC of 0.76 (0.69-0.82). Our study indicates that MS risk is determined by complex interactions between sex, environmental factors, and genotype where the milieu could provide the enabling proinflammatory environment that drives an autoimmune attack against myelin by self-reactive lymphocytes.

Antiviral, Immunomodulatory and Antiproliferative Activities of Recombinant Soluble IFNAR2 without IFN-ß Mediation.

Soluble receptors of cytokines are able to modify cytokine activities and therefore the immune system, and some have intrinsic biological activities without mediation from their cytokines. The soluble interferon beta (IFN-ß) receptor is generated through alternative splicing of IFNAR2 and has both agonist and antagonist properties for IFN-ß, but its role is unknown. We previously demonstrated that a recombinant human soluble IFN-ß receptor showed intrinsic therapeutic efficacy in a mouse model of multiple sclerosis. Here we evaluate the potential biological activities of recombinant sIFNAR2 without the mediation of IFN-ß in human cells. Recombinant sIFNAR2 down-regulated the production of IL-17 and IFN-É£ and reduced the cell proliferation rate. Moreover, it showed a strong antiviral activity, fully protecting the cell monolayer after being infected by the virus. Specific inhibitors completely abrogated the antiviral activity of IFN-ß, but not that of the recombinant sIFNAR2, and there was no activation of the JAK-STAT signaling pathway. Consequently, r-sIFNAR2 exerts immunomodulatory, antiproliferative and antiviral activities without IFN-ß mediation, and could be a promising treatment against viral infections and immune-mediated diseases.

New Algorithms Improving PML Risk Stratification in MS Patients Treated With Natalizumab.

Overview: We assessed the role of age and disease activity as new factors contributing to establish the risk of progressive multifocal leucoencephalopathy in multiple sclerosis patients treated with natalizumab in 36 University Hospitals in Europe. We performed the study in 1,307 multiple sclerosis patients (70.8% anti-John Cunninghan virus positive antibodies) treated with natalizumab for a median time of 3.28 years. Epidemiological, clinical, and laboratory variables were collected. Lipid-specific IgM oligoclonal band status was available in 277 patients. Factors associated with progressive multifocal leucoencephalopathy onset were explored by uni- and multivariate logistic regression. Results: Thirty-five patients developed progressive multifocal leucoencephalopathy. The multivariate analysis identified anti-John Cunninghan virus antibody indices and relapse rate as the best predictors for the onset of this serious opportunistic infection in the whole cohort. They allowed to stratify progressive multifocal leucoencephalopathy risk before natalizumab initiation in individual patients [area under the curve (AUC) = 0.85]. The risk ranged from <1/3,300 in patients with anti-John Cunninghan virus antibody indices <0.9 and relapse rate >0.5, to 1/50 in the opposite case. In patients with lipid-specific IgM oligoclonal bands assessment, age at natalizumab onset, anti-John Cunninghan virus antibody indices, and lipid-specific IgM oligoclonal band status predicted progressive multifocal leucoencephalopathy risk (AUC = 0.92). The absence of lipid-specific IgM oligoclonal bands was the best individual predictor (OR = 40.94). The individual risk ranged from <1/10,000 in patients younger than 45 years at natalizumab initiation, who showed anti John Cunningham virus antibody indices <0.9 and lipid-specific IgM oligoclonal bands to 1/33 in the opposite case. Conclusions: In a perspective of personalized medicine, disease activity, anti-lipid specific IgM oligoclonal bands, anti Jonh Cunninghan virus antibody levels, and age can help tailor natalizumab therapy in multiple sclerosis patients, as predictors of progressive multifocal leucoencephalopathy.

Multi-centre validation of a flow cytometry method to identify optimal responders to interferon-beta in multiple sclerosis.

BACKGROUND AND OBJECTIVES: Percentages of blood CD19+CD5+ B cells and CD8+perforin+ T lymphocytes can predict response to Interferon (IFN)-beta treatment in relapsing-remitting multiple sclerosis (RRMS) patients. We aimed to standardize their detection in a multicenter study, prior to their implementation in clinical practice. METHODS: Fourteen hospitals participated in the study. A reference centre was established for comparison studies. Peripheral blood cells of 105 untreated RRMS patients were studied. Every sample was analyzed in duplicate in the participating centre and in the reference one by flow cytometry. When needed, participating centres corrected fluorescence compensations and negative cut-off position following reference centre suggestions. Concordance between results obtained by participating centres and by reference one was evaluated by intraclass correlation coefficients (ICC) and Spearman correlation test. Centre performance was measured by using z-scores values. RESULTS: After results review and corrective actions implementation, overall ICC was 0.86 (CI: 0.81-0.91) for CD19+CD5+ B cell and 0.89 (CI: 0.85-0.93) for CD8+ perforin+ T cell quantification; Spearman r was 0.92 (0.89-0.95; p <0.0001) and 0.92 (0.88-0.95; p <0.0001) respectively. All centres obtained z-scores≤0.5 for both biomarkers. CONCLUSION: Homogenous percentages of CD19+CD5+ B cells and CD8 perforin+ T lymphocytes can be obtained if suitable compensation values and negative cut-off are pre-established.

Cross-reactivity of antibodies against interferon beta in multiple sclerosis patients and interference of the JAK-STAT signaling pathway.

Interferon beta (IFNß) therapy has immunogenic properties and induces the development of neutralizing antibodies (NAbs). From the extensive literature focused in the development of NAbs in multiple sclerosis (MS) patients, their ability to cross-react has been deficiently evaluated, despite having important consequences in the clinical practice. Here, the relation between the cross-reactivity and the NAbs titers has been evaluated in MS patients, by inhibition of the antiviral activity of IFNß by bioassay and through the interference with the activation of the IFNß pathway (JAK-STAT), by phosphoflow. Thus, patients with intermediate-high titers of NAbs, determined by bioassay, had a 79-fold increased risk of cross-reactivity compared to patients with low titers. The cross-reactivity is also demonstrated because NAbs positive sera were able to decrease significantly the activation of pSTAT1 achieved by other different IFNß molecules in the cells patients. Besides, a linear relationship between the STAT1 phosphorylation and NAbs titers was found. The study demonstrates that cross-reactivity increases with the titer of antibodies, which has important implications in clinical practice when switching the treatment. The direct relationship between the NAbs titer and the activation of STAT1 suggest that its determination could be an indirect method to identify the presence of NAbs.

Activation of the JAK-STAT Signaling Pathway after In Vitro Stimulation with IFNß in Multiple Sclerosis Patients According to the Therapeutic Response to IFNß.

Interferon beta (IFNß) is a common treatment used for multiple sclerosis (MS) which acts through the activation of the JAK-STAT pathway. However, this therapy is not always effective and currently there are no reliable biomarkers to predict therapeutic response. We postulate that the heterogeneity in the response to IFNß therapy could be related to differential activation patterns of the JAK-STAT signaling pathway. Our aim was to evaluate the basal levels and the short term activation of this pathway after IFNß stimulation in untreated and IFNß treated patients, as well as according to therapeutic response. Therefore, cell surface levels of IFNAR subunits (IFNAR1 and IFNAR2) and the activated forms of STAT1 and STAT2 were assessed in peripheral blood mononuclear cells from MS patients by flow cytometry. Basal levels of each of the markers strongly correlated with the expression of the others in untreated patients, but many of these correlations lost significance in treated patients and after short term activation with IFNß. Patients who had undergone IFNß treatment showed higher basal levels of IFNAR1 and pSTAT1, but a reduced response to in vitro exposure to IFNß. Conversely, untreated patients, with lower basal levels, showed a greater ability of short term activation of this pathway. Monocytes from responder patients had lower IFNAR1 levels (p = 0.039) and higher IFNAR2 levels (p = 0.035) than non-responders just after IFNß stimulation. A cluster analysis showed that levels of IFNAR1, IFNAR2 and pSTAT1-2 in monocytes grouped 13 out of 19 responder patients with a similar expression pattern, showing an association of this pattern with the phenotype of good response to IFNß (p = 0.013). Our findings suggest that an activation pattern of the IFNß signaling pathway in monocytes could be associated with a clinical phenotype of good response to IFNß treatment and that a differential modulation of the IFNAR subunits in monocytes could be related with treatment effectiveness.

Decreased soluble IFN-ß receptor (sIFNAR2) in multiple sclerosis patients: A potential serum diagnostic biomarker.

BACKGROUND: The soluble isoform of the interferon-ß (IFN-ß) receptor (sIFNAR2) could modulate the activity of both endogenous and systemically administered IFN-ß. Previously, we described lower serum sIFNAR2 levels in untreated multiple sclerosis (MS) than in healthy controls (HCs). OBJECTIVE: To assess sIFNAR2 levels in a new cohort of MS patients and HCs, as well as in patients with clinically isolated syndrome (CIS) and with other inflammatory neurological disorders (OIND) and to assess its ability as a diagnostic biomarker. METHODS: The cross-sectional study included 148 MS (84 treatment naive and 64 treated), 87 CIS, 42 OIND, and 96 HCs. Longitudinal study included 94 MS pretreatment and after 1 year of therapy with IFN-ß, glatiramer acetate (GA), or natalizumab. sIFNAR2 serum levels were measured by a quantitative ELISA developed and validated in our laboratory. RESULTS: Naive MS and CIS patients showed significantly lower sIFNAR2 levels than HCs and OIND patients. The sensitivity and specificity to discriminate between MS and OIND, for a sIFNAR2 cutoff value of 122.02 ng/mL, were 70.1%, and 79.4%, respectively. sIFNAR2 increased significantly in IFN-ß-treated patients during the first year of therapy in contrast to GA- and natalizumab-treated patients who showed non-significant changes. CONCLUSION: The results suggest that sIFNAR2 could be a potential diagnostic biomarker for MS.

Respuesta al tratamiento con interferón beta en pacientes con esclerosis múltiple. Validación del Rio Score / Response to treatment with interferon beta in patients with multiple sclerosis. Validation of the Rio Score

Introducción. Se han propuesto diferentes criterios de respuesta al tratamiento con interferón beta, y el Rio Score es uno de los más utilizados. El objetivo de este estudio fue validar la utilidad del Rio Score en una cohorte independiente. Pacientes y métodos. Estudio multicéntrico, prospectivo y longitudinal de pacientes con esclerosis múltiple remitente recurrente tratados con interferón beta. Los pacientes fueron clasificados basándose en la presencia de brotes, lesiones activas (nuevas en T2 o lesiones que captaban gadolinio) en la resonancia magnética, incremento confirmado de la discapacidad o combinaciones de estas variables (brotes, incremento en la Expanded Disability Status Scale y lesiones activas) tras un año de tratamiento. Se utilizó un análisis de regresión con el fi n de identificar las variables de predicción de respuesta después de un seguimiento de tres años. Resultados. Se incluyó a 249 pacientes con esclerosis múltiple remitente recurrente. El modelo logístico confirmó que la presencia de dos (odds ratio = 6,6; IC 95% = 2,7-16,1; p < 0,0001) o tres (odds ratio = 8,5; IC 95% = 1,6-46; p < 0,01) variables positivas durante el primer año de tratamiento confería un riesgo significativo de actividad (brotes o progresión) en los siguientes dos años. Conclusiones. Se confirma, en una cohorte independiente, la utilidad del Rio Score para identificar a pacientes con un mayor riesgo de desarrollar actividad clínica o progresión de la discapacidad durante el tratamiento con interferón beta (AU)

Introduction. Different criteria have been proposed for the response to treatment with interferon beta, and the Rio Score is one of the most widely used. The aim of this study was to validate the usefulness of the Rio Score in an independent cohort. Patients and methods. A multi-centre, prospective, longitudinal study was conducted on patients with relapsing-remitting multiple sclerosis treated with interferon beta. The patients were classified according to the presence of attacks, active lesions (new in T2 or gadolinium enhancing lesions) in magnetic resonance imaging, a confirmed increase in disability or combinations of these variables (attacks, increase on the Expanded Disability Status Scale and active lesions) after one year’s treatment. Regression analysis was used in order to identify the response-predicting variables after a three-year follow-up. Results. The sample consisted of 249 patients with relapsing-remitting multiple sclerosis. The logistic model confirmed that the presence of two (odds ratio = 6.6; CI 95% = 2.7-16.1; p < 0.0001) or three (odds ratio = 8.5; CI 95% = 1.6-46; p < 0.01) positive variables during the first year of treatment were indicative of a significant risk of activity (attacks or progression) in the next two years. Conclusions. The usefulness of the Rio Score is confirmed, in an independent cohort, as a means of identifying patients with a higher risk of developing clinical activity or progression of disability during treatment with interferon beta (AU)

Analysis of Plasminogen Genetic Variants in Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117), despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93-1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility.

TRAIL and TRAIL receptors splice variants during long-term interferon ß treatment of patients with multiple sclerosis: evaluation as biomarkers for therapeutic response.

OBJECTIVE: We aimed to assess the effects of interferon ß (IFNß) treatment on the expression of the splice variants of the Tumour necrosis factor-Related Apoptosis Inducing Ligand (TRAIL) and its receptors in different cell subpopulations (CD14+, CD4+ and CD8+) from patients with multiple sclerosis (MS), and to determine whether this expression discriminated responders from non-responders to IFNß therapy. METHODS: We examined mRNA expression of the TRAIL and TRAIL receptors variants in patients with MS, at baseline and after one year of IFNß therapy, according to responsiveness to this drug. RESULTS: Long-term therapy with IFNß increased the expression of TRAIL-α in T cell subsets exclusively from responders and decreased the expression of the isoform 2 of TRAILR-2 in monocytes from responders as well as non-responders. Lower expression of TRAIL-α, and higher expression of TRAIL-ß in monocytes and T cells, was found before the onset of IFNß therapy in patients who will subsequently become responders. Baseline expression of TRAILR-1 was also significantly higher in monocytes and CD4+ T cells from responders. CONCLUSIONS: The present study shows that long-term IFNß treatment has a direct influence on TRAIL-α and TRAILR-2 isoform 2 expression. Besides, receiver operating characteristic analysis revealed that the baseline expression of TRAIL-α in monocytes and T cells, and that of TRAILR-1 in monocytes and CD4+ T cells, showed a predictive value of the clinical response to IFNß therapy, pointing to a role of TRAIL system in the mechanism of action of IFNß in MS that will need further investigation.