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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

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.

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.

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.

We measured kappa/lambda light chain ratios of Ig and IgG in 41 serum and 34 cerebrospinal fluid (CSF) samples from 47 patients at different clinical stages of human immunodeficiency virus type 1 (HIV-1) infection and in serum and CSF samples from control subjects. Both ratios were more elevated in HIV-1 seropositive subjects than controls. The elevation was more evident in samples from asymptomatic seropositive patients (ASP) than those from patients with acquired immunodeficiency syndrome (AIDS). In addition, there was a statistically significant elevation of Ig kappa/lambda ratios in ASP CSF compared to serum. We also delineated the light chain composition of oligoclonal IgG bands (OCB) by isoelectric focusing followed by immunofixation in CSF and serum samples from selected ASP and patients with AIDS who had neurological involvement. Five of six AIDS and all seven ASP samples had IgG OCB exclusively or predominantly of the kappa type. Four IgG OCB of the lambda type and one free lambda chain band were seen in CSF from a pediatric AIDS patient. The presence of an abnormally elevated kappa/lambda ratio correlated with the presence of IgG kappa OCB (p less than 0.02). We conclude that HIV-1 infection is associated with a kappa light chain predominance and with OCB mainly composed of kappa light chains.

Interferon-gamma (IFN-gamma) contributes to the early events leading to T cell activation in relapsing-remitting (RR) multiple sclerosis (MS) by activating a transplasmalemma calcium influx, the detection of which is closely associated with clinical and MRI evidence of disease activity. The appearance of this influx represents one of the earliest peripheral events in the pathogenesis of RRMS. It is still questioned whether the same immune mediated mechanisms also operate in primary progressive (PP)MS. Fluorimetric evidence of the IFN-gamma activated calcium influx was sought in 16 patients with PPMS and 39 patients with secondary progressive (SP)MS. To compare peripheral versus CNS evidence of immune activation 11 of the patients with PPMS and 27 of the patients with SPMS underwent gadolinium enhanced brain MRI. The IFN-gamma activated influx was detected in peripheral blood lymphocytes from eight of 16 (50%) patients with PPMS, and 20 of 39 (51%) patients with SPMS, a frequency similar to that previously reported in patients with RRMS during phases of disease stability. Gadolinium enhancing brain MRI lesions were found in only one of 11 (9%) patients with PPMS and 12 of 27 (41%) with SPMS. Our study shows that peripheral blood lymphocytes from patients with PPMS and patients with SPMS express with the same frequency as patients with RRMS, an IFN-gamma dependent intracellular process leading to T cell activation able to trigger disease activity.

IL-12 has been shown to be involved in the pathogenesis of Th1-mediated autoimmune diseases, but its role in antibody-mediated autoimmune pathologies is still unclear. We investigated the effects of exogenous and endogenous IL-12 in experimental autoimmune myasthenia gravis (EAMG). EAMG is an animal model for myasthenia gravis, a T cell-dependent, autoantibody-mediated disorder of neuromuscular transmission caused by antibodies to the muscle nicotinic acetylcholine receptor (AChR). Administration of IL-12 with Torpedo AChR (ToAChR) to C57BL/6 (B6) mice resulted in increased ToAChR-specific IFN-gamma production and increased anti-ToAChR IgG2a serum antibodies compared with B6 mice primed with ToAChR alone. These changes were associated with earlier and greater neurophysiological evidence of EAMG in the IL-12-treated mice, and reduced numbers of AChR. By contrast, when IL-12-deficient mice were immunized with ToAChR, ToAChR-specific Th1 cells and anti-ToAChR IgG2a serum antibodies were reduced compared to ToAChR-primed normal B6 mice, and the IL-12-deficient mice showed almost no neurophysiological evidence of EAMG and less reduction in AChR. These results indicate an important role of IL-12 in the induction of an antibody-mediated autoimmune disease, suggest that Th1-dependent complement-fixing IgG2a anti-AChR antibodies are involved in the pathogenesis of EAMG, and help to account for the lack of correlation between anti-AChR levels and clinical disease seen in many earlier studies.

BACKGROUND—Interferon (IFN)-γ exerts a multiplicity of actions potentially relevant for the pathogenesis of multiple sclerosis, including the expression of a transplasmalemma calcium (Ca2+) influx leading to an intracellular Ca2+ ([Ca2+]i) increase able to lower T lymphocyte threshold of excitability. It has been previously shown in a cross sectional cumulative study that this influx is associated with clinical and MRI evidence of disease activity. METHODS—To evaluate the temporal relation between disease activity and the IFN-γ activated Ca2+ influx in individual patients, a fluorimetric analysis was performed on peripheral blood lymphocytes from eight patients with relapsing-remitting multiple sclerosis every 15 days for one year. Results—Fluctuations of the influx were correlated with clinical events and monthly enhanced brain MRI. The influx was detected a mean of 10.4 (range 7-17) times per patient during our analysis. In 61% of the occasions, influx induced [Ca2+]i increases were recorded in each patient in more than two consecutive measurements, determining sustained [Ca2+]i increases lasting for a mean of 31.5 days. Peak [Ca2+]i increases preceded clinical attacks (P=0.04) or maximal detection of brain MRI enhancing lesions (P=0.05) by a mean of 30.8 and 34.2 days respectively. Spectral analysis of time series further indicated that the fluctuation frequency of [Ca2+]i increases due to the influx over time were superimposable on the appearance of new MRI lesions in all patients and confirmed that in two thirds of the patients these [Ca2+]i increases occurred significantly before (P

Antibodies to glutamic acid decarboxylase (GAD) are found in about 40% of patients with stiff man syndrome. A new assay involving immunoprecipitation of (125)I-glutamic acid decarboxylase was used to measure anti-GAD antibodies in 18 patients with stiff man syndrome. Of the eight serum samples from patients with stiff man syndrome, that had previously been found positive by immunoprecipitation of (35)S-GAD, seven were strongly positive with (125)I-GAD and one gave an equivocal result. Other serum samples from patients with stiff man syndrome and from controls were negative except one from a patient who had a thymoma, acquired neuromyotonia, and myasthenia gravis. Nine of 35 serum samples referred for testing were positive; in two of these the serum titre was 20-50 times higher than that in the CSF. This assay should prove useful in the diagnosis, management, and investigation of stiff man syndrome.

The study of the origin and pathogenetic relevance of the oligoclonal antibodies present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients has been hampered by a lack of specific ligands. We recently reported a general strategy, based on phage-displayed random peptide libraries, to identify ligands for disease-specific antibodies even in the absence of any information on the nature of the pathologic antigen. With this procedure, we identified several peptides specifically recognized by antibodies present in the CSF of MS patients. Using these peptides as reagents, we demonstrated that they mimic different natural epitopes and react with antibodies enriched in the CSF of MS patients. Antibodies recognizing the selected peptides are commonly found with equal frequency in the sera of MS patients and of normal individuals. In contrast, the repertoire of CSF antibodies appears to be individual-specific and is probably the result of a nonspecific immunodysregulation rather than a stereotyped response to a single antigen/agent.

The pathogenetic role of anti-glutamic acid decarboxylase (GAD) antibodies found in up to 60% of patients with stiff-man syndrome (SMS) is still controversial. GAD, in fact, is also one of the major target antigen of insulin-dependent diabetes mellitus (IDDM), a disease affecting one third of anti-GAD antibody-positive patients with SMS. To better define the role of autoimmunity in SMS we looked for molecular and immunological evidence of an autoimmune recognition of a second IDDM-associated autoantigen, the pancreatic 37/40 kDa IDDM-autoantigen, whose gene called ICA 105 has been recently cloned. By Northern blot analysis we found that tissue distribution of human ICA 105 is restricted to pancreas and brain and within the central nervous system (CNS) its distribution is similar to GAD. We also measured anti-ICA 105 antibodies in 11 SMS patients and 56 control patients with other neurological diseases (OND). Anti-ICA 105 antibodies were found in 4/11 (36%) patients with SMS (a frequency similar to that of anti-GAD-antibodies in our SMS population) but in only 2/56 (3%) patients with OND (P0.001). Anti-ICA 105 and anti-GAD antibodies were associated in 3/4 (75%) patients with SMS but in none of the 2 anti-ICA 105 antibody-positive OND patients. Among anti-ICA 105 antibody-positive patients with SMS, only 1 suffered also from IDDM. In contrast, the only 2 anti-ICA 105 antibody-positive with OND had IDDM. Our results indicate that ICA 105 represents another putative neuroendocrine autoantigen in SMS. The presence of circulating anti-GAD and/or anti-ICA 105 antibodies might help the diagnosis of SMS. The absence, however, of antibodies recognising specific CNS autoantigens (e.g. GAD, ICA 105) does not rule out SMS.

T lymphocytes are the main cellular mediators in MS pathogenesis, and their activity is modulated by a complex cytokine network in which gamma-interferon (gamma-IFN) is considered essential. We have recently identified a new transplasmalemma Ca2+ influx activated by gamma-IFN in T lymphocytes (mainly CD4+) from patients with MS that makes T cells more susceptible to proliferation. To define the possible role of this Ca2+ influx as a marker of disease activity, we correlated its appearance with clinical and MRI findings in a cross-sectional study of 67 patients with relapsing-remitting MS (RR-MS). We also conducted a short-term longitudinal evaluation (every 15 days over a 5- to 7-month period) in three of the RR-MS patients. Sixty-five percent of all clinically active RR-MS patients showed the gamma-IFN-activated Ca2+ influx. However, positivity was higher in the first week (78%) after the onset of a clinical exacerbation than the second (57%) and third (44%) weeks. The influx was also detected in 45% of clinically stable RR-MS patients, 30% of RR-MS patients with a "benign" course of the disease, 14% of the other active autoimmune or neurologic disease patients, and 9% of healthy subjects (RR-MS versus control subjects, p0.001). Brain-MRI gadolinium-enhancing lesions were more frequently found in influx-positive (72%) than in influx-negative (47%) patients (p0.005). In the longitudinal study, we recorded five intracellular Ca2+ ([Ca2+]i) elevations and three clinical attacks (one per patient). A peak increase of [Ca2+]i due to the gamma-IFN-activated Ca2+ influx always preceded the clinical attacks from 4 to 45 days and coincided to MRI evidence of inflammation. [Ca2+]i had returned to baseline levels by the time of the onset of two clinical attacks. This finding may account for the lack of detection of the gamma-IFN-activated Ca2+ influx in some RR-MS patients during the first week after clinical onset. The strong association between the influx and clinical and MRI evidence of disease activity supports its role in the early phases of cellular immune activation leading to demyelination in MS. The detection of [Ca2+]i elevations due to the gamma-IFN-activated Ca2+ influx may represent a valuable prognostic marker of disease activity and may be useful to monitor immunologic studies of MS patients in future clinical trials.

To evaluate whether hemispheric lesion distribution in multiple sclerosis is related to the uneven interhemispheric localisation of cerebral function, a 10 item self administered questionnaire evaluating hand preference and supratentorial brain MRI was obtained in 23 patients with clinically definite multiple sclerosis. The mean degree of hand preference was +74 (range -86 to +100). The median lesion volumes were 7275 (range 1045-22 440) mm(3) for the left hemisphere and 5385 (range 1010-19 490) mm(3) for the right hemisphere. The degree of hand preference correlated with the index of interhemispheric lesion distribution (P = 0.02). The data suggest that local events, possibly related to specialisation of hemispheric function, might be responsible for the increased vulnerability of the dominant hemisphere to the pathological process of multiple sclerosis.

In order to reproduce some of the pathological features of multiple sclerosis (MS) we transplanted peripheral blood lymphocytes (PBLs) from seven patients with MS into the peritoneal activity of 28 severe combined immunodeficiency (MS-SCID) mice. Seven SCID mice were also transplanted with PBLs from two healthy subjects (hu-SCID). Animals were sacrificed between 2 and 8 weeks after transplantation (a.t.). Polymerase chain reaction(PCR) using primers able to amplify the HLA-DQα region showed presence of human cells in neural tissues of MS-SCID mice. Immunocytochemical analysis revealed the scattered appearance of human lymphocytes (mostly CD45RO + T cells) in the meningeal space and choroid plexuses of MS-SCID brains. However, human lymphocytes were similarly found in brains of hu-SCID mice. Both groups of mice never showed signs or symptoms of neurological impairment. Our results indicate that the simple transplantation of lymphocytes from MS patients into SCID mice is not likely to produce an MS-like pathology.

To study the extent of systemic immunodysregulation in multiple sclerosis (MS) we measured immunoglobulin (Ig)G, A, and M levels and studied their migrational properties after agarose isoelectric focusing in serum, cerebrospinal fluid (CSF) and tear samples from 18 MS patients, 17 patients with other neurological diseases (OND), and tears and serum samples from ten normal controls (NC). A slight elevation of total IgG, IgM and IgA levels was detected in tears from patients with MS and OND compared to NC. Of the five patients (two MS, three OND) that showed IgG oligoclonal bands (OCB) in tears, only one MS patient showed unique bands in tears not seen in the paired CSF and serum. We never found IgA, and IgM OCB in serum, CSF or tear samples. Our results suggest that polyclonal Igs are systemically elevated during chronic neurological inflammatory diseases. Oligoclonal Ig in MS, although occasionally detectable in tears, is mainly confined to the central nervous system and appears restricted to class G.

Stiff-man syndrome is a rare neurological disorder characterized by skeletal muscle rigidity and spasms in which detection of circulating anti-glutamic acid decarboxylase antibodies has suggested an autoimmune pathogenesis. To further define the role of autoimmunity in the pathogenesis, we studied anti-glutamic acid decarboxylase antibodies, as well as organ- and non-organ-specific autoantibodies in 13 patients with stiff-man syndrome and 127 patients with other neurological disorders. Thyrogastric antibodies were more frequent in patients with stiff-man syndrome (46%) than in those with other neurological disorders (12%) (p < 0.05). Non-organ-specific antibodies were found at a similar frequency in the patients with stiff-man syndrome (61%) and those with other neurological disorders (65%). Islet-cell autoantibodies and anti-glutamic acid decarboxylase antibodies were more common in stiff-man syndrome patients (38% and 31%) compared to the patients with other neurological disorders (6% and 3%, respectively; p < 0.001). With the exception of 1 patient in the other neurological disorders group, anti-glutamic acid decarboxylase antibodies were always associated with islet-cell autoantibodies. Four patients with stiff-man syndrome had an associated solid tumor: 3 of them had antibodies recognizing a 125/130-kd protein and not glutamic acid decarboxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

It has recently been proposed that the islet 64,000 Mr protein autoantigen (64K) of insulin-dependent diabetes mellitus (IDDM) is glutamic acid decarboxylase (GAD). We evaluated, by means of a newly developed immunotrapping enzyme activity assay (ITEAA), the prevalence of circulating GAD-autoantibodies (Ab) in a large population of IDDM patients (n = 168), blood donors (n = 87) and non-diabetic autoimmune patients (n = 40). The latter two groups were used as controls. Overall, GAD-Ab were found in 22% of IDDM patients, but in none of the two control groups (P = 0.007). These specificities were invariably associated with islet cell antibodies (ICA) (31.6% in IDDM with ICA vs 0 in IDDM without ICA, P = 0.0001), and this prevalence was higher in sera with high titer ICA (54.5% in IDDM with ICA > 80 JDF-units vs 22.6% of IDDM with ICA 5–80 JDF units; P = 0.002). Moreover, GAD-Ab were associated with the female sex (P = 0.002) and the concomitant presence of thyroid and/or gastric antibodies (P = 0.002). No correlation was observed between GAD-Ab and age of the patients, duration of IDDM, or associated non-organ specific antibodies. Our study indicates that GAD-Ab measured by ITEAA are: (1) detected in a proportion of IDDM patients; (2) strongly associated with ICA; (3) preferentially found in IDDM female patients with autoimmune polyendocrine serology; and (4) detected with lower frequency than that reported for 64K-Ab in IDDM.

To assess the role of alpha-tumor necrosis factor in the pathogenesis of central nervous system involvement during human immunodeficiency virus type 1 infection, we recorded clinical data and measured alpha-tumor necrosis factor levels in serum and cerebrospinal fluid samples from 45 patients infected with human immunodeficiency virus type 1, classified as group II/III (10), group IV A (5), group IV B (10), and group IV C-1 (20) of the Centers for Disease Control acquired immunodeficiency syndrome classification system and 42 controls. Alpha-tumor necrosis factor was above the limit of detection in only 3 of 15 sera and 3 of 15 cerebrospinal fluid samples from patients in group II/III and group IV A, whereas it was detected in 17 of 30 sera (p less than 0.05) and 22 of 30 cerebrospinal fluid (p less than 0.0002) samples from clinically more advanced patients (group IV B and group IV C-1). Alpha-tumor necrosis factor mean values were 21.5 pg/ml in sera and 50.0 pg/ml in cerebrospinal fluid from group IV B patients and 30.4 pg/ml in sera and 24 pg/ml in cerebrospinal fluid from group IV C-1 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

To test the effect of sampling on the detection of immunoglobulin (Ig) cerebrospinal fluid (CSF) abnormalities, we analyzed the first and last 1 ml fraction of 10 ml obtained during a single CSF removal from 27 multiple sclerosis (MS) patients and six patients with other neurological diseases. IgG index, hyperbolic function, and IgG synthesis rate decreased between the first and the last CSF aliquot. Discordant results were found in 4 27 (15%) MS patients. In 2 27 (7.5%) clinically definite MS patients, the number of CSF oligoclonal bands (OCB) decreased between the first and the last fraction. In one of the two patients, the three OCB visualized in the first fraction were not found in the last. We conclude that fractionated sampling may partially account for the absence of OCB in the CSF of some definite MS patients.

Article abstract The A1/A1 genotype of the anti-inflammatory cytokine interleukin 1 receptor antagonist (IL-1Ra) polymorphism was more frequent in 339 Italian MS patients than in healthy controls (HCs) (odds ratio = 1.83). A more aggressive disease course was also associated with A1+ genotypes and might reflect the reduced ability of mononuclear cell cultures of A1+ HCs to produce IL-1Ra. We conclude that an IL-1Ra gene polymorphism is associated with occurrence of disease and clinical course variability in Italian MS patients.

The cysteine protease caspase-1 plays a crucial part in the inflammatory process due to its ability to proteolitically activate proinflammatory cytokine precursors, such as interleukin (IL)-1β and IL-18. Multiple sclerosis is a chronic inflammatory demyelinating disease of the CNS in which the pathogenic process is mainly orchestrated by proinflammatory cytokines. The role of caspase-1 in multiple sclerosis was evaluated by measuring its mRNA levels in peripheral blood mononuclear cells (PBMCs) from seven patients with relapsing-remitting multiple sclerosis every 15 days over a 1 year period. The recorded levels were compared with clinical and MRI evidence of disease activity. Brain MRI was performed monthly in each patient. Caspase-1 mRNA levels were significantly increased in PBMCs from patients with multiple sclerosis compared with healthy controls (p