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Background Blood is a common source of RNA for gene expression studies. However, it is known to be vulnerable to pre-analytical variables. Although RNA stabilization systems have been shown to reduce such influence, traditional EDTA tubes are still widely used since they are less expensive and enable to study specific leukocyte populations. This study aimed to assess the influence of storage time and temperature between blood sampling and handling on RNA from peripheral blood mononuclear cells (PBMCs). Methods and results Nine blood samples were collected in EDTA tubes from 10 healthy donors. One tube from each donor was immediately processed for PBMC isolation, while the others were first incubated at either 4 degrees Celsius (°C) or room temperature for 2, 4, 6 and 24 h. RNA yield and quality and the expression level of fourt housekeeping (B2M, CASC3, GAPDH, HPRT1) and 8 target genes (CD14, CD19, CD20, IL10, MxA, TNF, TNFAIP3, NR4A2) were compared between samples. RNA yield, quality and integrity did not vary significantly with time and temperature. B2M was the most stable housekeeping gene, while the others were increasingly influenced by storing time, especially at 4 °C. Even when normalized to B2M, the expression level of some target genes, particularly TNFAIP3 and NR4A2, was highly affected by delays in blood processing at either temperature, already from 2 h. Conclusion Pre-analytical processing has a great impact on transcript expression from blood collected in EDTA tubes, especially on genes related to inflammation. Standardized procedure of blood handling are needed to obtain reliable results.

Background MSQOL-54 is a multidimensional, widely-used, health-related quality of life (HRQOL) instrument specific for multiple sclerosis (MS). Findings from the validation study suggested that the two MSQOL-54 composite scores are correlated. Given this correlation, it could be assumed that a unique total score of HRQOL may be calculated, with the advantage to provide key stakeholders with a single overall HRQOL score. We aimed to assess how well the bifactor model could account for the MSQOL-54 structure, in order to verify whether a total HRQOL score can be calculated. Methods A large international database (3669 MS patients) was used. By means of confirmatory factor analysis, we estimated a bifactor model in which every item loads onto both a general factor and a group factor. Fit of the bifactor model was compared to that of single and two second-order factor models by means of Akaike information and Bayesian information criteria reduction. Reliability of the total and subscale scores was evaluated with Mc Donald’s coefficients (omega, and omega hierarchical). Results The bifactor model outperformed the two second-order factor models in all the statistics. All items loaded satisfactorily (≥ 0.40) on the general HRQOL factor, except the sexual function items. Omega coefficients for total score were very satisfactory (0.98 and 0.87). Omega hierarchical for subscales ranged between 0.22 to 0.57, except for the sexual function (0.70). Conclusions The bifactor model is particularly useful when it is intended to acknowledge multidimensionality and at the same time take account of a single general construct, as the HRQOL related to MS. The total raw score can be used as an estimate of the general HRQOL latent score.

Extended interval dosing (EID) of Natalizumab (NAT) has been proposed to reduce progressive multifocal leukoencephalopathy (PML) risk associated with standard interval dosing (SID) in people with multiple sclerosis (MS). Previous studies have suggested that NAT effectiveness is maintained in the great majority of patients who switch from SID to EID; monitoring of disease activity is currently based exclusively on clinical and MRI parameters. Frequent MRI are expensive and not always applicable, underlining the need for biological markers able to detect central nervous system lesions. Serum Neurofilament-light chain (sNFL) currently represents the most promising biomarker of disease activity, prognosis and treatment response in MS, and their clinical suitability is increasingly evident. The objective of the present study is to assess the applicability of sNFL as additional/alternative measure of treatment efficacy during EID regimen.We measured sNFL by Simoa technology in longitudinal samples from 63 Relapsing Remitting (RR) MS patients switched from SID to EID.diagnosis of RRMS, age 18-60 years; NAT SID for at least 12 months; NEDA-3 (no evidence of disease activity) for at least 12 months; availability of at least 2 serum samples collected 6 months apart. Patients' follow-up time during EID was at least 12 months and 2 blood samples were collected after at least 6 and 12 months. Clinical examination was performed before each infusion, while MRI 6 and 12 months after NAT initiation and according to PML risk during the whole study.No patients showed clinical or MRI activity during the whole follow-up. sNFL levels measured during SID and EID were comparable, without significant difference between groups. The effect of EID on NFL levels did not show significant effects (LMM, p0.05) and sNFL levels did not vary with time during SID or EID protocols (LMM, p0.05). Intra-individual sNFL levels demonstrated overall stability during SID and EID (median CV=11% between SID and EID samples). According to our previously published reference values, sNFL levels were in the normal range in all samples, both during SID and EID.Our results suggest that sNFL quantification can be used as an alternative/additional approach to MRI in managing individual patients. The present work provides a new clinical application of sNFL to monitor NAT efficacy.

ObjectiveTo determine whether autologous hematopoietic stem cell transplantation (aHSCT) is able to induce durable disease remission in people with multiple sclerosis (MS), we analyzed the long-term outcomes after transplantation in a large cohort of patients with MS.MethodsTo be included, a minimum dataset (consisting of age, MS phenotype, Expanded Disability Status Scale [EDSS] score at baseline, information on transplantation technology, and at least 1 follow-up visit after transplantation) was required.ResultsTwo hundred ten patients were included (relapsing-remitting [RR] MS 122 [58%]). Median baseline EDSS score was 6 (1–9); mean follow-up was 6.2 (±5.0) years. Among patients with RRMS, disability worsening–free survival (95% confidence interval [CI]) was 85.5% (76.9%–94.1%) at 5 years and 71.3% (57.8%–84.8%) at 10 years. In patients with progressive MS, disability worsening–free survival was 71.0% (59.4%–82.6%) and 57.2% (41.8%–72.7%) at 5 and 10 years, respectively. In patients with RRMS, EDSS significantly reduced after aHSCT (p = 0.001; mean EDSS change per year −0.09 [95% CI −0.15% to −0.04%]). In patients with RRMS, the use of the BCNU+Etoposide+Ara-C+Melphalan (BEAM) + anti-thymocyte globulin (ATG) conditioning protocol was independently associated with a reduced risk of no evidence of disease activity 3 failure (hazard ratio 0.27 [95% CI 0.14–0.50], p < 0.001). Three patients died within 100 days from aHSCT (1.4%); no deaths occurred in patients transplanted after 2007.ConclusionsaHSCT prevents disability worsening in the majority of patients and induces durable improvement in disability in patients with RRMS. The BEAM + ATG conditioning protocol is associated with a more pronounced suppression of clinical relapses and MRI inflammatory activity.Classification of EvidenceThis study provides Class IV evidence that for people with MS, aHSCT induces durable disease remission in most patients.

ObjectiveAssessing the risk of clinical and radiological reactivation during pregnancy and post partum in women with multiple sclerosis (MS) treated with natalizumab (NTZ) throughout pregnancy (LONG_EXP) compared with women interrupting treatment before (NO_EXP) and within >−30 days and ≤90 days from conception (SHORT_EXP), and describing newborns’ outcomes.MethodsMaternal clinical and radiological outcomes and obstetric and fetal outcomes were retrospectively collected and compared among groups (NO_EXP, SHORT_EXP, LONG_EXP). Predictors of clinical and radiological reactivation were investigated through univariable and multivariable analysis.Results170 eligible pregnancies from 163 women referring to 29 Italian MS centres were included. Annualised relapse rate (ARR) was significantly lower in LONG_EXP (n=66, 0.02 (0.001–0.09)) compared with NO_EXP (n=31, 0.43 (0.21–0.75), p=0.002) and SHORT_EXP (n=73, 0.46 (0.30–0.66), p=0.0004) during pregnancy, and in LONG_EXP (0.12 (0.05–0.24)) compared with SHORT_EXP (0.30 (0.17–0.50), p=0.008) during post partum. Gadolinium-enhancing (Gd+) lesions were less frequent in LONG_EXP (n=6/50, 2.00%) compared with NO_EXP (n=9/21, 42.86%) and SHORT_EXP after delivery (n=17/49, 34.69%, p=0.010).Delaying NTZ resumption after delivery significantly increased the risk of relapses (OR=1.29 (95% CI 1.07 to 1.57), p=0.009) and Gd+ lesions (OR=1.49 (95% CI 1.17 to 1.89, p=0.001). Newborns’ weight, length, head circumference and gestational age did not differ among groups after adjusting for confounders. Anaemia was tracked in 4/69 LONG_EXP newborns. Congenital anomaly rate was within the expected range for the untreated MS population.ConclusionsOur findings indicate that in women with MS treated with NTZ before conception, continuation of NTZ throughout pregnancy and its early resumption after delivery mitigate the risk of clinical and radiological reactivation. This approach has no major impact on newborns’ outcomes.

The transcription factor NURR1 is essential to the generation and maintenance of midbrain dopaminergic (mDA) neurons and its deregulation is involved in the development of dopamine (DA)-associated brain disorders, such as Parkinson's disease (PD). The old male NURR1 heterozygous knockout (NURR1-KO) mouse has been proposed as a model of PD due to its altered motor performance that was, however, not confirmed in a subsequent study. Based on these controversial results, we explored the effects of the NURR1 deficiency on locomotor activity, motor coordination, brain and plasma DA levels, blood pressure and heart rate of old mice, also focusing on the potential effect of sex. As a probable consequence of the role of NURR1 in DA pathway, we observed that the old NURR1-KO mouse is characterized by motor impairment, and increased brain DA level and heart rate, independently from sex. However, we also observed an alteration in spontaneous locomotor activity that only affects males. In conclusion, NURR1 deficiency triggers sex- and age-specific alterations of behavioral responses, of DA levels and cardiovascular abnormalities. Further studies in simplified systems will be necessary to dissect the mechanism underlying these observations.

Fingolimod (FTY720) and siponimod (BAF312) are selective agonists for sphingosine-1-phosphate (S1P) receptors approved for the treatment of relapsing–remitting (RR) and secondary progressive (SP) multiple sclerosis (MS), respectively. BAF312 exerts pro-myelination and neuro-protective functions on CNS resident cells, although the underlying molecular mechanism is not yet fully understood. NR4A2 is an anti-inflammatory gene, belonging to the NR4A family, whose expression is reduced in blood from treatment-naïve patients with RRMS, but is restored in patients treated with FTY720 for more than two years. We performed an in vitro study to investigate the potential involvement of the NR4A genes in the protective and restorative effects of BAF312. We showed that BAF312 enhances the expression of NR4A1 and NR4A2 in the N9 microglial cell line, but has no effect in the peripheral blood mononuclear cells and oligodendrocytes. This study suggests a novel molecular mechanism of action for the selective agonists for S1P receptors within the CNS.

Multiple Sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) in which inflammation plays a key pathological role. Recent evidences showed that systemic inflammation induces increasing cell infiltration within meninges and perivascular spaces in the brain parenchyma, triggering resident microglial and astrocytic activation. The anti‐inflammatory enzyme A20, also named TNF associated protein 3 (TNFAIP3), is considered a central gatekeeper in inflammation and peripheral immune system regulation through the inhibition of NF‐kB. The TNFAIP3 locus is genetically associated to MS and its transcripts is downregulated in blood cells in treatment‐naïve MS patients. Recently, several evidences in mouse models have led to hypothesize a function of A20 also in the CNS. Thus, here we aimed to unveil a possible contribution of A20 to the CNS human MS pathology. By immunohistochemistry/immunofluorescence and biomolecular techniques on post‐mortem brain tissue blocks obtained from control cases (CC) and progressive MS cases, we demonstrated that A20 is present in CC brain tissues in both white matter (WM) regions, mainly in few parenchymal astrocytes, and in grey matter (GM) areas, in some neuronal populations. Conversely, in MS brain tissues, we observed increased expression of A20 by perivascular infiltrating macrophages, resident‐activated astrocytes, and microglia in all the active and chronic active WM lesions. A20 was highly expressed also in the majority of active cortical lesions compared to the neighboring areas of normal‐appearing grey matter (NAGM) and control GM, particularly by activated astrocytes. We demonstrated increased A20 expression in the active MS plaques, particularly in macrophages and resident astrocytes, suggesting a key role of this molecule in chronic inflammation., The anti‐inflammatory enzyme A20, codified by the TNFAIP3 gene, is genetically associated to MS and its transcripts is down‐regulated in blood cells of treatment‐naïve MS patients. By immunohistochemistry/immunofluorescence and biomolecular techniques on post‐mortem brain tissues of progressive MS cases, we observed an increased expression of A20 in the active and chronic active white matter lesions and in the active cortical lesions, particularly in activated astrocytes. Our results suggest a key role of A20 also in chronic inflammation of the central nervous system.

Introduction In clinical trials of alemtuzumab, autoimmune thyroid adverse events (AEs) were frequent. Here, we assess the impact of thyroid AEs on health-related quality of life (HRQL) in alemtuzumab-treated patients with relapsing-remitting multiple sclerosis (RRMS). Methods In phase 3 CARE-MS I (NCT00530348) and II (NCT00548405) trials, patients with RRMS were administered alemtuzumab 12 mg/day on 5 consecutive days at baseline and on 3 consecutive days 12 months later. Patients could participate in an extension study (NCT00930553) through year 6. HRQL was assessed at baseline and annually using the Functional Assessment of Multiple Sclerosis (FAMS), EuroQoL-5 Dimension Visual Analog Scale (EQ-5D VAS), and 36-Item Short-Form Survey (SF-36) questionnaires. Outcomes were analyzed in patients with or without thyroid AEs (nonserious or serious). A subset of patients with thyroid AEs was analyzed to assess HRQL before and during the onset of thyroid AEs. Results A total of 811 CARE-MS patients were treated with alemtuzumab. Of these, 342 (42%) patients experienced thyroid AEs over 6 years; serious thyroid AEs occurred in 44 (5%) patients. At year 6, HRQL outcomes generally remained slightly improved or similar to core study baseline in alemtuzumab-treated patients with or without thyroid AEs: FAMS (least-squares mean change from baseline without thyroid AEs, 0.7; with nonserious thyroid AEs, 5.1; with serious thyroid AEs, − 5.3), EQ-5D VAS (2.0; 3.0; − 6.8), SF-36 mental component summary (MCS [0.6; 1.6; − 2.8]), SF-36 physical component summary (PCS [0.8; 1.0; 1.1]). Over 6 years, 63–82% of patients in each group had improved/stable SF-36 MCS and PCS scores. Among patients with thyroid AE onset in year 3 (peak incidence), there were minimal differences between HRQL outcomes before onset (year 2) and after onset (year 3). Conclusion Autoimmune thyroid AEs (serious and nonserious) had minimal impact on HRQL in alemtuzumab-treated patients. These data may aid therapeutic decisions in patients with relapsing MS. Electronic supplementary material The online version of this article (10.1007/s40120-020-00191-7) contains supplementary material, which is available to authorized users., Plain Language Summary This study looked at alemtuzumab, an approved treatment for multiple sclerosis (MS). People who receive alemtuzumab may develop thyroid problems. The researchers wanted to know whether people who developed thyroid problems with alemtuzumab had a worse quality of life compared with those who did not. The researchers measured quality of life using a questionnaire. The questionnaire looked at people’s physical, social, and psychological well-being over 6 years. A total of 811 people with MS treated with alemtuzumab took part in this study. Of these, 469 people (58%) did not develop thyroid problems and 342 people (42%) developed thyroid problems. The thyroid problems were serious in 44 people. The researchers observed that thyroid problems during alemtuzumab treatment did not make quality of life worse in most people. Some people with serious thyroid problems had worsened quality of life; this was mostly among people who required certain treatments for their thyroid problems. Quality of life did not change much in people while the thyroid problems were ongoing. This study shows that thyroid problems after alemtuzumab treatment for MS have little negative impact on quality of life for most people. These findings may help healthcare providers make decisions about MS treatment. Electronic supplementary material The online version of this article (10.1007/s40120-020-00191-7) contains supplementary material, which is available to authorized users.

In the last decade, microRNAs have been increasingly recognized as key modulators of glial development. Recently, we identified miR-125a-3p as a new player in oligodendrocyte physiology, regulating in vitro differentiation of oligodendrocyte precursor cells (OPCs). Here, we show that miR-125a-3p is upregulated in active lesions of multiple sclerosis (MS) patients and in OPCs isolated from the spinal cord of chronic experimental autoimmune encephalomyelitis (EAE) mice, but not in those isolated from the spontaneously remyelinating corpus callosum of lysolecithin-treated mice. To test whether a sustained expression of miR-125a-3p in OPCs contribute to defective remyelination, we modulated miR-125a-3p expression in vivo and ex vivo after lysolecithin-induced demyelination. We found that lentiviral over-expression of miR-125a-3p impaired OPC maturation, whereas its downregulation accelerated remyelination. Transcriptome analysis and luciferase reporter assay revealed that these effects are partly mediated by the direct interaction of miR-125a-3p with Slc8a3, a sodium-calcium membrane transporter, and identified novel candidate targets, such as Gas7, that we demonstrated necessary to correctly address oligodendrocytes to terminal maturation. These findings show that miR-125a-3p upregulation negatively affects OPC maturation in vivo, suggest its role in the pathogenesis of demyelinating diseases and unveil new targets for future promyelinating protective interventions.

Fingolimod (FTY720) and siponimod (BAF312) are selective agonists for sphingosine-1-phosphate (S1P) receptors approved for the treatment of relapsing-remitting (RR) and secondary progressive (SP) multiple sclerosis (MS), respectively. BAF312 exerts pro-myelination and neuro-protective functions on CNS resident cells, although the underlying molecular mechanism is not yet fully understood.

Objective: This study was undertaken to describe and compare disease course and prognosis of early (ie, disease onset before age 11 years) and late (ie, disease onset after age 11 years) onset pediatric multiple sclerosis. Methods: Prospectively collected clinical information from Italian Multiple Sclerosis Register of 1993 pediatric multiple sclerosis patients, of whom 172 had early onset, was analyzed. Cox models adjusted for sex, baseline Expanded Disability Status Scale score, and disease-modifying treatments and stratified for diagnostic criteria adopted (Poser vs McDonald) were used to assess the risk of reaching irreversible Expanded Disability Status Scale scores of 3, 4, and 6, and conversion to secondary progressive phenotype in early versus late onset pediatric patients. Prognostic factors were also evaluated. Results: A greater proportion of males, isolated brainstem involvement, and longer time interval between first and second clinical episode were observed in early versus late onset pediatric patients. Compared to late onset, early onset pediatric patients took longer from disease onset to convert to secondary progressive phenotype and to reach all disability milestones. Recovery from first demyelinating event, time to first relapse, annualized relapse rate during the first 3 years of disease, and disease-modifying treatment exposure were independent predictors for long-term disability in early onset pediatric patients. In late onset pediatric patients, isolated optic neuritis, multifocal symptoms, and progressive course at disease onset were additional predictors for long-term disability. Interpretation: These findings point toward the existence of a different natural history in early versus late onset pediatric multiple sclerosis patients. ANN NEUROL 2022.

Amyotrophic lateral sclerosis (ALS) is known to represent a collection of overlapping syndromes. Various classification systems based on empirical observations have been proposed, but it is unclear to what extent they reflect ALS population substructures. We aimed to use machine-learning techniques to identify the number and nature of ALS subtypes to obtain a better understanding of this heterogeneity, enhance our understanding of the disease, and improve clinical care.In this retrospective study, we applied unsupervised Uniform Manifold Approximation and Projection [UMAP]) modelling, semi-supervised (neural network UMAP) modelling, and supervised (ensemble learning based on LightGBM) modelling to a population-based discovery cohort of patients who were diagnosed with ALS while living in the Piedmont and Valle d'Aosta regions of Italy, for whom detailed clinical data, such as age at symptom onset, were available. We excluded patients with missing Revised ALS Functional Rating Scale (ALSFRS-R) feature values from the unsupervised and semi-supervised steps. We replicated our findings in an independent population-based cohort of patients who were diagnosed with ALS while living in the Emilia Romagna region of Italy.Between Jan 1, 1995, and Dec 31, 2015, 2858 patients were entered in the discovery cohort. After excluding 497 (17%) patients with missing ALSFRS-R feature values, data for 42 clinical features across 2361 (83%) patients were available for the unsupervised and semi-supervised analysis. We found that semi-supervised machine learning produced the optimum clustering of the patients with ALS. These clusters roughly corresponded to the six clinical subtypes defined by the Chiò classification system (ie, bulbar, respiratory, flail arm, classical, pyramidal, and flail leg ALS). Between Jan 1, 2009, and March 1, 2018, 1097 patients were entered in the replication cohort. After excluding 108 (10%) patients with missing ALSFRS-R feature values, data for 42 clinical features across 989 patients were available for the unsupervised and semi-supervised analysis. All 1097 patients were included in the supervised analysis. The same clusters were identified in the replication cohort. By contrast, other ALS classification schemes, such as the El Escorial categories, Milano-Torino clinical staging, and King's clinical stages, did not adequately label the clusters. Supervised learning identified 11 clinical parameters that predicted ALS clinical subtypes with high accuracy (area under the curve 0·982 [95% CI 0·980-0·983]).Our data-driven study provides insight into the ALS population substructure and confirms that the Chiò classification system successfully identifies ALS subtypes. Additional validation is required to determine the accuracy and clinical use of these algorithms in assigning clinical subtypes. Nevertheless, our algorithms offer a broad insight into the clinical heterogeneity of ALS and help to determine the actual subtypes of disease that exist within this fatal neurodegenerative syndrome. The systematic identification of ALS subtypes will improve clinical care and clinical trial design.US National Institute on Aging, US National Institutes of Health, Italian Ministry of Health, European Commission, University of Torino Rita Levi Montalcini Department of Neurosciences, Emilia Romagna Regional Health Authority, and Italian Ministry of Education, University, and Research.For the Italian and German translations of the abstract see Supplementary Materials section.

The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.

Background and ObjectivesPatients with pediatric-onset multiple sclerosis (MS) can be especially vulnerable to cognitive impairment (CI) due to the onset of MS during a critical period for CNS development and maturation. The objective of this longitudinal study was to assess long-term cognitive functioning and socioprofessional attainment in the Italian pediatric MS cohort, previously assessed at baseline and 2 and 5 years.MethodsThe 48 patients evaluated at the 5-year assessment were screened for inclusion. All participants were assessed with a cognitive test battery exploring 4 different cognitive abilities. Depression, fatigue, and socioprofessional attainment were also assessed. Mean cognitivezscores were calculated for the whole cohort, and their evolution over time was analyzed with an analysis of variance for repeated measurements test. Predictors of cognitive worsening or improvement were assessed with a linear mixed-model analysis.ResultsThirty-three participants were included (mean follow-up 12.8 ± 0.8 years). The global cognitive performance worsened at year 2 and improved at year 5, although thezscore remained significantly lower than at baseline (−0.9 ± 1.2 vs −0.3 ± 0.9,p= 0.002). There was no significant variation between years 5 and 12 (−0.7 ± 1.1,p= 0.452). Higher IQ (>90) at baseline (effect 0.3, 95% CI 0.1–0.5,p= 0.017) and lower number of relapses in the 2 years before baseline (effect −0.1, 95% CI −0.1 to 0.1,p= 0.025) predicted better cognitive performances. Eighteen (54.5%) patients failed at least 2 tests compared with healthy controls and were defined as cognitively impaired. The presence of CI predicted worse socioprofessional attainment (β = 4.8, 95% CI 1.4–8.2,p= 0.008).DiscussionThe longitudinal cognitive trajectory in pediatric-onset MS has a heterogeneous course over time, with a decline in the first years followed by a partial recovery over the long term. However, at the last follow-up evaluation, the proportion of impaired patients was more than double compared with baseline, with a negative impact on the individual’s socioprofessional attainment in adulthood. This study underscores how cognitive reserve may partially mitigate the negative effects of brain damage, highlighting the critical importance of intellectual enrichment early during the disease course.

none 38 no BACKGROUND: Natalizumab (NTZ) is an effective treatment for relapsing-remitting multiple sclerosis (RRMS). However, patients and physicians may consider discontinuing NTZ therapy due to safety or efficacy issues. The aim of our study was to evaluate the NTZ discontinuation rate and reasons of discontinuation in a large Italian population of RRMS patients. MATERIALS AND METHODS: The data were extracted from the Italian MS registry in May 2018 and were collected from 51,845 patients in 69 Italian multiple sclerosis centers. MS patients with at least one NTZ infusion in the period between June 1st 2012 to May 15th 2018 were included. Discontinuation rates at each time point were calculated. Reasons for NTZ discontinuation were classified as "lack of efficacy", "progressive multifocal leukoencephalopathy (PML) risk" or "other". RESULTS: Out of 51,845, 5151 patients, 3019 (58.6%) females, with a mean age of 43.6 ± 10.1years (median 40), were analyzed. Out of 2037 (39.5%) who discontinued NTZ, a significantly higher percentage suspended NTZ because of PML risk compared to lack of efficacy [1682 (32.7% of 5151) vs 221 (4.3%), p

Hepatocyte growth factor (HGF) and its tyrosine kinase receptor, encoded by the MET cellular proto-oncogene, are expressed in the nervous system from pre-natal development to adult life, where they are involved in neuronal growth and survival. In this review, we highlight, beyond the neurotrophic action, novel roles of HGF-MET in synaptogenesis during post-natal brain development and the connection between deregulation of MET expression and developmental disorders such as autism spectrum disorder (ASD). On the pharmacology side, HGF-induced MET activation exerts beneficial neuroprotective effects also in adulthood, specifically in neurodegenerative disease, and in preclinical models of cerebral ischemia, spinal cord injuries, and neurological pathologies, such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). HGF is a key factor preventing neuronal death and promoting survival through pro-angiogenic, anti-inflammatory, and immune-modulatory mechanisms. Recent evidence suggests that HGF acts on neural stem cells to enhance neuroregeneration. The possible therapeutic application of HGF and HGF mimetics for the treatment of neurological disorders is discussed.

Objectives The aim of this study is to quantitatively investigate, at the preclinical level, the extent of Gd retention in the CNS, and peripheral organs, of immune-mediated murine models (Experimental Autoimmune Encephalomyelitis –EAE) of Multiple Sclerosis, compared to control animals, upon the injection of gadodiamide. The influence of the Gadolinium Based Contrast Agent administration timing during the course of EAE development is also monitored. Methods EAE mice were injected with three doses (1.2 mmol/kg each) of gadodiamide at three different time points during the EAE development and sacrificed after 21 or 39 days. Organs were collected and the amount of Gd was quantified through Inductively Coupled Plasma-Mass Spectrometry. Transmission electron microscopy (TEM) and MRI techniques were applied to add spatial and qualitative information to the obtained results. Results In the spinal cord of EAE group, 21 days after gadodiamide administration, a significantly higher accumulation of Gd occurred. Conversely, in the encephalon, a lower amount of Gd retention was reached, even if differences emerged between EAE and controls mice. After 39 days, the amounts of retained Gd markedly decreased. TEM validated the presence of Gd in CNS. MRI of the encephalon at 7.1T did not highlight any hyper intense region. Conclusion In the spinal cord of EAE mice, which is the mostly damaged region in this specific animal model, a preferential but transient accumulation of Gd is observed. In the encephalon, the Gd retention could be mostly related to inflammation occurring upon immunization rather than to demyelination.

Pregnancy is a unique situation of physiological immunomodulation, as well as a strong Multiple Sclerosis (MS) disease modulator whose mechanisms are still unclear. Both maternal (decidua) and fetal (trophoblast) placental cells secrete extracellular vesicles (EVs), which are known to mediate cellular communication and modulate the maternal immune response. Their contribution to the MS disease course during pregnancy, however, is unexplored. Here, we provide a first phenotypic and functional characterization of EVs isolated from cultures of term placenta samples of women with MS, differentiating between decidua and trophoblast. In particular, we analyzed the expression profile of 37 surface proteins and tested the functional role of placental EVs on mono-cultures of CD14+ monocytes and co-cultures of CD4+ T and regulatory T (Treg) cells. Results indicated that placental EVs are enriched for surface markers typical of stem/progenitor cells, and that conditioning with EVs from samples of women with MS is associated to a moderate decrease in the expression of proinflammatory cytokines by activated monocytes and in the proliferation rate of activated T cells co-cultured with Tregs. Overall, our findings suggest an immunomodulatory potential of placental EVs from women with MS and set the stage for a promising research field aiming at elucidating their role in MS remission.

Objective: This study was undertaken to assess the impact of immunosuppressive and immunomodulatory therapies on the severity of coronavirus disease 2019 (COVID-19) in people with multiple sclerosis (PwMS). Methods: We retrospectively collected data of PwMS with suspected or confirmed COVID-19. All the patients had complete follow-up to death or recovery. Severe COVID-19 was defined by a 3-level variable: mild disease not requiring hospitalization versus pneumonia or hospitalization versus intensive care unit (ICU) admission or death. We evaluated baseline characteristics and MS therapies associated with severe COVID-19 by multivariate and propensity score (PS)-weighted ordinal logistic models. Sensitivity analyses were run to confirm the results. Results: Of 844 PwMS with suspected (n = 565) or confirmed (n = 279) COVID-19, 13 (1.54%) died; 11 of them were in a progressive MS phase, and 8 were without any therapy. Thirty-eight (4.5%) were admitted to an ICU; 99 (11.7%) had radiologically documented pneumonia; 96 (11.4%) were hospitalized. After adjusting for region, age, sex, progressive MS course, Expanded Disability Status Scale, disease duration, body mass index, comorbidities, and recent methylprednisolone use, therapy with an anti-CD20 agent (ocrelizumab or rituximab) was significantly associated (odds ratio [OR] = 2.37, 95% confidence interval [CI] = 1.18–4.74, p = 0.015) with increased risk of severe COVID-19. Recent use (

Background and aim In the scientific literature, there is unanimous consensus that hospitalization in stroke unit (SU) is the most important treatment for stroke patients. In this regard, the Act number 70/2015 by the Italian government identified specific skills that contribute to a classification of SU and outlined a “hub and spoke” stroke network. The aim of our study was to check the coverage of requirements of first and second level SU in the national territory and to shed light on any deficit or misdistribution of resources. Material and methods In 2019, a survey on the current situation related to stroke care in Italy was carried out by the Italian Society of Neurology (SIN), The Italian Stroke Organization (ISO), and the Association for the Fight against Stroke (A.L.I.Ce). Results First level SU was found to be 58 against a requirement, according to the Act 70/2015, of 240. Second level SU was found to be 52 compared with an expected requirement of 60. Neurointerventionists were 280 nationally, with a requirement of 240. A misdistribution of resources within individual regions was often seen. Conclusions The survey demonstrated a severe shortage of beds dedicated to cerebrovascular diseases, mainly because of lack of first level SU, especially in central and southern Italy. It also suggests that the current hub and spoke system is not yet fully implemented across the country and that resources should be better distributed in order to ensure uniform and fair care for all stroke patients on the whole territory.

Epidemiological studies show a strong association between exposure to air pollution - and particularly to particulate matter (PM) -, increased prevalence of Multiple Sclerosis (MS) and higher rates of hospital admissions for MS and MS relapses. Besides having immunomodulatory effects and sustaining a systemic oxidative-inflammatory response, PM may participate in MS pathogenesis by targeting also Central Nervous System (CNS)-specific processes, such as myelin repair. Here we show that, in a mouse model of lysolecithin-induced demyelination of the subcortical white matter, post-injury exposure to fine PM hampers remyelination, disturbs oligodendroglia differentiation dynamics and promotes astroglia and microglia reactivity. These findings support the view that exposure to fine PM can contribute to demyelinating pathologies by targeting the endogenous regenerative capability of the CNS tissue.

Introduction: Italy is considered a high-risk country for multiple sclerosis (MS). Exploiting electronic health archives (EHAs) is highly useful to continuously monitoring the prevalence of the disease, as well as the care delivered to patients and its outcomes. The aim of this study was to validate an EHA-based algorithm to identify MS patients, suitable for epidemiological purposes, and to estimate MS prevalence in Piedmont (North Italy). Methods: MS cases were identified, in the period between January 1, 2012 and December 31, 2017, linking data from 4 different sources: hospital discharges, drug prescriptions, exemptions from co-payment to health care, and long-term care facilities. Sensitivity of the algorithm was tested through record linkage with a cohort of 656 neurologist-confirmed MS cases; specificity was tested with a cohort of 2,966,293 residents presumably not affected by MS. Undercount was estimated by a capture-recapture method. We calculated crude, and age- and gender-specific prevalence. We also calculated age-adjusted prevalence by level of urbanization of the municipality of residence. Results: On December 31, 2017, the algorithm identified 8,850 MS cases. Sensitivity was 95.9%, specificity was 99.97%, and the estimated completeness of ascertainment was 91.9%. The overall prevalence, adjusted for undercount, was 152 per 100,000 among men and 286 among women; it increased with increasing age and reached its peak value in the 45- to 54-year class, followed by a progressive reduction. The age-adjusted prevalence of residents in cities was 15% higher than in those living in the countryside. Discussion/Conclusion: We validated an algorithm based on EHAs to identify cases of MS for epidemiological use. The prevalence of MS, adjusted for undercount, was among the highest in Italy. We also found that the prevalence was higher in highly urbanized areas.

Background Few data exist concerning conversion to secondary progressive MS in patients treated with disease-modifying therapies. Objective Determine the proportion of alemtuzumab-treated patients converting from relapsing-remitting to secondary progressive MS during the CARE-MS core and extension studies. Methods Patients ( N = 811) were analyzed post hoc for secondary progressive MS conversion. Optimal conversion definition: Expanded Disability Status Scale (EDSS) score ≥4, pyramidal functional system score ≥2, and confirmed progression over ≥3 months including confirmation within the functional system leading to progression, independent of relapse. Results Over 6.2 years median follow-up, 20 alemtuzumab-treated patients converted (Kaplan-Meier estimate, 2.7%; 95% confidence interval, 1.8%–4.2%). Sensitivity analysis accounting for dropouts showed similar results (3%), as did analyses using alternative definitions with different EDSS thresholds and/or confirmation periods, and analysis of core study subcutaneous interferon beta-1a-treated patients who received alemtuzumab in the extension. Patients converting to secondary progressive MS were older, and had higher EDSS scores and greater brain lesion volumes at baseline, but did not need additional alemtuzumab or other therapies. Conclusions The 6-year conversion rate to secondary progressive MS was low for alemtuzumab-treated patients, supporting further study of the role alemtuzumab may play in reducing risk of secondary progression. ClinicalTrials.gov identifiers: NCT00530348, NCT00548405, NCT00930553.

Pediatric-onset multiple sclerosis (MS) has a highly active and aggressive course, which can have a devastating effect on the physical and cognitive functioning of a child if not treated appropriately with effective disease-modifying drugs. The optimal treatment strategy of pediatric MS is currently unknown and debate continues as to whether treatment escalation or initiation of a highly active therapy provides a better outcome. Here, we present the case of a 16-year-old female diagnosed with highly active relapsing-remitting MS (age at onset: 14 years) who received first-line treatment with fingolimod within 1 year of the first recorded symptom. Since starting fingolimod, the course of the disease has essentially been stable. No new or active lesions were observed in magnetic resonance imaging scans performed at 3 and 12 months after starting fingolimod, and treatment was well tolerated. These data suggest that, in this case, early treatment with first-line fingolimod was able to slow disease progression.

Epidemiological studies show a strong association between exposure to air pollution - and particularly to particulate matter (PM) -, increased prevalence of Multiple Sclerosis (MS) and higher rates of hospital admissions for MS and MS relapses. Besides having immunomodulatory effects and sustaining a systemic oxidative-inflammatory response, PM may participate in MS pathogenesis by targeting also Central Nervous System (CNS)-specific processes, such as myelin repair. Here we show that, in a mouse model of lysolecithin-induced demyelination of the subcortical white matter, post-injury exposure to fine PM hampers remyelination, disturbs oligodendroglia differentiation dynamics and promotes astroglia and microglia reactivity. These findings support the view that exposure to fine PM can contribute to demyelinating pathologies by targeting the endogenous regenerative capability of the CNS tissue.

Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins. ispartof: NEUROBIOLOGY OF AGING vol:92 ispartof: location:United States status: published

COVID‐19 infection can cause a severe pneumonia which, in some cases, can lead to admission in intensive care unit for respiratory support.1 In severe cases, systemic thrombotic complication has been described, including cerebrovascular disease (5.7‐23% of cases).2,3

The intracellular-ubiquitin-ending-enzyme tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is a potent inhibitor of the pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway. Single nucleotide polymorphisms in TNFAIP3 locus have been associated to autoimmune inflammatory disorders, including Multiple Sclerosis (MS). Previously, we reported a TNFAIP3 down-regulated gene expression level in blood and specifically in monocytes obtained from treatment-na&iuml, ve MS patients compared to healthy controls (HC). Myeloid cells exert a key role in the pathogenesis of MS. Here we evaluated the effect of specific TNFAIP3 deficiency in myeloid cells including monocytes, monocyte-derived cells (M-MDC) and microglia analyzing lymphoid organs and microglia of mice. TNFAIP3 deletion is induced using conditional knock-out mice for myeloid lineage. Flow-cytometry and histological procedures were applied to assess the immune cell populations of spleen, lymph nodes and bone marrow and microglial cell density in the central nervous system (CNS), respectively. We found that TNFAIP3 deletion in myeloid cells induces a reduction in body weight, a decrease in the number of M-MDC and of common monocyte and granulocyte precursor cells (CMGPs). We also reported that the lack of TNFAIP3 in myeloid cells induces an increase in microglial cell density. The results suggest that TNFAIP3 in myeloid cells critically controls the development of M-MDC in lymphoid organ and of microglia in the CNS.

Autologous hematopoietic stem cell transplantation (AHSCT) has been used in the treatment of highly active multiple sclerosis (MS) for over two decades. It has been demonstrated to be highly efficacious in relapsing–remitting (RR) MS patients failing to respond to disease-modifying drugs (DMDs). AHSCT guarantees higher rates of no evidence of disease activity (NEDA) than those achieved with any other DMDs, but it is also associated with greater short-term risks which have limited its use. In the 2019 updated EBMT and ASBMT guidelines, which review the clinical evidence of AHSCT in MS, AHSCT indication for highly active RRMS has changed from “clinical option” to “standard of care”. On this basis, AHSCT must be proposed on equal footing with second-line DMDs to patients with highly active RRMS, instead of being considered as a last resort after failure of all available treatments. The decision-making process requires a close collaboration between transplant hematologists and neurologists and a full discussion of risk–benefit of AHSCT and alternative treatments. In this context, we propose a standardized protocol for decision-making and informed consent process.

Background Fingolimod is the first oral agent approved for treatment of relapsing-remitting multiple sclerosis (RRMS). We aimed to evaluate fingolimod effectiveness in a real-world sample of RRMS patients. Methods A retrospective, multicentre study in patients treated with fingolimod, whom clinical and radiological data were collected in the 2 years preceding and following the initiation of fingolimod. Results Out of 414 patients, 56.8% received prior first-line injectable disease-modifying therapies, 25.4% were previously treated with natalizumab, 1.2% with immunosuppressant agents, and 16.7% were treatment naive. The annualized relapse rate decreased by 65% in the first year and by 70% after two years of treatment. Age ≤ 40 years, ≥ 1 relapse in the 24 months before fingolimod initiation and previous treatment with natalizumab were risk factors for relapses. Overall, 67.9% patients had no evidence of disease activity (NEDA-3) after 1 year and 54.6% after 2 years of treatment. A higher proportion of naïve (81.2% in 1 year and 66.7% after 2 years) or first-line injected patients (70.2% and 56.6%) achieved NEDA-3 than those previously treated with natalizumab (54.3% and 42.9%). Conclusions Fingolimod appeared to be effective in naive patients and after first-line treatment failure in reducing risk of relapse and disease activity throughout the 2-year follow-up.

Fatigue is one of the most distressing symptoms of Multiple Sclerosis, impairing quality of life, work performance and social interactions. Moreover, it is difficult to objectively assess, and is often evaluated via subjective questionnaire; however, objective metrics are highly desirable. Objective of this paper is to implement a differential fatigue measure based on evoked potentials, and assess its reliability and coherence with walking tests and subjective questionnaires. Method: the Regional Multiple Sclerosis Reference Centre, San Luigi Gonzaga Hospital, Italy, is carrying out tests in order to assess the effectiveness of 4-amino-pyridine for fatigue relief. This work takes as input evoked potential waveforms and implements an algorithm to perform suitable signal processing and provide a fatigue index and a related reliability metric. This information is put in relationship with the walking test results and the subjective fatigue scores. Preliminary results reveal that fatigue measures based on evoked potentials, subject to proper data processing, are not always coherent with subjective questionnaires and walking tests. This may be due to the fact that fatigue cannot be reduced to a mere muscular/conduction problem, and the walking tests are heavily conditioned by the disability degree. Moreover, the reliability of such measures carries not trivial information that should be carefully considered. As a conclusion, evoked potentials and gait analysis represent a good complement of subjective questionnaires as for fatigue assessment. The significance of our work lies in the fact that reliable fatigue measures can help improving the patients’ quality of life, allowing assessment of the therapy effectiveness and posology.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects both lower and upper motor neurons (MNs) in the central nervous system. ALS etiology is highly multifactorial and multifarious, and an effective treatment is still lacking. Neuroinflammation is a hallmark of ALS and could be targeted to develop new therapeutic approaches. Interestingly, the transcription factor Nurr1 has been demonstrated to have an important role in the inflammatory process in several neurological disorders, such as Parkinson's disease and multiple sclerosis. In the present paper, we demonstrate for the first time that Nurr1 expression levels are upregulated in the peripheral blood of ALS patients. Moreover, we investigated Nurr1 function in the SOD1-G93A mouse model of ALS. Nurr1 was strongly upregulated in the spinal cord during the asymptomatic and early symptomatic phases of the disease, where it promoted the expression of brain-derived neurotrophic factor mRNA and the repression of NFκB pro-inflammatory targets, such as inducible nitric oxide synthase. Therefore, we hypothesize that Nurr1 is activated in an early phase of the disease as a protective endogenous anti-inflammatory mechanism, although not sufficient to reverse disease progression. On the basis of these observations, Nurr1 could represent a potential biomarker for ALS and a promising target for future therapies., Summary: We hypothesize that the transcription factor Nurr1 is activated in the early phase of the neurodegenerative disease amylotrophic lateral sclerosis (ALS), probably as a neuroprotective endogenous mechanism. Nurr1 might represent a promising target for ALS therapy.

Background. The intracellular ubiquitin-ending enzyme TNFAIP3 is one of the most potent inhibitor of the pro-inflammatory NF-kB pathway. Single nucleotide polymorphisms in the TNFAIP3 locus have been associated to autoimmune inflammatory disorders, including Multiple Sclerosis (MS). Previously, we reported a TNFAIP3 down-regulated gene expression level in blood and specifically in CD14+ monocytes obtained from treatment naïve MS patients in comparison to healthy controls (HC). Notably, myeloid cells which include monocytes, exert a key role in the neuro-inflammatory pathogenic process of MS.Methods. Here, we evaluated the effect of the specific TNFAIP3 deficiency in myeloid cells including monocytes, monocyte-derived cells (M-MDC) and microglia analyzing the lymphoid organs and central nervous system (CNS) of experimental mice. The TNFAIP3 deletion is induced using conditional knock-out mice for the myeloid lineage. Flow cytometry and histological procedures were applied to evaluate the immune cell population of spleen, lymph nodes and bone marrow and the microglial cell density in CNS, respectively.Results. Here, we found that the deletion of TNFAIP3 in myeloid cells induces a reduced body weight, a decrease in the percentage number of M-MDC and of common monocyte and granulocyte precursor cells. We also reported that the deletion of TNFAIP3 in myeloid cells reports an increased microglial cell density in brain.Conclusions. Collectively, the results suggest that the presence of TNFAIP3 in myeloid cells critically controls the development of M-MDC in lymphoid organ and of microglia in brain.

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: We recently devised a shortened version of the 54-item Multiple Sclerosis Quality of Life (MSQOL-54) in paper (MSQOL-29, consisting of 25 items forming 7 subscales and 4 single items, and one filter question for 3 ‘sexual function’ items) and electronic format (eMSQOL-29). Objectives: To prospectively assess eMSQOL-29 psychometric properties, acceptability/equivalence versus MSQOL-29. Methods: Multiple sclerosis (MS) patients ( n = 623; Expanded Disability Status Scale (EDSS) range 0.0–9.0) completed eMSQOL-29, Hospital Anxiety and Depression Scale, Functional Assessment of MS (FAMS), European Quality of life Five Dimensions-3L, and received EDSS and Symbol Digit Modalities Test (SDMT). Equivalence versus MSQOL-29 was assessed in 242 patients (randomized cross-over design). Results: ‘Sexual function’ items were filtered out by 273 patients (47%). No multi-item scale had floor effect, while five had ceiling effect. Cronbach’s alpha range was 0.88–0.90. Confirmatory factor analysis showed good overall fit and the two-factor solution for composite scores was confirmed. Criterion validity was sub-optimal for ‘cognitive function’ (vs SDMT, r = 0.25) and ‘social function’ (vs FAMS social function, r = 0.38). eMSQOL-29 equivalence was confirmed and its acceptability was good. Conclusion: eMSQOL-29 showed good internal consistency, factor structure and no floor effect, while most subscales had some ceiling effect. Criterion validity was sub-optimal for two subscales. Equivalence and acceptability were good.

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.

This document presents the guidelines for the cerebrospinal fluid (CSF) analysis and the determination of oligoclonal bands (OCBs) as pivotal tests in neuroin flammatory pathologies of the central nervous system. The guidelines have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Essential clinical information on the pathologies in which the CSF analysis is indicated, and, particularly, on those characterized by the presence of OCBs in the intrathecal compartment, indications and limits of CSF analysis and OCB determination, instructions for result interpretation, and agreed laboratory protocols (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

Prognostic markers of primary progressive multiple sclerosis evolution are needed. We investigated the added value of magnetic resonance imaging measures of brain and cervical cord damage in predicting long-term clinical worsening of primary progressive multiple sclerosis compared to simple clinical assessment. In 54 patients, conventional and diffusion tensor brain scans and cervical cord T1-weighted scans were acquired at baseline and after 15 months. Clinical evaluation was performed after 5 and 15 years in 49 patients. Lesion load, brain and cord atrophy, mean diffusivity and fractional anisotropy values from the brain normal-appearing white matter and grey matter were obtained. Using linear regression models, we screened the clinical and imaging variables as independent predictors of 15-year disability change (measured on the expanded disability status scale). At 15 years, 90% of the patients had disability progression. Integrating clinical and imaging variables at 15 months predicted disability changes at 15 years better than clinical factors at 5 years (R2 = 61% versus R2 = 57%). The model predicted long-term disability change with a precision within one point in 38 of 49 patients (77.6%). Integration of clinical and imaging measures allows identification of primary progressive multiple sclerosis patients at risk of long-term disease progression 4 years earlier than when using clinical assessment alone.

International audience; Multiple sclerosis is a heterogenous disease. Although several EMA-approved disease-modifying treatments including biopharmaceuticals are available, their efficacy is limited, and a certain percentage of patients are always nonresponsive. Drug efficacy monitoring is an important tool to identify these nonresponsive patients early on. Currently, detection of antidrug antibodies and quantification of biological activity are used as methods of efficacy monitoring for interferon beta and natalizumab therapies. For natalizumab and alemtuzumab treatments, drug level quantification could be an essential component of the overall disease management. Thus, utilization and development of strategies to determine treatment response are vital aspects of multiple sclerosis management given the tremendous clinical and economic promise of this tool.

This is the accepted version of the following article: "Rationale for Therapeutic Drug Monitoring of Biopharmaceuticals in Inflammatory Diseases", which has been published in final form at https://insights.ovid.com/crossref?an=00007691-201708000-00007; International audience; Biopharmaceuticals bring together a number of specific characteristics as compared with other drugs. However, as it is done for most drugs, an individual adjustment of their dose may be necessary. Similar to “chemical” drugs, biopharmaceuticals used in immunoinflammatory diseases have a rather narrow therapeutic range, lack good early clinical or biological marker of response, have variable pharmacokinetics, and their serum concentrations are most often related with response. Monoclonal antibodies have additional specific sources of pharmacokinetic variability. Low concentrations may increase the risks of immunization, plasmapheresis may increase their elimination, and subcutaneous formulations may be associated with decreased adherence. For all these reasons, pharmacokinetic therapeutic drug monitoring may be useful. However, few randomized controlled therapeutic drug monitoring studies have been published. For monoclonal antibodies, a precise definition of the therapeutic concentrations is challenging because of the interindividual variability in their concentration–effect relationship.

Economic sustainability is of paramount importance in the rapidly evolving therapeutic scenario of multiple sclerosis (MS). Glatiramoids are a class of drugs whose forefather, glatiramer acetate, has been used as a disease modifying drug (DMD) in patients with MS for over 20 years. Its patent expired in 2015; new versions of such drug are nowadays available on the market, potentially contributing to lowering prices and enhancing a better allocation of economic resources. In this review, we analyze the recommendations underlying the approval of both generic drugs and biosimilars by regulatory authorities, and we provide methodological tools to contextualize the design of studies on these new classes of drugs. We examine in more detail the preclinical and clinical data of Copemyl®, a new member of the glatiramoid class, focusing on its biological and immunological properties and illustrating randomized controlled trials that led to its authorization.

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.

Chronic inflammation significantly contributes to the pathogenesis of several neurodegenerative disorders. In physiological conditions, a chronic inflammatory state is prevented through the termination of the acute inflammatory response once the triggering insult is eliminated. Several mechanisms regulate the resolution of inflammation. Among these, a potent inhibitor of the pro-inflammatory NF-kB signaling known as A20 has emerged as a key player. Recent studies have shown reduced blood levels of A20 in the patients of diverse chronic inflammatory diseases. Similar results have also been demonstrated in patients of multiple sclerosis (MS), a neurodegenerative disease characterized by persisting inflammation. In the present study, we investigate whether other similar neurodegenerative disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) also demonstrate deregulated levels of A20 expression as compared to healthy controls (HC) and treatment-naive MS patients. Our results confirm previous data that the A20 expression is reduced in whole blood of MS patients as compared to HC. Additionally, we demonstrate that significantly diminished A20 expression is also evident in PD patients. The dysregulation of the A20 pathway could then contribute to the persistence of inflammation in these disorders. It would thus be interesting to investigate further whether such commonly deregulated pathways between different inflammatory diseases could represent novel targets for therapy.