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Background Knowledge concerning the predictors of social security benefits and the proportion of Multiple Sclerosis (MS) patients receiving these benefits is very limited. Objective To estimate the likelihood of receiving social security benefits for Italian MS patients. Methods From September 2014 to November 2015, we interviewed MS outpatients from two Italian MS clinics to collect information regarding their personal data, clinical and working history, and access to social security benefits. We performed both univariate and multivariable analyses to evaluate the predictors for receiving social security benefits. Results We interviewed 297 patients, with a mean age of 49.5 (± 10.7) years; 71.4% were females. About 73% of patients had a relapsing-remitting (RR) course and the median EDSS score was 2.5 (IQR 1.5–6). About 75% of MS patients received a full exemption from co-payments, while the proportions of people who enjoyed each of the other social security benefits were lower, ranging from 8.8% (car adaptation) to 32% (disable badge). At multivariable analysis, the probability of obtaining each of the benefits was significantly associated with the EDSS score: walking aids (OR 3.9), care allowance (OR 3.6), disabled badge (OR 2.4), exemption from co-payment (OR 1.6) and allowed off work permit (OR 1.7). Only the probability of obtaining an allowed off work permit was also influenced by comorbidities (OR 2.9) and a higher education (OR 2.2). Conclusion Except for full exemption from co-payments, the proportions of MS patients who enjoyed social security benefits seem to be limited in our study sample. The EDSS score is the strongest predictor of the probability of receiving all the benefits. Only a small proportion of patients received care allowance and working permits, probably because such benefits are only granted to people with a high level of disability. On the other hand, the low proportion of patients who enjoyed fiscal benefits for home and car adaptations could have been influenced by the way such benefits are granted in our country.

Background Serum Neurofilament Light (sNFL) is the most promising marker for patient's monitoring in Multiple Sclerosis (MS). However, operating reference values for use in clinical practice are still lacking. Here, we defined sNFL reference cut-off values in a cohort of healthy controls (HC) and assessed their performance in Multiple Sclerosis (MS) patients, as well as the intra-individual sNFL variability. Methods We measured sNFL by single molecule array (Simoa) assay in 79 HC assessing their correlation with age. Changes of sNFL levels were evaluated during a short-term follow-up (median 67 days between consecutive samples) in a subgroup of 27 participants. sNFL were tested in 23 untreated MS patients, at both diagnostic time and start of therapy (median 80 days after), considering disease activity. Results Findings confirmed a correlation between sNFL levels and age in HC, thus cut-off values specific for age decades were calculated. sNFL did not vary significantly with time during short-term follow-up (median CV 13%). sNFL levels in MS patients were higher and demonstrated a higher variability between diagnostic time and treatment start (median CV 39%). According to cut-off values, “pathologic” sNFL levels were found in 57% of MS patients at diagnostic time, and in 30% of samples at treatment start. In particular, “pathologic” sNFL levels were found in 80% of samples (16/20) obtained during a phase of disease activity, while a total of 85% of samples (22/26) associated with inactive disease showed sNFL in the normal range. Conclusion This study demonstrates an overall intra-individual stability of sNFL values in the short-term in HC and suggests age-dependent reference cut-off values that could be beneficial for sNFL implementation in clinical practice.

The presence of KIR4.1 antibodies has been proposed to be a characteristic of Multiple Sclerosis (MS). This could have a significant impact on disease management. However, the validation of the initial findings has failed till date. Conflicting results have been attributed to difficulties in isolating the lower-glycosylated (LG) KIR4.1 expressed in oligodendrocytes, the putative target antigen of autoantibodies. The aim of this study is to verify the presence of KIR4.1 antibodies in MS patients, by independently replicating the originally-described procedure. Assay procedure consisted of KIR4.1 expression in HEK293 cells, 3-step elution to isolate LG-KIR4.1 in elution fraction 3, and ELISA. Sera of 48 MS patients and 46 HCs were studied in 21 working sessions. In a preliminary analysis, we observed different KIR4.1 antibody levels between MS patients and Healthy Controls (HCs). However, a high variability across working sessions was observed and the sensitivity of the assay was very low. Thus, stringent criteria were established in order to identify working sessions in which the pure LG-KIR4.1 was isolated. As per these criteria, we detected LG-KIR4.1 antibodies in 28% of MS patients and 5% of HCs. Unlike previous findings, this study is in agreement with the original report. We propose further efforts be made towards the development of a uniform method to establish the detection of KIR4.1 antibodies in MS patients.

Balance impairment, frequent in Multiple Sclerosis patients (MS), is difficult to detect promptly with routine clinical examination. Computerized platforms can measure subtle deficit but, given the complexity of postural system, multiple tests should be adopted. To evaluate whether platform was more sensitive than Romberg Test (RT) in detecting balance abnormalities, we 1) chose a battery of posturographic tests, 2) collected normative data from 58 healthy subjects 3) applied the tests to Clinically Isolated Syndrome (n=42) and minimally impaired MS (n=76). Subjects underwent 3 trials of quiet standing with eyes open and closed (modified Clinical Test of Sensory Interaction on Balance, mCTSIB) and 4 trials of voluntary anterior and lateral maximal leaning on right and left sides (Limits of Stability, LOS), giving 10 postural indexes. For every subject, the best trials were selected for subsequent analysis. Normative values were established in a range from 1st to 99th percentile, defining balance impairment by the presence of at least 2 indexes out of range. Even adopting the above mentioned strict definition of balance impairment, the forceplate resulted more sensitive than RT, detecting abnormalities in 25% of patients, while RT was abnormal in 7% only. In RT-negative patients with 1-year follow-up (n =67) the detection of a single abnormal index was able to predict a subsequent onset of symptomatic balance impairment. The proposed procedure is quick, easy to perform and can improve the assessment of the clinical course of MS, from a pre-clinical stage up to medium degree of disability.

Myelin Oligodendrocyte Glycoprotein Antibody Disease (MOGAD) represents a demyelinating disorder for which tocilizumab, an anti-IL6 receptor, has been tested to prevent disabling relapses. In a subgroup of patients affected with novel Coronavirus disease (COVID-19), tocilizumab has also increased the survival rate. We present the case of a 31-years-old Caucasian patient who experienced an almost asymptomatic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) infection during treatment with tocilizumab, which was continued due to the very high risk of relapses of the patient. According to this case, tocilizumab might be not discontinued during COVID-19.

We studied the baseline expression level of 25 interferon-regulated genes (MxA, GPR3, IL17RC, ISG15, TRAIL, OASL, IFIT1, IFIT2, RSAD2, OAS3, IFI44L, TRIM22, IL10, CXCL10, STAT1, OAS1, OAS2, IFNAR1, IFNAR2, IFNβ, ISG20, IFI6, PKR, IRF7, USP18), recurrently proposed in the literature as predictive biomarkers of interferon-beta treatment response, in whole blood of 10 "responders" and 10 "non-responders" multiple sclerosis relapsing-remitting patients, retrospectively selected on the basis of stringent clinical criteria after a five years follow-up. However, we cannot confirm the predictive value of these candidate biomarkers.

Objective To develop a neurophysiological method to explore central motor pathways to proximal and distal muscles of lower limbs. Methods MEPs to transcranial magnetic stimulation using the double cone coil were bilaterally and simultaneously recorded from vastus medialis, tibialis anterior and flexor hallucis brevis. Voluntary facilitation was controlled using a predefined sequence of movements of constant amplitude. Compound motor action potentials elicited by maximal high voltage electrical stimulation of lumbosacral roots (root-CMAPs) were recorded from the same muscles to obtain the corresponding peripheral conduction times. We studied 28 healthy subjects and 28 multiple sclerosis (MS) patients with no or mild motor impairment. Results The described facilitation procedure and the averaging of 5 MEPs reduced area variability to about 10%. In MS patients conduction slowing and/or MEP area reduction in at least one muscle was found in 91.7% of cases, with significant correlation with individual motor impairment. Conclusions Combined use of stable MEPs and maximal root-CMAPs was able to detect both conduction slowing and conduction failure in central motor pathways to proximal and distal districts of lower limbs in MS patients. Significance The proposed method provides an extensive electrophysiological mapping of central motor impairment of lower limbs in clinical application.

The anti-CD52 monoclonal antibody alemtuzumab reduces disease activity in previously untreated patients with relapsing-remitting multiple sclerosis. We aimed to assess efficacy and safety of alemtuzumab compared with interferon beta 1a in patients who have relapsed despite first-line treatment.In our 2 year, rater-masked, randomised controlled phase 3 trial, we enrolled adults aged 18-55 years with relapsing-remitting multiple sclerosis and at least one relapse on interferon beta or glatiramer. Eligible participants were randomly allocated in a 1:2:2 ratio by an interactive voice response system, stratified by site, to receive subcutaneous interferon beta 1a 44 μg, intravenous alemtuzumab 12 mg per day, or intravenous alemtuzumab 24 mg per day. Interferon beta 1a was given three-times per week and alemtuzumab was given once per day for 5 days at baseline and for 3 days at 12 months. The 24 mg per day group was discontinued to aid recruitment, but data are included for safety assessments. Coprimary endpoints were relapse rate and time to 6 month sustained accumulation of disability, comparing alemtuzumab 12 mg and interferon beta 1a in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT00548405.202 (87%) of 231 patients randomly allocated interferon beta 1a and 426 (98%) of 436 patients randomly allocated alemtuzumab 12 mg were included in the primary analyses. 104 (51%) patients in the interferon beta 1a group relapsed (201 events) compared with 147 (35%) patients in the alemtuzumab group (236 events; rate ratio 0·51 [95% CI 0·39-0·65]; p0·0001), corresponding to a 49·4% improvement with alemtuzumab. 94 (47%) patients in the interferon beta 1a group were relapse-free at 2 years compared with 278 (65%) patients in the alemtuzumab group (p0·0001). 40 (20%) patients in the interferon beta 1a group had sustained accumulation of disability compared with 54 (13%) in the alemtuzumab group (hazard ratio 0·58 [95% CI 0·38-0·87]; p=0·008), corresponding to a 42% improvement in the alemtuzumab group. For 435 patients allocated alemtuzumab 12 mg, 393 (90%) had infusion-associated reactions, 334 (77%) had infections (compared with 134 [66%] of 202 patients in the interferon beta 1a group) that were mostly mild-moderate with none fatal, 69 (16%) had thyroid disorders, and three (1%) had immune thrombocytopenia.For patients with first-line treatment-refractory relapsing-remitting multiple sclerosis, alemtuzumab could be used to reduce relapse rates and sustained accumulation of disability. Suitable risk management strategies allow for early identification of alemtuzumab's main adverse effect of secondary autoimmunity.Genzyme (Sanofi) and Bayer Schering Pharma.

Introduction Fampridine is a slow-release potassium channel blocker that ameliorates the impaired conduction in CNS demyelinated axons. In multiple sclerosis (MS) patients, wide fluctuations in fatigue perception make difficult to assess the highly variable clinical response to treatment. Previous study reported that central motor conduction time (CMCT) improvement may predict fampridine response. Nevertheless, more than conduction slowdown, central motor conduction failure (CMCF), i.e. conduction block and axonal damage, reflects walking impairment in MS patients. Decrease of Motor Evoked Potentials (MEP) area, much more than amplitude, may detect CMCF. Serial MEP area recordings and analysis, according to a protocol that reduces MEP area random variability, may detect CMCF variation. We evaluated the effects of fampridine on CMCT and CMCF by serial recordings of MEPs from several proximal and distal muscle districts of lower limbs in a MS patients cohort and we correlated the obtained data with clinical outcome. Methods In 10 MS patients, MEPs to TMS by double-cone coil and maximal Compound Motor Action Potentials (CMAPs) to High Voltage Electrical Stimulation of lumbosacral roots were recorded from Vastus Medialis and Lateralis, Tibialis Anterior, Peroneus Longus and Flexor Hallucis Brevis of both limbs (T1) according to the published method (Di Sapio et al., 2014). Stimulation and recorded sites were marked. In 3 patients the test was repeated after one week (T1b), as control. After 14 days of fampridine treatment, before stopping therapy (T2), the procedure was repeated, strictly maintaining the same stimulation and recording sites and stimulation intensities. The 25-foot walking test (25FWT), 6-min walking test (6’-WT), the Fatigue Score Scale (FSS) and the Modified Fatigue Inventory Scale (MFIS) were administered at each time point. In each patient CMCT, MEP area and MEP Area/CMAP area ratios (ARs) were compared, and if appropriate their variability normalized according to Troni et al. (2016). Patients were classified as neurophysiological responders when 2 or more area/latency indexes significantly improved, remaining the others stable. Moreover, pooled data from the whole cohort in different time-points were compared using T test (Wilcoxon). Results CMCT, MEP area and AR did not change significantly in patients not receiving fampridine. After fampridine administration 3 patients were classified as responders; the same patients reported fatigue reduction and showed significant improvement in FSS score and 25FWT, while MFIS and 6’WT improved only in 1 patient. By analysing pooled data obtained from responders only, a significant reduction of CMCT (p = 0.0057) and increase in MEP area (p = 0.0014) and AR (p = 0.048) were noted. Conclusion The CMCF evaluation, together with CMCT, both made more sensitive by adopting multiple recording sites, correlate with self-reported and clinical outcome of fampridine treatment and may help to clarify doubt cases.

Therapeutic proteins like recombinant human interferon-beta (rhIFNβ) may induce neutralising antibodies (NABs), which inhibit their efficacy. Hence, there is a great need for strategies to predict whether a formulation will induce an immune response. Immune tolerant transgenic animals are important tools to study this phenomenon. This article describes a bioassay for NABs detection in mouse sera. The bioassay corresponds to the MxA Gene expression Assay (MGA) for human sera and measures the inhibition by mouse serum of the IFNβ induced MxA mRNA. Samples from 6 non-immunised and 14 IFNβ-immunised mice were tested for both binding antibodies (BABs) and NABs using the bioassay. All 16 mouse sera tested positive for NABs were also positive for BABs; BAB and NAB levels were correlated with a coefficient of 0.62 (p = 0.0186). The intra-assay variations ranged from 1.38% to 5.26% (mean 3.03%). Effects of cytotoxicity against A549 cells were slightly evident at low serum dilutions (i.e. 1/10, 1/20), but levels of damaged cells were easily evaluated based on the threshold cycle (Ct) values of the housekeeping gene 18SrRNA. The possibility of measuring NABs, in addition to BABs, in mouse sera increases the usefulness of the animal model, in studying the many factors influencing the immunogenicity of rhIFNβ.

Many multiple sclerosis (MS) patients treated with interferon-beta (IFNbeta) develop anti-IFNbeta antibodies (BAbs), which can interfere with both in vitro and in vivo bioactivity of the injected cytokine. Objective of this study was to correlate these measures. Among the 256 enrolled patients, 11 (4.3%) showed a significant inhibition of the IFNbeta activity in vitro, but no measurable BAbs. As a whole, in vivo bioactivity was inhibited in 9/11 (82%) of these patients. A minority of IFNbeta treated patients have a non-antibody mediated neutralising activity, which competitively inhibits the bioactivity both in vitro and in vivo.

Recently we reported that the keratan sulphate epitope recognised by the monoclonal antibody 5D4 is expressed by a population of ramified microglia in adult rats. As ramified microglia is believed to differentiate from ameboid microglia during postnatal development, we studied the rat brain from birth to 90 postnatal days of life with the monoclonal antibody 5D4. Contrary to all the other microglia markers until now described, keratan sulphate is not expressed by ameboid microglia and by macrophages but appears on the surface of microglia only when the cells are differentiated and show ramified processes. The keratan sulphate positive cells become evident at different times in different central nervous system areas; the first were localised in the pyriform cortex and brainstem from the end of the second postnatal week. These observations suggest that keratan sulphate expression on microglia cells is induced by differentiation and by a resting functional state. Moreover the 5D4 monoclonal antibody showed a strong diffuse positive staining of some cortical, thalamic and white matter areas during the first two postnatal weeks. This staining was transient and it does not seem biologically correlated with the expression of the keratan sulphate on differentiated microglia.

Anti-chondroitin unsulfated proteoglycan (COS-PG) 1B5 monoclonal antibody (MAb) and Vicia villosa agglutinin (VVA) recognize the same neuronal subset. Moreover, when in double immunofluorescence study the sections were digested with chondroitinase ABC (ChABC), a procedure necessary to create the epitope of 1B5 MAb, and incubated with VVA, no VVA positive neurons were detected. This finding suggests that VVA binds terminal N -acetyl-galactosamine present in the glycidic chains of COS-PG or of glycoproteins that form perineuronal molecular aggregates with COS-PG.

Balance impairment is common in Multiple Sclerosis (MS) patients. We administered the modified Clinical Test of Sensory Interaction on Balance (mCTSIB) to 73 MS patients and 57 healthy controls, with the aim to collect normative data and compare sensitivity of mCTSIB and clinical examination in detecting balance impairment. Subjects were asked to stand quietly upright for 10 s with eyes opened and then eyes closed (EC) (3 times per condition) on a firm surface on a fixed dual forceplate. The centre of gravity (COG) sway was expressed as velocity (deg/s) and path length (mm); MS patients were examined with the Romberg Test (RT), scored according to Neurostatus. mCTSIB-EC clearly allowed to differentiate patients with positive RT (0.38 deg/s, CI 0.32–0.43) from those with negative RT (0.19 deg/s, 95% CI 0.17–0.21, p

We studied the relationship between CMAP area variability and CMAP area size in an experimental setting in which any other possible cause of variability was removed. In 10 normal volunteers we recorded from Abductor Digiti Minimi a CMAP of large area and one of small area simultaneously after maximal stimulation of the ulnar nerve at wrist. Ulnar nerve stimulation was repeated 5 times, 4-6 s apart, and two sets of 5 CMAPs were obtained: one of large area and one of small area. The same procedure was performed with stimulation of the ulnar nerve at the elbow. Non-polarizable, ring-shaped sintered Ag/AgCl Multitrode® surface electrodes were adopted. For each set of rectified responses (large and small) Coefficient of Variation (COV, %) was calculated. Areas and COVs from all subjects were expressed as mean ± SD. Mean intrasession variability of small CMAPs elicited at wrist was significantly greater than that of large CMAPs elicited at the same site (paired t test: p

We investigated the Intertrial Variability and Relative Intertrial Variation (RIV) of latency and area of motor responses to High Voltage Electric Stimulation (HVES) of lumbo-sacral roots and to Transcranial Magnetic Stimulation (TMS) recorded from several sites of lower limbs. The purpose was to obtain normative variability values to detect significant changes in the individual patient in follow up studies. Maximal CMAPs to HVES of lumbo-sacral roots were obtained from 10 proximal and distal muscle districts of lower limbs; MEPs to TMS using the double cone coil were recorded from the same sites. The procedure was repeated twice, 1–2 days apart, in 30 subjects, including healthy volunteers and clinically stable multiple sclerosis patients. The use of stable recording and stimulation sites with maximal root-CMAPs and adequately stabilized MEPs allowed a significant reduction of ITV for all latency and area parameters. Mean RIV values (5th–95th percentile) for Central Motor Conduction Time and Area Ratio (MEP/CMAP) did not exceeded −14.8 and +16.0 ms and −26.0 and +33.7 mV ms, respectively. The combined monitoring of latency and areas indexes allows assessment of both conduction slowing and conduction failure, i.e the variable association of axonal damage and conduction block, which directly correlates with motor impairment.

Summary Chondroitin 4-sulfate proteoglycan (C4S-PG) was localized both in rat and human central nervous system (CNS) by monoclonal and polyclonal antisera recognizing the 4-sulfate disaccharide (C4S). In the rat the whole CNS was studied in serial coronal sections. A positive extracellular meshwork was observed both in white and grey matters. In the white matter (WM) C4S-PG formed a network around myelinated axons, sparing myelin sheaths and axoplasms. The neuropil of the grey matter (GM) showed a positive meshwork constituted by delicate intermingling filaments. The cytoplasms of neuronal, glial and endothelial cells were negative. Stronger straining than in the neuropil was observed around the soma and the proximal part of the cell processes of some neurons located in the cortex, in the deep cerebellar nuclei and in some other CNS nuclei. A similar pattern was also observed in human CNS, the only difference being a smaller amount of cortical neurons surrounded by a rim of C4S-PG. This study shows that a PG bearing C4S disaccharide is located extracellularly in the rodent and human CNS and that C4S disaccarides can be present in different types of CNS proteoglycans (PGs).

We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [ 3 H ]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes (recognized by the monoclonal antibody LB1 at early stages of their development) synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [ 3 H ]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bipotential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.

We have used an antibody raised against the bovine nasal cartilage proteoglycan chondroitin sulfate (CS) digested with chondroitinase ABC (anti-CS serum) to stain cerebellar glial cells maintained in culture. In cultures grown in the presence of serum, the antibody stained a subclass of GFAP+ astrocytes which we have previously shown to selectively bind the monoclonal antibodies A2B5 and LB1. Also the direct bipotential precursors of these cells, capable of differentiating into GFAP+ astrocytes or into Gal-C+, O1+ oligodendrocytes depending on the culture conditions, were stained, but stopped to produce CS when they differentiated into oligodendrocytes.