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Abstract: There are two commonly employed types of bioassays for the detection of neutralizing antibodies (NAbs) against interferon-beta (IFNβ): the cytopatic effect assay (CPE), and the MxA (myxovirus resistance protein A) protein assay (MPA). This article describes a bioassay based on the real time PCR measurement of mRNA that results from the induction, in cultured human cells, of the MxA gene by IFNβ. Serum samples from 104 patients with multiple sclerosis (MS) treated with IFNβ were tested for NAbs using our real time PCR bioassay. NAbs also were measured in the same specimens by the MPA assay and CPE assay. The calibration range of the real time PCR bioassay is 0.125–30 LU/mL. The range of the intra- and inter-assay variations (coefficients of variation in log10) were 4.05% (range 0.88%–7.90%) and 4.42% (range 0.31%–9.15%), respectively. Samples of the three commercial preparations of IFNβ-1a and -1b were measured showing dose–response curves parallel to that of the NIH reference IFNβ (mean SD at the midpoint of the dose–response curve=5%). In addition, the assay was robust with respect to number of cells plated (i.e., increasing cell densities from 12×103/well to 384×103/well resulted in 3.03% variability in MxA expression normalized with glyceraldehyde-3 phosphate dehydrogenase). NAbs titers measured were closely comparable to those obtained by the MPA [r spearman =0.899; 89% of observed agreements; K =0.779] and the CPE [r spearman =0.7899); 86%; K =0.729] assays. Despite the obvious disadvantage of cost, when carried out according to quality assurance guidelines for molecular diagnostics the new MxA gene-expression assay (MGA) has significant advantages over the other methods for testing NAbs: it has excellent reliability and reproducibility, and utilizes equipment and methodologies already accessible in many clinical laboratories. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: This study is the first to evaluate biological response to first injections of interferon-beta (IFNβ) in patients with multiple sclerosis. MxA mRNA was measured in 96 patients receiving IFNβ-1a (Avonex, n=32), IFNβ-1b (Betaferon, n=19), IFNβ-1a (Rebif) 22 μg (n=30), or IFNβ-1a 44 μg (n=15). Patients were analysed before, 3 and 24 h after the first injection, and 12 h after the second administration. Results showed that up-regulation was evident within 3 h of IFNβ injection, peaked 12 h after injection, and progressively declined 24 h after administration. The cumulative responses were similar after a single administration, regardless of product/dose. Moreover, data indicate that the abolition of the biological activity detected during IFNβ therapy is due to underlying phenomena (e.g., neutralizing antibodies), because all patients were constitutively responders to IFNβ at treatment initiation. [Copyright &y& Elsevier]

Background and purpose: Fingolimod is a drug approved for treatment of relapsing‐remitting multiple sclerosis (RRMS) that exerts its effects via sequestering lymphocytes within the lymph nodes. The drug, acting on the sphingosine‐1‐phosphate pathway, is involved in a plethora of processes and, to date, its mechanism of action is not completely understood. Recently, it has been demonstrated that Fingolimod increases the expression of transcription factor NR4A2 in murine brain. NR4A2 belongs to nuclear receptor family 4, group A (NR4A) along with NR4A1 and NR4A3. The role of NR4A2 in the pathogenesis of multiple sclerosis is already known and supported by its down‐regulation observed in blood obtained from patients with RRMS compared with healthy controls (HCs). It is notable that NR4A2 impairment is reversed in patients with RRMS during pregnancy, which represents a transitory state of immune tolerance, associated with reduced disease activity. An inverse correlation between NR4A2 gene expression levels and clinical parameters indicates that more aggressive forms of the disease are characterized by lower levels of NR4A2. Methods: Gene expression levels of NR4A in blood obtained from HCs, treatment‐naive (T0) and Fingolimod‐treated patients with RRMS were evaluated to determine their contribution to drug response. Results: Gene expression levels of NR4A were down‐regulated in T0 patients compared with HCs. Patients treated with Fingolimod for >2 years were characterized by higher levels of NR4A2 compared with the T0 group, approaching those of HCs. NR4A1 and NR4A3 levels were not altered. Conclusions: Involvement of the NR4A family in the pathogenesis of multiple sclerosis and a role of Fingolimod in the recovery from NR4A2 deficit can be hypothesized based on our data. [ABSTRACT FROM AUTHOR]

Multiple Sclerosis (MS) is a heterogeneous disease and a variable percentage of patients are non-responders to common treatment. Early diagnosis of non-responders allows change to a more useful therapy for the patient and better allocates a large amount of financial resources. Quantification of Neutralizing antibodies (Nabs) and of biological activity of IFN-β are recognized approaches to identify immuno-pharmacological non-responders. A consistent number of studies have demonstrated that quantification of Myxovirus-induced protein A (MxA) is a valid biomarker to detect immune-pharmacological non responders after one year of treatment. Persistent high titre of Nabs and absence of biological activity predict abolition of IFN-β effects in disease activity measured through MRI, number of relapses and disability. Guidelines and flow-charts including both Nabs and MxA quantification are presented. [ABSTRACT FROM AUTHOR]

Background Neutralising antibodies (NAb) to interferon beta (IFNβ) are associated with a reduced bioactivity and efficacy of IFNβ in multiple sclerosis (MS). Unclear is how to apply IFNβ bioactivity measurements (quantification of Myxovirus resistance protein A (MxA) mRNA) in clinical practice. Objectives To evaluate value and feasibility of IFNβ bioactivity measurement with a single MxA mRNA measurement for screening and a second measurement before and after IFNβ administration for definite confirmation of IFNβ bioactivity status. Methods In 79 MS patients MxA mRNA expression was determined 4 hours after IFNβ administration. If inadequate, MxA mRNA expression testing was repeated 3 months afterwards, comparing post- and pre injection samples to determine whether IFNb bioactivity was persistently lacking. MxA mRNA expression was compared to NAβ titres, determined by the cytopathic effect assay (CPE). Results NAb titres correlated significantly with MxA mRNA expression and MxA mRNA induction. Of all screened patients, only one patient had adequate MxA mRNA expression and high NAb titres simultaneously. Of the biological non-responders at second measurement (21/55), 17 (81%) were high-titre NAb positive, 1 (5%) was low-titre NAb positive and 3 (14%) were NAb negative. Without considering the pre-injection measurement, two more NAb negative patients would have tested negative for IFNβ bioactivity, emphasizing the need of a pre-injection sample. Conclusions Our data suggest that for IFNβ bioactivity screening a single post-injection measurement seems reasonable. However, MxA induction measurement based on both pre- and post-IFNβ injection samples at second measurement is somewhat more precise in determining ultimate IFNβ bioactivity status. [ABSTRACT FROM AUTHOR]

To date, inter- and intra-laboratory consistency of binding assays for measuring anti-interferon (IFN)β antibodies has not been assessed. In this investigation, two independent laboratories tested a library of 80 serum specimens obtained from multiple sclerosis (MS) patients treated with IFNβ. For binding antibodies (BAbs) evaluations, each laboratory used both a capture-ELISA (cELISA) and an enzyme-immuno-assay (EIA), which is commercially available. Samples were also tested for neutralizing antibodies (NAbs). Data demonstrated good intra-laboratory reliability (rpearson ≥ 0.86), and a good overall agreement between the results obtained from the two centers, using both the cELISA (69/80 of observed agreements) and the EIA (67/80). Accordingly, kappa coefficients (K) showed good concurrence (K ≥ 0.651). There was also substantial agreement between cELISA and EIA measurements, as performed in both centers (Orbassano, 66/80, K = 0.631; Basel, 70/80, K = 0.717). However, by comparing NAbs and BAbs titers obtained with both assays, we found that a high degree of BAb-negative samples were positive in NAb-assay. Thus, our study does not support the usefulness of ELISA-based BAb assays as a screening tool for NAbs. Otherwise, BAb-assays can be used as a confirmation test, indicating that the decrease of the biological effects is due to antibodies. In this context, both ELISA-based assays are equally reliable techniques. [ABSTRACT FROM AUTHOR]

Relapsing-remitting multiple sclerosis (MS) has a very fluctuating course and responsiveness to interferon beta (IFN-β) treatment in each patient is extremely difficult. Agreement exists about the negative role of neutralising antibodies (NAbs) on clinical efficacy and markers of IFN-β bioavailability have been studied; no guidelines exist yet about what to do when a patient becomes NAbs positive or IFN biological activity is lost. In this study 405 MS patients have been longitudinally studied for NAbs and MxA expression. A spontaneous disappearance of NAbs was observed in a few patients with low antibody titre; according to the clinical course, a therapeutic modification has been made in patients persistently NAbs positive; in these patients NAbs persisted over time despite the interruption of IFN therapy. [ABSTRACT FROM AUTHOR]