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The past decade has seen extraordinary increase in worldwide availability of and access to several large multiple sclerosis (MS) databases and registries. MS registries represent powerful tools to provide meaningful information on the burden, natural history, and long-term safety and effectiveness of treatments. Moreover, patients, physicians, industry, and policy makers have an active interest in real-world observational studies based on register data, as they have the potential to answer the questions that are most relevant to daily treatment decision-making. In 2014, the Italian MS Foundation, in collaboration with the Italian MS clinical centers, promoted and funded the creation of the Italian MS Register, a project in continuity with the existing Italian MS Database Network set up from 2001. Main objective of the Italian MS Register is to create an organized multicenter structure to collect data of all MS patients for better defining the disease epidemiology, improving quality of care, and promoting research projects in high-priority areas. The aim of this article is to present the current framework and network of the Italian MS register, including the methodology used to improve the quality of data collection and to facilitate the exchange of data and the collaboration among national and international groups. The past decade has seen extraordinary increase in worldwide availability of and access to several large multiple sclerosis (MS) databases and registries. MS registries represent powerful tools to provide meaningful information on the burden, natural history, and long-term safety and effectiveness of treatments. Moreover, patients, physicians, industry, and policy makers have an active interest in real-world observational studies based on register data, as they have the potential to answer the questions that are most relevant to daily treatment decision-making. In 2014, the Italian MS Foundation, in collaboration with the Italian MS clinical centers, promoted and funded the creation of the Italian MS Register, a project in continuity with the existing Italian MS Database Network set up from 2001. Main objective of the Italian MS Register is to create an organized multicenter structure to collect data of all MS patients for better defining the disease epidemiology, improving quality of care, and promoting research projects in high-priority areas. The aim of this article is to present the current framework and network of the Italian MS register, including the methodology used to improve the quality of data collection and to facilitate the exchange of data and the collaboration among national and international groups.

In this retrospective, multicenter, real-world study we collected clinical and magnetic resonance imaging (MRI) data of all patients (n = 40) with relapsing-remitting multiple sclerosis (RRMS) treated with alemtuzumab according to a "free-of-charge" protocol available before the drug marketing approval in Italy. Almost all (39/40) started alemtuzumab after discontinuing multiple disease-modifying treatments (DMTs) because of either lack of response or safety concerns. We considered the proportion of alemtuzumab-treated patients who had no evidence of disease activity (NEDA-3) and disability improvement over a 36-month follow-up period. NEDA-3 was defined as absence of relapses, disability worsening, and MRI activity. Disability improvement was defined as a sustained reduction of ≥ 1-point in Expanded Disability Status Scale (EDSS) score. At follow-up, 18 (45%) patients achieved NEDA-3, 30 (75%) were relapse-free, 33 (82.5%) were EDSS worsening-free, and 25 (62.5%) were MRI activity-free. Eleven (27.5%) patients had a sustained disability improvement. We found no predictor for the NEDA-3 status, while the interaction of higher EDSS score by higher number of pre-alemtuzumab relapses was associated with a greater chance of disability improvement (odds ratio 1.10, p = 0.049). Our study provides real-world evidence that alemtuzumab can promote clinical and MRI disease remission, as well as disability improvement, in a significant proportion of patients with RRMS despite prior multiple DMT failures. The drug safety profile was consistent with data available from clinical trials.

Background: Natalizumab is a promising option for pediatric multiple sclerosis (MS) patients with active evolution and a poor response to Interferon-beta or Glatiramer Acetate. However, no data are available in large cohorts of patients and after a long-term follow up. Our study was planned to shed lights on this topic. Methods: A registry was established in 2007 in Italy to collect MS cases treated with Natalizumab (NA) before 18 years of age. Results: 101 patients were included (69 females), mean age of MS onset 12.9±2.7 years, mean age at NA initiation 14.7±2.4 years. Mean treatment duration was 34.2±18.3 months. During NA treatment, a total of 15 relapses were recorded in 9 patients, annualized relapse rate was 2.3±1.0 in the year prior to NA and decreased to 0.1±0.3 (p

We measured the concentrations of 21 chemical elements (TE) in hair of 48 Sicilian patients with RRMS compared with 51 controls (HC). MS patients showed a significantly (p

Tissue inhibitor of metalloproteinases I (TIMP-1) inhibits several proteinases including a disintegrin and metalloproteinase 10 (ADAM10), a major alpha-secretase that cleaves the beta-amyloid precursor protein within its amyloidogenic Abeta domain. The gene encoding TIMP-1 (TIMP1) maps to the short arm of the X chromosome, in a region previously suggested as conferring genetic susceptibility for Alzheimer’s disease (AD). To determine whether genetic variability of TIMP1 contributes to the pathogenesis of AD, we analysed one single nucleotide polymorphism within TIMP1 and one single nucleotide polymorphism in the 5’-untranslated region of TIMP1 in patients with AD and control subjects from two independent and ethnically different populations. We did not observe any association between TIMP1 genotypes and the diagnosis of AD in men or women. We also measured TIMP-1 protein levels in the cerebrospinal fluid of patients with AD, healthy control subjects, and patients with other neurological disorders. TIMP-I levels were similar in all groups. In addition, no significant differences were observed after stratification for TIMP1 genotypes. Our data show that neither genetic variability nor protein levels of TIMP-1 are associated with AD. (C) 2002 Lippincott Williams Wilkins.

Plasma levels of al-antichymotrypsin (ACT) and interleukin-1 beta (IL-1 beta) were increased in patients with probable Alzheimer's disease (AD). A common polymorphism within ACT and IL-IP genes affected plasma levels of ACT or IL-1 beta, and AD patients with the ACT T,T or IL-1 beta T,T genotype show-ed the highest levels of plasma ACT or IL-1 beta, respectively. The concomitant presence of the ACT T,T and IL-1 beta T,T genotypes increased the risk of AD (odds ratio: 5.606, confidence interval: 1.654-18.996) and decreased the age at onset of the disease

Overexpression of the pluripotent cytokine interleukin-l (IL-I) by microglial cells correlates with formation of neuritic P-amyloid plaques in Alzheimer's disease (AD). We evaluated polymorphisms in the genes coding for the IL-1 alpha, IL-IP, and IL-1 receptor antagonist cytokines, and tested their association with the occurrence and age at onset of sporadic AD. We found a strong association between the IL-IA T/T genotype and AD onset before 65 years of age (odds ratio, 4.86), with carriers of this genotype showing an onset of disease 9 years earlier than IL-1A CIC carriers. A weaker association with the age at onset was also shown for the IL-1B and IL-1RN genes. These data suggest either a direct effect of the IL-I gene family, mainly IL-1A, on the clinical onset of AD, or a linkage dysequilibrium with an unknown locus relevant to AD on chromosome 2.

Teriflunomide (TRF) and Dimethyl fumarate (DMF) are licensed drugs for relapsing-remitting Multiple Sclerosis (RRMS). We aimed to compare the rate and the time to discontinuation among persons with RRMS (pwRRMS), newly treated with TRF and DMF. A retrospective study on prospectively collected data was performed in nine tertiary MS centers, in Italy. The 24-month discontinuation rate in the two cohorts was the primary study outcome. We also assessed the time to discontinuation and reasons of therapy withdrawn. Discontinuation of TRF and DMF was defined as a gap of treatment ≥ 60 days. A cohort of 903 pwRRMS (316 on TRF and 587 on DMF) was analyzed. During 24 months of follow-up, pwRRMS on TRF and DMF showed similar discontinuation rates. The analysis of predictors with Cox regression model showed differences between the two groups (p for log-rank test = 0.007); male gender [HR 2.21 (1.00–4.90); p = 0.01] and the number of previous switches [HR 1.47 (1.16–1.86); p = 0.01] were associated with higher hazard of discontinuation in the DMF group. In a real-world setting, pwRRMS on TRF and DMF had similar discontinuation rates over 24 months. Male pwRRMS on DMF with a previous history of therapeutic failure are at more risk of discontinuation therapy.

BACKGROUND: In patients with relapsing-remitting MS (RRMS) fingolimod prevents disease relapses and delays disability progression. First dose administration of fingolimod is associated with a transient, dose-dependent decrease in heart rate (HR) in the 6 hours after drug intake.The aim of the study is to to assess safety and tolerability of the first dose of fingolimod in a cohort of Italian patients with RRMS without alternative therapeutic options. METHODS: Open-label, single arm, multicentre study. After the first dose of fingolimod, patients were observed for 6 hours and had their vital signs monitored hourly. Extended on-site monitoring was provided when required. RESULTS: Of the 906 patients enrolled in the study, most (95.2%) did not experience any adverse event (AE) following fingolimod administration. Cardiovascular AEs occurred in 18 patients and included bradycardia (1.3%), first-and second-degree atrioventricular block (0.1% and 0.2%), palpitations (0.1%), sinus arrhythmia (0.1%) and ventricular premature beats (0.1%). All events were self-limiting and did not require any intervention. Extended monitoring was required in 34 patients. CONCLUSIONS: These results, in a population who better resembled real-world clinical practice in terms of concomitant diseases and medications, are consistent with previous clinical trials and confirmed that the first dose administration of fingolimod is generally safe and well tolerated. TRIAL REGISTRATION: EudraCT 2011-000770-60.

Objective Thiamine or vitamin B 1 deficiency diminishes thiamine-dependent enzymatic activity, alters mitochondrial function, impairs oxidative metabolism, and causes selective neuronal death. We analyzed for the first time, the role of all known mutations within three specific thiamine carrier genes, SLC19 A2, SLC19 A3 , and SLC25 A19 , in a patient with atrophic beriberi, a multiorgan nutritional disease caused by thiamine deficiency. Methods A 44-year-old male alcoholic patient from Morocco developed massive bilateral leg edema, a subacute sensorimotor neuropathy, and incontinence. Despite normal vitamin B 1 serum levels, his clinical picture was rapidly reverted by high-dose intramuscular thiamine treatment, suggesting a possible genetic resistance. We used polymerase chain reaction followed by amplicon sequencing to study all the known thiamine-related gene mutations identified within the Human Gene Mutation Database. Results Thirty-seven mutations were tested: 29 in SLC19 A2, 6 in SLC19 A3, and 2 in SLC25 A19 . Mutational analyses showed a wild-type genotype for all sequences investigated. Conclusion This is the first genetic study in beriberi disease. We did not detect any known mutation in any of the three genes in a sporadic dry beriberi patient. We cannot exclude a role for other known or unknown mutations, in the same genes or in other thiamine-associated genes, in the occurrence of this nutritional neuropathy.

Multiple sclerosis (MS) is a T cell-mediated organ-specific inflammatory disease leading to central nervous system (CNS) demyelination. On the basis of results obtained in experimental autoimmune encephalomyelitis (EAE) models, MS treatment by administration of antiinflammatory cytokines such as interleukin 4 (IL-4) is promising but is hampered by the limited access of the cytokines to the CNS and by the pleiotropic effects of systemically administered cytokines. We established a cytokine delivery system within the CNS using non-replicative herpes simplex type 1 (HSV-1) viral vectors engineered with cytokine genes. These vectors injected into the cisterna magna (i.c.) of mice diffuse in all ventricular and subarachnoid spaces and infect with high efficiency the ependymal and leptomeningeal cell layers surrounding these areas, without obvious toxic effects. Heterologous genes contained in the vectors are efficiently transcribed in infected ependymal cells, leading to the production of high amounts of the coded proteins. For example, 4.5 ng of interferon gamma (IFN-gamma) per milliliter is secreted into the cerebrospinal fluid (CSF) up to day 28 postinjection (p.i.) and reaches the CNS parenchyma in bioactive form, as demonstrated by upregulation of MHC class I expression on CNS-resident cells. We then exploited the therapeutic potential of the vectors in EAE mice. An HSV-1-derived vector containing the IL-4 gene was injected i.c. in Biozzi AB/H mice at the time of EAE induction. We found the following in treated mice: (1) delayed EAE onset, (2) a significant decrease in clinical score, (3) a significant decrease in perivascular inflammatory infiltrates and in the number of macrophages infiltrating the CNS parenchyma and the submeningeal spaces, and (4) a reduction in demyelinated areas and axonal loss. Peripheral T cells from IL-4-treated mice were not affected either in their antigen-specific proliferative response or in cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1 vectors is feasible and might represent an approach for the treatment of demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached in the CNS, the absence of effects on the peripheral immune system, and the long-lasting cytokine production in the CNS after a single vector administration.

A successful gene therapy approach in organ-specific autoimmune diseases, such as multiple sclerosis (MS), encompasses the inhibition of the autoreactive T cells or the modification of the target organ cells by the introduction of exogenous `protective' genes. In MS, an autoimmune disease of the central nervous system (CNS), the inciting autoantigen is still unknown and therefore the isolation of autoreactive T cells may only be inferential. At present, gene therapy approaches in MS should therefore aim to the modification of the target organ. Possible candidate genes to be transferred within the CNS of MS patients are those coding for anti-inflammatory cytokines (i.e. interleukin-4, interleukin-10, transforming growth factor b) which have been shown to ameliorate demyelinating diseases at least in experimental models. However, a limiting factor for this therapy is the difficulty to reach the CNS. A gene therapy approach using viral vectors able to infect post-mitotic cells, such as those present within the CNS, without inducing toxic reactions, may overcome this limitation. We propose to use non-replicative herpetic vectors, which represent a viable gene-transfer alternative to the classical retroviral and adenoviral vectors. Key advantages are their size, able to accommodate multiple foreign genes, and their ability to infect post-mitotic cells such as those present within the CNS. We first transferred a gene coding for interleukin-4 within the CNS of mice undergoing experimental allergic encephalomyelitis, an animal model for MS, using non-replicative Herpes Simplex Virus type 1- derived vectors. We found that this approach ameliorates the disease course and delays the disease onset. The establishment of this technique to deliver anti-inflammatory cytokines within the CNS using herpetic vectors should clarify the role of individual cytokines in the demyelinating process and allow assessment of whether gene therapy using herpetic vectors is a feasible and safe approach to treat human demyelinating disorders.

Central nervous system (CNS) lesions typical of multiple sclerosis (MS) are characterized by demyelinating inflammatory infiltrates that contain few CNS antigen-specific autoreactive T cells and a multitude of pathogenic non-antigen-specific mononuclear cells. Here, we report that in patients with MS the combined action of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-2, and IL-6 leads to the activation of most peripheral T cells (mainly CD4 memory) by promoting a persistent intracellular calcium increase via two independent signaling pathways. The activation of these pathways, one activated by IFNgamma and the other by the combination TNFalpha/IL-2/IL-6, is independent from myelin antigens and precedes by 2 weeks phases of disease activity (eg, clinical relapses and/or appearance of gadolinium-enhancing lesions on brain magnetic resonance imaging scans during 1 year of follow-up). Our results indicate that an appropriate combination of the four cytokines, three with a proinflammatory profile and one necessary for T-cell growth and differentiation, can activate in an antigen-independent fashion most peripheral T cells from MS patients. This mechanism is likely to contribute to the recruitment of nonspecific lymphocytes into the cellular activation processes leading to CNS demyelination and may represent a major target for immune intervention in MS.

In the attempt to further characterize the extent and timing of tumor necrosis factor (TNF)alpha-system activation during multiple sclerosis (MS), we performed a cross-sectional and a longitudinal study in a total of 73 relapsing-remitting MS patients. We assessed serum levels of soluble TNF alpha, soluble TNF alpha receptor 1 (R1) and soluble TNF alpha receptor 2 (R2) in 65 relapsing-remitting MS patients in different phases of disease. TNF alpha, R1 and R2 serum levels measured in MS patients did not differ from those measured in healthy individuals and did not correlate with (a) clinical relapses, (b) presence of gadolinium-enhancing brain-magnetic resonance imaging (MRT) lesions, and (c) bioactivity of TNF alpha. We also measured in 8 additional relapsing-remitting MS patients peripheral blood mononuclear cells (PBMC) mRNA levels of TNF alpha, R1 and R2 every 15 days for one year. In 4 of these patients we also measured levels of soluble TNF alpha, R1 and R2 every 15 days for 5 months across a clinical exacerbation. PBMC TNF alpha, R1 and R2 mRNA levels and serum levels of soluble R1 and R2, but not TNF alpha, fluctuated concordantly (P

Human T-cell lymphotropic virus type-I (HTLV-I) is the etiologic agent of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL). HAM/TSP and ATL occur infrequently among HTLV-I-infected individuals, and rarely develop in the same individual. To study host and viral factors involved in the induction, tissue tropism, as well as pathogenesis of HAM/TSP, peripheral blood lymphocytes (PBL) from 14 patients with HAM/TSP and from 9 controls were introduced into severe combined immunodeficiency (SCID) mice by intraperitoneal injection. Mice were followed for up to 26 weeks. Human IgG was produced from 2 to 14 weeks after reconstitution in all animals. Thirty-two of 44 mice (72%) showed circulating human antibody against the major viral protein products of HTLV-I. Analysis of viral sequences by polymerase chain reaction (PCR) demonstrated HTLV-I sequences in 21/38 (55%) brains and in 7/17 (41%) spinal cords from HTLV-I-hu SCID mice. No animal had clinical evidence of neurological impairment or pathological findings similar to those seen in HAM/TSP. Seven mice who received PBL from Epstein Barr virus (EBV)-seropositive patients developed an intraperitoneal lymphoma. In 2 mice an infiltration of brain by a lymphoblastic tumor of B/T cell type was observed. By PCR, all the tumors were EBV-positive; HTLV-I sequences were detected in 5 of them. Our study suggests that the HTLV-I-hu-SCID mouse provides a potentially valuable system for studying the production, kinetics, and pathogenicity of anti-HTLV-I antibody, and may help clarify the interaction of EBV and retroviruses in the development of disease.

Intracellular Ca2+ stores were investigated in resting and activated splenic T lymphocytes from Lewis rats. Activation was obtained either in vitro (spleen cells isolated from "naive" rats exposed to concanavalin A for 24 h) or in vivo (spleen cells from rats with fully developed symptoms of experimental allergic encephalomyelitis). In both experimental conditions several changes of Ca2+ homeostasis were observed with respect to resting lymphocytes: (1) a threefold increase of the total intracellular calcium (from 1.15 to 3.5 mmol/l); (2) a moderate increase of the pool sensitive to inositol 1,4,5-trisphosphate (IP3), investigated both in intact T lymphocytes (fura-2 and 45Ca(2+)-release techniques in cells challenged with phytohemagglutinin) and in T lymphocytes permeabilized with beta-escin (45Ca2+ release induced by saturating concentrations of IP3); and (3) the appearance of a pool released by the endoplasmic reticulum (ER) Ca2+ ATPase inhibitor thapsigargin (Tg), but insensitive to IP3, which, therefore, appears to be localized in areas of the ER devoid of the cognate receptor. The latter two findings were paralleled in activated lymphocytes by an increase of expression of ER markers, involved (calreticulin; Ca2+ ATPase) or not (protein disulfide isomerase) in the regulation of Ca2+ homeostasis. In contrast, calnexin (another ER marker) and the receptor for IP3 were increased to only a moderate extent. Finally, an enlargement of non-ER Ca2+ pools was observed in the cells pretreated with Tg in which 45Ca2+ release was induced by the Ca2+ ionophore ionomycin. Our results document structural and functional changes of intracellular Ca2+ stores which might play an important regulatory role in activated T lymphocytes.

We have established a new chimeric human-mouse model of myasthenia gravis in severe combined immunodeficiency mice, using human peripheral blood lymphocytes that survive in the mouse and produce specific antibodies that mediate pathological changes typical of human myasthenia gravis. Mice given peripheral blood lymphocytes from both anti-acetylcholine receptor (AChR) antibody-positive and -negative patients with myasthenia gravis showed circulating anti-acetylcholine receptor antibodies, deposition of human IgG at muscle end-plates, and simplification of the postsynaptic membrane, findings characteristic of human myasthenia gravis. Mice given human peripheral blood lymphocytes from healthy donors and simultaneously immunized with Torpedo acetylcholine receptor showed the same changes. This chimeric model, utilizing human cells to reproduce the immunopathological findings of human myasthenia gravis in a nonhuman environment, offers new opportunities to study immune regulation in autoimmunity.

BackgroundAlbeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis.Methodology/principal findingsImmunoproteasomes and PA28-alphabeta regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP(111-119).Conclusion/significanceThe immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis.

OBJECTIVE: To describe the effect of natalizumab in the treatment of subjects with active multiple sclerosis (MS) treated before the age of 18 years. METHODS: Nineteen pediatric subjects with MS (mean age 14.6 +/- 2.2 years, mean number of attacks 5.2 +/- 1.9 during the pretreatment phase of 27.7 +/- 19.7 months, median pretreatment Expanded Disability Status Scale score [EDSS] 2.5, range 1.0-5.0) were treated with natalizumab at the dose of 300 mg every 28 days. After treatment initiation, patients were reassessed clinically every month; brain MRI was performed at baseline and every 6 months. RESULTS: Patients received a median number of 15 infusions (range 6-26). A transient reversible worsening of preexisting symptoms occurred in 1 subject during and following the first infusion. All the patients remained relapse-free during the whole follow-up. The median EDSS decreased from 2.5 to 2.0 at the last visit (p < 0.001). EDSS remained stable in 5 cases, decreased by at least 0.5 point in 6 cases, and decreased by at least 1 point in 8 cases. At baseline, the mean number of gadolinium-enhancing lesions was 4.1 (range 1-20). During the follow-up, no gadolinium-enhancing lesions were detected (p = 0.008); 3 patients developed new T2-visible lesions at month 6 scan but the overall number of T2 lesions remained stable during the subsequent follow-up. Transient and mild side effects occurred in 8 patients. CONCLUSIONS: Natalizumab was well-tolerated in all subjects. A strong suppression of disease activity was observed in all subjects during the follow-up. Classification of evidence: This study provides Class IV evidence that natalizumab, 300 mg IV once every 28 days, decreased EDSS scores in pediatric patients with MS over a mean treatment period of 15.2 months.

Cells secreting antibodies against guinea pig myelin and synthetic myelin basic protein (MBP) peptides were evaluated in cerebrospinal fluid (CSF) and blood from patients with multiple sclerosis (MS) and a variety of other neurological diseases (OND). The peptides used, reproducing amino acid sequences 1-20, 70-89, 108-126, or 157-166 of MBP, were selected on the basis of their hydrophilic and encephalitogenic properties. Low numbers of cells secreting IgG antibodies against myelin or each of the MBP peptides (about 1 per 50,000) were detected in peripheral blood, with no difference between MS and OND. In CSF, cells secreting IgG antibodies to MBP 70-89 were more frequently (p = 0.007) detected in patients with MS (1/380 IgG-secreting cells on average) than in patients with OND (1/2083 IgG-secreting cells on average). The frequencies of cells secreting antibodies against myelin or the three other MBP peptides were similar in MS and OND. Thus, evaluation of B cell immunity at the cellular level indicates that MBP 70-89 is an immunodominant B cell epitope in MS. It is not clear whether this intrathecal anti-MBP 70-89 IgG antibody response has any pathogenetic relevance in MS or is the result of myelin breakdown.