Serum and cerebrospinal fluid neurofilament light chains measured by SIMOA™, Ella™, and Lumipulse™ in multiple sclerosis naïve patients.

BACKGROUND: Neurofilament light chains (NfL) are cytoskeletal biomarkers of axonal damage, about 40-fold higher in cerebrospinal fluid (CSF) compared to serum, and requiring ultrasensitive techniques to be measured in this latter fluid. OBJECTIVES: To compare CSF and serum NfL levels in multiple sclerosis (MS) patients using different platforms. METHODS: 60 newly diagnosed relapsing-remitting MS patients (38 females; median age: 36.5 years, range: 15-60) were enrolled before steroid or disease-modifying treatments. CSF and serum NfL were measured with: the commercial Ella™ microfluidic platform (Bio-Techne), the Lumipulse™ Chemiluminescent Enzyme ImmunoAssay (Fujirebio), and the SIMOA™ on the SR-X instrument using NF-light assays (Quanterix). RESULTS: CSF and serum NfL absolute levels strongly correlated between assays, although being more elevated with Ella™. Passing-Bablok regression showed high agreement in measuring CSF NfL between assays (with greater proportional difference using Ella™), and very high agreement for serum comparing SIMOA™ and Lumipulse™. Similarly, the Bland-Altman comparison evidenced lower biases for Lumipulse™ for both fluids. CONCLUSIONS: CSF and serum NfL in naïve MS patients are reliably measured with all assays. Although not interchangeable, SIMOA™ and Lumipulse™ showed high agreement for serum and CSF values.

Serum Neurofilaments are a reliable biomarker to early detect PML in Multiple Sclerosis patients.

BACKGROUND: The earliest detection of progressive multifocal leukoencephalopathy (PML) is crucial in Natalizumab (NTZ)-treated Multiple Sclerosis (MS) patients. This study aims to assess serum Neurofilaments (sNFL) ability to early detect PML in longitudinal patients' follow-up. METHODS: NFL were retrospectively measured in four PML cases occurred at the Regional Referring Center for MS (CRESM, Italy), in samples collected since one year before PML diagnosis, at PML diagnosis, during PML and in post-PML follow-up. sNFL levels were interpreted according to previously defined reference values. Clinical examination and EDSS were performed at each NTZ infusion. Routinary MRI was undertaken every six months; after PML diagnosis, MRI was performed according to clinical evaluation. sNFL were also measured in 45 NTZ-treated patients experiencing NEDA-3 status for at least 12 months. RESULTS: Patients showed different PML onsets and manifestations: in 3 patients routinary brain MRI revealed radiological signs of PML preceding different clinical manifestations, while in one patient brain MRI was performed after the clinical onset. PML diagnosis was defined at the time of the first detection of JCV DNA in cerebrospinal fluid. The following different PML phases were considered: 1. Basal (up to 4 months before PML diagnosis): sNFL values were in the normal range in all patients' samples, except for one (median 9.1 pg/ml, range 6.2-15.1 pg/ml) 2. Pre-PML (within 3 months before PML diagnosis): sNFL were elevated in all available samples (median 19.50 pg/ml, range 15.50-33.80 pg/ml). 3. PML diagnosis: sNFL were elevated in all patients (median 59.20 pg/ml, range 11.1-101.50 pg/ml). 4. PML/IRIS: during this phase, sNFL levels reached their peak (median 96.35 pg/ml, range 20.5-272.9) in all patients. 5. Post-PML (recovery phase, starting from the first MRI without enhancement, up to the end of follow-up): sNFL levels showed a decrease (median 12.80 pg/ml, range 9.30-30.60); however, based on reference values, sNFL were still elevated in 2 out of 4 patients at the end of their follow-up (622 and 887 days after PML diagnosis). sNFL were always elevated when MRI scan suggested a suspicious of PML. In NEDA-3 patients, sNFL levels were in the normal range in all patients' samples (median 4.7 pg/ml, range 1.4-8.6 pg/ml). CONCLUSION: Elevated sNFL were observed not only at PML diagnosis, but also in pre-PML phase. At PML recovery, sNFL weren't normalized in all patients' samples, suggesting ongoing neuronal degeneration. sNFL represent a reliable biomarker and should be introduced in clinical practice as an additional/alternative parameter to MRI to early detect and monitor PML.

Risk of multiple sclerosis relapses when switching from fingolimod to cell-depleting agents: the role of washout duration.

BACKGROUND: Fingolimod (FTY) induces sequestration of lymphocytes in secondary lymphoid organs and the average lymphocyte recovery following discontinuation takes 1-2 months. It has been hypothesized that the therapeutic effects of subsequent cell-depleting agents may be compromised if initiated before lymphocyte recovery has occurred. OBJECTIVE: To assess the risk of relapses following FTY discontinuation and the initiation of a B/T cell-depleting agent in relation to washout duration using data from the Italian MS Register. METHODS: The risk of relapses was assessed in relation to different washout durations (< 6, 6-11, 12-17 and > / = 18 weeks) in patients starting alemtuzumab, rituximab, ocrelizumab or cladribine following FTY discontinuation. RESULTS: We included 329 patients in the analysis (226F, 103 M; mean age 41 ± 10 years). During the cell-depleting treatment, the incidence rate ratio for a relapse was significantly greater in patients with a washout period of 12-17 and > / = 18 weeks compared to the reference period (< 6 weeks). The risk of a relapse was significantly influenced by the occurrence of relapses during FTY treatment and by washout length, with hazard ratios markedly increasing with the washout duration. CONCLUSION: The risk of relapses increases with the washout duration when switching from FTY to lymphocyte-depleting agents.

Informing MS patients on treatment options: a consensus on the process of consent taking.

In the last years, change in multiple sclerosis (MS) therapeutic scenario has highlighted the need for an improved doctor-patient communication in advance of treatment initiation in order to allow patient's empowerment in the decision-making process. AIMS: The aims of our project were to review the strategies used by Italian MS specialists to inform patients about treatment options and to design a multicentre shared document that homogenizes the information about disease-modifying treatment (DMTs) and the procedure of taking informed consent in clinical practice. RESULTS: The new resource, obtained by consensus among 31 neurologists from 27 MS Centres in Italy with the supervision of a medico-legal advisor, received the aegis of Italian Neurological Society (SIN) and constitutes a step toward a standardized decision process around DMTs in MS.

A multicenter, observational, prospective study of self- and parent-reported quality of life in adolescent multiple sclerosis patients self-administering interferon-ß1a using RebiSmart™-the FUTURE study.

Besides the impact of disease per se, the use of immunomodulatory therapies in adolescents with relapsing-remitting multiple sclerosis (RRMS) may have an effect on quality of life (QL). The FUTURE (Quality of liFe in adolescent sUbjecTs affected by mUltiple sclerosis treated with immunomodulatoRy agEnt using self-injecting device) study was designed to evaluate the changes in QL of Italian adolescents with RRMS receiving treatment with IFN-ß1a (Rebif; 22 µg), administered subcutaneously three times weekly using the RebiSmart™ electronic autoinjection device over a 52-week period. Fifty adolescents with RRMS were enrolled and 40 completed the study. Changes from baseline to end of treatment (EoT) in adolescent self-reported and parent-reported QL were assessed using the Pediatric Quality of Life Inventory Multidimensional Fatigue Scale (PedsQL), which has been validated for use in pediatric MS and for which an Italian version is available. The adolescent self-reported total PedsQL4.0 score and all of its subscales tended to increase from baseline to EoT, the only exception being "Emotional functioning." In parent-reported measures, the total PedsQL4.0 score increased significantly from baseline to EoT (+ 5.27 points, p = 0.041). Significant increases were also evident for parent-reported "Psychosocial health summary score" (+ 5.90 points; p = 0.015) and "School functioning" (+ 7.84 points; p = 0.029). Our results indicate that adolescents with RRMS using the electronic injection device RebiSmart™ for self-administration of Rebif® can experience long-term improvements in QL.

Anti-inflammatory genes associated with multiple sclerosis: a gene expression study.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system caused by a complex interaction between multiple genes and environmental factors. HLA region is the strongest susceptibility locus, but recent huge genome-wide association studies identified new susceptibility genes. Among these, BACH2, PTGER4, RGS1 and ZFP36L1 were highlighted. Here, a gene expression analysis revealed that three of them, namely BACH2, PTGER4 and ZFP36L1, are down-regulated in MS patients' blood cells compared to healthy subjects. Interestingly, all these genes are involved in the immune system regulation with predominant anti-inflammatory role and their reduction could predispose to MS development.

Biological monitoring of IFN-ß therapy in Multiple Sclerosis.

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-ß 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-ß effects in disease activity measured through MRI, number of relapses and disability. Guidelines and flow-charts including both Nabs and MxA quantification are presented.

No impact of current therapeutic strategies on disease reactivation after natalizumab discontinuation: a comparative analysis of different approaches during the first year of natalizumab discontinuation.

BACKGROUND AND PURPOSE: Natalizumab discontinuation induces the recurrence of multiple sclerosis disease activity: currently no therapeutic approach has been found able to abolish disease reactivation. METHODS: The recurrence of disease activity after natalizumab discontinuation was retrospectively evaluated in 79 patients who had been treated with immunomodulating agents, other first-line therapies, fingolimod or not treated. RESULTS: No differences have been found in clinical or magnetic resonance imaging recurrence of disease activity amongst the groups. Interestingly, no disease reactivation was observed only in one patient treated for 6 months with monthly pulses of cyclophosphamide. CONCLUSION: Disease modifying treatment or 'no treatment' is unable to abolish disease activity reactivation after natalizumab discontinuation.

Monocytes and CD4+ T cells contribution to the under-expression of NR4A2 and TNFAIP3 genes in patients with multiple sclerosis.

We recently found a gene signature for multiple sclerosis (MS) that reverted to normal during pregnancy in MS patients and included NR4A2 and TNFAIP3, key molecules in anti-inflammatory processes. Here we focus on the expression levels of these two genes in monocytes and CD4+ T cells from healthy controls and treatment-naïve RRMS patients. Our findings show that monocytes play a key role in the dysregulated anti-inflammatory response, being the expression of both genes down-regulated in these cells in RRMS patients with respect to healthy individuals. CD4+ T cells seem to have only a marginal part, because we can observe only a slight down-regulation in NR4A2.

Early detection of neutralizing antibodies to interferon-beta in multiple sclerosis patients: binding antibodies predict neutralizing antibody development.

BACKGROUND: Neutralizing antibodies (NAb) affect efficacy of interferon-beta (IFN-b) treatment in multiple sclerosis (MS) patients. NAbs evolve in up to 44% of treated patients, usually between 6-18 months on therapy. OBJECTIVES: To investigate whether early binding antibody (BAb) titers or different IFN-b biomarkers predict NAb evolution. METHODS: We included patients with MS or clinically isolated syndrome (CIS) receiving de novo IFN-b treatment in this prospective European multicenter study. Blood samples were collected at baseline, before and after the first IFN-b administration, and again after 3, 12 and 24 months on that therapy; for determination of NAbs, BAbs, gene expression of MxA and protein concentrations of MMP-9, TIMP-1, sTRAIL, CXCL-10 and CCL-2. RESULTS: We found that 22 of 164 (13.4%) patients developed NAbs during a median time of 23.8 months on IFN-b treatment. Of these patients, 78.9% were BAb-positive after 3 months. BAb titers ≥ 1:2400 predicted NAb evolution with a sensitivity of 74.7% and a specificity of 98.5%. Cross-sectionally, MxA levels were significantly diminished in the BAb/NAb-positive samples; similarly, CXCL-10 and sTRAIL concentrations in BAb/NAb-positive and BAb-positive/NAb-negative samples, respectively, were also diminished compared to BAb/NAb-negative samples. CONCLUSIONS: BAb titers reliably predict NAbs. CXCL-10 is a promising sensitive biomarker for IFN-b response and its abrogation by anti-IFN-b antibodies.

Endovascular treatment of CCSVI in patients with multiple sclerosis: clinical outcome of 462 cases.

Although it is still debated whether chronic cerebro-spinal venous insufficiency (CCSVI) plays a role in multiple sclerosis (MS) development, many patients underwent endovascular treatment (ET) of CCSVI. The objective of the study is to evaluate the outcome and safety of ET in Italian MS patients. Italian MS centers that are part of the Italian MS Study Group were all invited to participate to this retrospective study. A structured questionnaire was used to collect detailed clinical data before and after the ET. Data from 462 patients were collected in 33 centers. ET consisted of balloon dilatation (93 % of cases) or stent application. The mean follow-up duration after ET was 31 weeks. Mean EDSS remained unchanged after ET (5.2 vs. 4.9), 144 relapses occurred in 98/462 cases (21 %), mainly in RR-MS patients. Fifteen severe adverse events were recorded in 3.2 % of cases. Given the risk of severe adverse events and the lack of objective beneficial effects, our findings confirm that at present ET should not be recommended to patients with MS.

Expression and regulation of IFNalpha/beta receptor in IFNbeta-treated patients with multiple sclerosis.

BACKGROUND: The cytokine interferon beta (IFNbeta) is successfully used in the treatment of multiple sclerosis (MS), although there is a high degree of variability in the response. A common mechanism involved in the modulation of responsiveness to cytokines is represented by regulation of their receptor expression through autocrine ligand-mediated loops. The present study is aimed at investigating the regulation of IFNalpha/beta receptor (IFNAR) during IFNbeta therapy in patients with MS and at correlating it with the biologic responsiveness to the cytokine. METHODS: Quantitative PCR measurements of IFNAR-1 and the three IFNAR-2 isoforms were performed in 141 patients after short-term and long-term treatment. Patients were also regularly screened for anti-IFNbeta neutralizing antibodies (NAbs). IFN-inducible myxovirus resistance protein A messenger RNA was used as an indicator of bioactivity. RESULTS: Pretreatment levels of IFNAR-2 in patients were lower overall than in controls (p = 0.038), and high levels correlated with greater bioactivity. Upon prolonged treatment, NAb-negative patients displayed a state of decreased transmembrane IFNAR-2 expression (p < or = 0.025), whereas levels of soluble IFNAR-2 were slightly increased (p < 0.0001). The presence of NAbs reversed these effects (p < or = 0.0056). In NAb-positive patients, pretreatment expression levels of both transmembrane IFNAR-2 isoforms were significantly lower than in NAb-negative patients (p < or = 0.0089). CONCLUSIONS: Findings show that interferon-alpha/beta receptor (IFNAR)-2 isoforms are important regulators of the responsiveness to endogenous and systemically administered interferon beta (IFNbeta). They show a dual action, agonistic and antagonistic, that influences both the magnitude and the nature of the biologic response to IFNbeta. Levels of IFNAR-2 are regulated with the aim of keeping the body in a state of equilibrium, even when nonphysiologic stimuli are present.

Predictive markers for response to interferon therapy in patients with multiple sclerosis.

BACKGROUND: Prolonged therapy with interferon beta (IFN beta) often leads to the development of anti-IFN beta binding antibodies (BAbs). A subset of the BAbs is of a neutralizing nature (neutralizing antibodies, NAbs) and is associated with reduced clinical efficacy of therapy. Myxovirus-resistance-protein A (MxA) has proven to be a reliable biomarker of IFN beta bioactivity. We analyzed the prognostic value of MxA mRNA, NAbs, and BAbs on the risk of having a new relapse in IFN beta-treated patients. METHODS: A 3-year study was conducted in 137 IFN beta-treated patients. Blood samples for BAbs, NAbs, and MxA mRNA measurements were taken after 12 +/- 3 months of therapy. Analysis of relapse-free survival (RFS) was performed for all measures by using known thresholds, generating "positive" and "negative" groups. Also, time between sampling and following relapse and risk of new relapses were calculated. RESULTS: The MxA-negative group showed poorer RFS rates than the MxA-positive group [p < 0.0001, hazard ratio (HR) = 2.87]. Likewise, the NAb-positive group showed poorer RFS rates than the NAb-negative group (p =0.0013; HR = 2.49). On the contrary, BAb measurement did not show a clear clinical significance. CONCLUSIONS: Findings indicate that measurements of both myxovirus-resistance-protein A (MxA) and neutralizing antibodies (NAbs) predict the risk of new relapses; however, the slightly stronger prognostic significance of MxA mRNA and the easier method for it measurement make MxA mRNA the preferred biomarker for monitoring interferon beta (IFN beta)-treated patients. This information can be used to better tailor treatment to the individual patient with MS.

Variable responses to rituximab treatment in neuromyelitis optica (Devic's disease).

We have described two cases of Devic's disease patients treated with rituximab with different outcomes. The results indicate that there may be early unresponsiveness in very aggressive cases. Well designed clinical trials are needed to assess treatment effects in such a rare disease.

Development and validation of a real time PCR-based bioassay for quantification of neutralizing antibodies against human interferon-beta.

There are two commonly employed types of bioassays for the detection of neutralizing antibodies (NAbs) against interferon-beta (IFNbeta): 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 IFNbeta. Serum samples from 104 patients with multiple sclerosis (MS) treated with IFNbeta 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 log(10)) were 4.05% (range 0.88%-7.90%) and 4.42% (range 0.31%-9.15%), respectively. Samples of the three commercial preparations of IFNbeta-1a and -1b were measured showing dose-response curves parallel to that of the NIH reference IFNbeta (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 12x10(3)/well to 384x10(3)/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.

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

Biological activity of interferon-beta (IFNbeta) 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 IFNbeta. 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 IFNbeta, although there is no evidence for a functional role of it in MS.

Biological responsiveness to first injections of interferon-beta in patients with multiple sclerosis.

This study is the first to evaluate biological response to first injections of interferon-beta (IFNbeta) in patients with multiple sclerosis. MxA mRNA was measured in 96 patients receiving IFNbeta-1a (Avonex, n=32), IFNbeta-1b (Betaferon, n=19), IFNbeta-1a (Rebif) 22 microg (n=30), or IFNbeta-1a 44 microg (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 IFNbeta 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 IFNbeta therapy is due to underlying phenomena (e.g., neutralizing antibodies), because all patients were constitutively responders to IFNbeta at treatment initiation.

Fate of multiple sclerosis patients positive for neutralising antibodies towards interferon beta shifted to alternative treatments.

Relapsing-remitting multiple sclerosis (MS) has a very fluctuating course and responsiveness to interferon beta (IFN-beta) treatment in each patient is extremely difficult. Agreement exists about the negative role of neutralising antibodies (NAbs) on clinical efficacy and markers of IFN-beta 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.

Biological activity of interferon betas in patients with multiple sclerosis is affected by treatment regimen and neutralising antibodies.

BACKGROUND: MxA gene expression is one of the most appropriate markers of biological activity of exogenous interferon (IFN) beta. METHODS: We quantified MxA mRNA for five consecutive days in 62 patients treated with IFN beta (16, Avonex; 10, Betaferon; 24, Rebif 22; 12, Rebif 44), by quantitative-competitive polymerase chain reaction. Every three months, IFN beta induced neutralising antibodies (NAbs) were evaluated in sera using a cytopathic effect assay. RESULTS: Two categories of patients were identified: one group (49/62) had a sharp post-injection increase in MxA expression (defined as "IFN beta biological responder"), whereas the other group (13/62) had no MxA induction after IFN beta administrations (defined as "IFN beta biological non-responder"). In 11/13 biological non-responders, the persistent presence of NAbs correlated with abolished biological activity, independently of treatment regimen. The two remaining IFN beta biological non-responders were NAb-. Among the 49 IFN beta biological responders, biological activity was comparable between the four preparations on day 2 and 3 (+12 and +36 hours post-injection), but it was greater in Betaferon and both Rebif preparations on day 1, 4, and 5. In biological responders treated three times a week, only 82% (59/72) of injections were considered effective, compared with 100% (13/13) of Avonex injections. CONCLUSION: Our results suggest that an optimal IFN beta regimen is not yet available: Avonex, given once a week, shows lower cumulative biological activity. On the other hand, both Betaferon and Rebif, given three times a week, show 18% biologically ineffective injections and higher risk of developing NAbs, which abolish biological activity.

Neutralizing antibodies reduce the efficacy of betaIFN during treatment of multiple sclerosis.

OBJECTIVE: To analyze the impact of neutralizing antibodies (NAbs) on the clinical efficacy of IFNbeta. METHODS: This was an open-label study involving 78 patients with multiple sclerosis treated with Betaferon 8 million IU (MIU) subcutaneously (SC) every other day (n = 20), Rebif 22 micro g SC 3 times weekly (n = 25), or Avonex 30 micro g IM once weekly (n = 33). Every 3 months, blood samples were collected and sera were analyzed for NAbs using an antiviral cytopathic effect assay. Patients were categorized according to their NAb status: NAb negative (NAb-); isolated NAb positive (NAb+), patients with > or =1 positive sample (titer > or = 20); and persistent NAb+, patients with > or =2 consecutive positive samples (titer > or = 20). Patients who were NAb- and persistent NAb+ were compared for relapse rate, time between first and second relapse, percentage of relapse-free patients, and percentage of patients who had a sustained progression of > or =1 point on the Expanded Disability Status Scale (EDSS). RESULTS: The incidence of persistent NAb+ patients was 35% for Betaferon, 20% for Rebif, and 3% for Avonex. During IFNbeta treatment, both NAb+ and NAb- patients showed a reduction in relapse rate; this reduction (25%) was not significant in NAb+ patients but was significant (67%; p < 0.0001) in NAb- patients. In addition, the mean relapse rate was higher (p = 0.039), mean time between first and second relapse was shorter (13 vs 21 months; p = 0.0064), and there was a trend suggesting that NAbs affected the probability of remaining relapse free (p = 0.08). A higher percentage of NAb+ patients versus NAb- patients had worsening of EDSS scores during follow-up (p = 0.013). CONCLUSION: Persistent NAbs significantly reduce the clinical efficacy of IFNbeta.