Epstein-Barr virus nuclear antigen-1 B-cell epitopes in multiple sclerosis twins.

BACKGROUND: Compared with quantitative observations, the search for qualitative changes that may characterize the immune response to Epstein-Barr virus (EBV) in multiple sclerosis (MS) has been less intense. OBJECTIVE: To examine the B-cell epitopes of antibodies against the Epstein-Barr nuclear antigen-1 (EBNA-1) and their relevance for MS, through a study in disease-discordant identical twins. METHODS: We evaluated the antibodies to all unique, maximally overlapping octapeptides of EBNA-1 in 12 pairs of monozygotic (MZ) twins (9 MS-discordant, 3 healthy), 3 non-twin patients and 2 healthy subjects. All except one of the patients were untreated. The EBV serology of these individuals had been assessed in advance using commercially available and in-house enzyme-linked immunosorbent assay (ELISA) kits, including assays for antibodies against select peptides of EBNA-1: EBNA-72 (GAGGGAGAGG) and EBNA-206 (EADYFEYHQEGGPDGE). RESULTS: The glycine-alanine rich domain of EBNA-1 was immunodominant in all subjects. Compared with healthy individuals, and similarly to what has been described in infectious mononucleosis (IM) patients, affected co-twins and non-twin patients had a significantly increased response to another EBNA-1 epitope (aa. 401-411). CONCLUSION: In a study that controls for confounders, our data focus an EBNA-1 specificity that may be associated with MS pathogenesis.

Disentangling the molecular mechanisms of multiple sclerosis: The contribution of twin studies.

Twin studies of disease concordance are useful to weight the relative contribution of genetic and environmental factors to the cause of common complex disorders. In multiple sclerosis (MS) different twinning rates from geographic areas at different prevalence suggested that heritable and non-heritable factors contribute in different proportions and ways to MS risk in diverse populations. This concept prompted genome-wide association studies, and the implementation of the co-twin control design, that allows stringent experimental approaches in MS-discordant identical pairs, controlling for genetic influences and many other known and unknown factors. The co-twin control design provided important clues on MS molecular model. These studies will be reviewed, focusing on those showing significant differences between affected and healthy co-twins. In some cases, differences that emerged in non-twin patients compared to matched controls were not confirmed in identical MS-discordant pairs, suggesting an 'MS subclinical trait'. Early patterns of magnetic resonance imaging and predictive biomarkers that characterize 'healthy' co-twins may be useful for the identification of a prodromal reversible phase of the disease.

Intestinal Permeability in Relapsing-Remitting Multiple Sclerosis.

Changes of intestinal permeability (IP) have been extensively investigated in inflammatory bowel diseases (IBD) and celiac disease (CD), underpinned by a known unbalance between microbiota, IP and immune responses in the gut. Recently the influence of IP on brain function has greatly been appreciated. Previous works showed an increased IP that preceded experimental autoimmune encephalomyelitis development and worsened during disease with disruption of TJ. Moreover, studying co-morbidity between Crohn's disease and MS, a report described increased IP in a minority of cases with MS. In a recent work we found that an alteration of IP is a relatively frequent event in relapsing-remitting MS, with a possible genetic influence on the determinants of IP changes (as inferable from data on twins); IP changes included a deficit of the active mechanism of absorption from intestinal lumen. The results led us to hypothesize that gut may contribute to the development of MS, as suggested by another previous work of our group: a population of CD8+CD161high T cells, belonging to the mucosal-associated invariant T (MAIT) cells, a gut- and liver-homing subset, proved to be of relevance for MS pathogenesis. We eventually suggest future lines of research on IP in MS: studies on IP changes in patients under first-line oral drugs may result useful to improve their therapeutic index; correlating IP and microbiota changes, or IP and blood-brain barrier changes may help clarify disease pathogenesis; exploiting the IP data to disclose co-morbidities in MS, especially with CD and IBD, may be important for patient care.

A staged screening of registered drugs highlights remyelinating drug candidates for clinical trials.

There is no treatment for the myelin loss in multiple sclerosis, ultimately resulting in the axonal degeneration that leads to the progressive phase of the disease. We established a multi-tiered platform for the sequential screening of drugs that could be repurposed as remyelinating agents. We screened a library of 2,000 compounds (mainly Food and Drug Administration (FDA)-approved compounds and natural products) for cellular metabolic activity on mouse oligodendrocyte precursors (OPC), identifying 42 molecules with significant stimulating effects. We then characterized the effects of these compounds on OPC proliferation and differentiation in mouse glial cultures, and on myelination and remyelination in organotypic cultures. Three molecules, edaravone, 5-methyl-7-methoxyisoflavone and lovastatin, gave positive results in all screening tiers. We validated the results by retesting independent stocks of the compounds, analyzing their purity, and performing dose-response curves. To identify the chemical features that may be modified to enhance the compounds' activity, we tested chemical analogs and identified, for edaravone, the functional groups that may be essential for its activity. Among the selected remyelinating candidates, edaravone appears to be of strong interest, also considering that this drug has been approved as a neuroprotective agent for acute ischemic stroke and amyotrophic lateral sclerosis in Japan.

IFN-ß and multiple sclerosis: from etiology to therapy and back.

Several immunomodulatory treatments are currently available for relapsing-remitting forms of multiple sclerosis (RRMS). Interferon beta (IFN) was the first therapeutic intervention able to modify the course of the disease and it is still the most used first-line treatment in RRMS. Though two decades have passed since IFN-ß was introduced in the management of MS, it remains a valid approach because of its good benefit/risk profile. This is witnessed by new efforts of pharmaceutical industry to improve this line: a PEGylated form of subcutaneous IFN-ß 1a, (Plegridy(®)) with a longer half-life, has been recently approved in RRMS. This review will survey the various stages of the use of type I IFN in MS, with special attention to the effect of the treatment on the supposed viral etiologic factors associated to the disease. The antiviral activities of IFN (that initially prompted its use as immunomodulatory agent in MS), and the mounting evidences in favor of a viral etiology in MS, allowed us to outline a re-appraisal from etiology to therapy and back.

Linkage analysis of multiple sclerosis with candidate region markers in Sardinian and Continental Italian families.

Previous genome screens in multiple sclerosis have shown some evidence of linkage in scattered chromosomal regions. Although in no case the evidence of each single study was compelling and although in general the linkage 'peaks' of the different studies did not coincide, some regions can be considered likely candidates for the presence of MS risk genes because of the clustering of MLS scores and homology with eae loci. We performed a linkage analysis of markers in these regions and of intragenic markers of some individual candidate genes (HLA-DRB1, CTLA-4, IL9, APOE, BCL2, TNFR2). For the first time, Southern European populations were targeted, namely Continental Italians and Sardinians. A total of 69 multiplex families were typed for 67 markers by a semi-automatic fluorescence-based assay. Results were analysed for linkage by two non-parametric tests: GENEHUNTER and SimIBD. In general, the linkage scores obtained were low, confirming the conclusion that no gene is playing a major role in the disease. However, some markers, in 2p11, 3q21.1, 7p15.2 and 22q13.1 stood out as promising since they showed higher scores with one or the other test. This stimulates further association analysis of a large number of simplex families from the same populations.

Use of Bacille Calmette-Guèrin (BCG) in multiple sclerosis.

We studied the effect of Bacille Calmette-Guerin (BCG) vaccine as an immunomodulator in MS. According to the guidelines for clinical trials in MS, a single crossover, MRI-monitored trial was performed in 14 patients with relapsing-remitting MS. After treatment, MRI activity was significantly reduced. No major adverse effects were reported. Adjuvant therapy with BCG vaccine was safe and merits study in MS.

Serum amyloid A protein is elevated in relapsing-remitting multiple sclerosis.

In multiple sclerosis (MS), the signs of inflammation that can be detected in the central nervous system are not mirrored by unequivocal markers of activation of the immune system in the periphery. We performed a serial monitoring of serum amyloid A protein (SAA), a major acute phase reactant, in peripheral blood of patients with relapsing-remitting MS over a 3-month period. Patients were monitored in parallel with gadolinium-enhanced magnetic resonance imaging (Gd-MRI) of the brain. The results show that signs of ongoing peripheral inflammation, reflected by elevations of SAA levels, can be detected in MS patients.

Myelin basic protein intramolecular spreading without disease progression in a patient with multiple sclerosis.

A case with stable multiple sclerosis (MS) and T cell responses which initially focused on peptide 16-38 of myelin basic protein (MBP) allowed us to investigate the dynamics of the MBP-specific T cell repertoire and its relationship with disease progression. Epitope mapping experiments and T cell receptor usage of MBP-reactive T cell lines (obtained at four distinct time points over a 7-year period) showed a spreading of the response. Transient expansions and persistence of T cells recognizing different MBP epitopes were also detected. The patient's expanded disability status scale and magnetic resonance imaging lesion load remained stable. Our case shows both persistent self-recognitions and determinant spreading in stable MS. This finding suggests that the relationship between dynamics of self-recognition and disease progression is highly complex.

The immune response to mycobacterial 70-kDa heat shock proteins frequently involves autoreactive T cells and is quantitatively disregulated in multiple sclerosis.

Heat shock proteins (HSP) are the most conserved molecules known to date that may also function as immune targets during infection. Hence, theoretically there is a high chance of cross-reactive responses to epitopes shared by host and microbe HSP. If not properly regulated, these responses may contribute to the pathogenesis of autoimmune disease. To determine if immune responses to HSP could contribute to the pathogenesis of multiple sclerosis, we raised T lymphocyte lines specific for the purified protein derivative of Mycobacterium tuberculosis (PPD) from patients with multiple sclerosis, patients with tuberculosis and from healthy individuals. These lines were then screened for their proliferative response to a M. tuberculosis 70-kDa heat shock protein (M.tb.HSP70). The relative frequency of the recognition of highly conserved sequences of M.tb.HSP70 compared to variable ones was also assessed by mapping experiments on those PPD specific T lymphocyte lines which also recognized the mycobacterial 70-kDa heat shock protein. In patients with multiple sclerosis, we observed a significantly higher estimated frequency of PPD-specific T lines responding to M.tb.HSP70 compared to healthy individuals and patients with tuberculosis. Furthermore, mapping experiments using recombinant proteins representing mycobacterial and human HSP70 sequences and a panel of synthetic peptides encompassing the whole sequence of Mycobacterium leprae HSP70, showed that the response to conserved epitopes of HSP70 is a frequent event in each of the three conditions studied, often leading to the cross-recognition of microbial and human sequences. These findings implicate the 70-kDa heat shock proteins as potential autoantigens in multiple sclerosis.

HLA-DM polymorphisms do not associate with multiple sclerosis: an association study with analysis of myelin basic protein T cell specificity.

The MHC region on 6p harbors at least one susceptibility gene for multiple sclerosis (MS). Within this region, HLA-DM loci are of interest being involved in class II antigen processing. We investigated the association of HLA-DM polymorphisms with MS. Sixty-three patients with MS and 46 healthy controls from continental Italy were typed for HLA-DM polymorphisms and HLA-DRB1 alleles. Besides, among the donors characterized for the DM polymorphisms, we considered 6 MS patients previously studied for the fine specificity of their MBP-specific T lymphocyte lines (TLL). The frequencies of allelic variants at the DMA and DMB loci were similar between MS patients and controls, even when HLA-DRB1*1501 positive and negative donors were analyzed separately. Patients with predominant responses to different MBP epitopes did not differ for their HLA-DM haplotype while patients with predominant responses to the same MBP epitope could present different HLA-DM haplotypes. HLA-DM polymorphisms do not associate with MS and may not affect specific patterns of T cell responses to MBP.

A major influence of the T cell receptor repertoire as compared to antigen processing-presentation in the selection of myelin basic protein epitopes in multiple sclerosis.

We selected two multiple sclerosis (MS) patients, compatible for HLA-DR2 subtype, and differing for HLA-DM haplotype as well as for the myelin basic protein (MBP) epitope recognized by the vast majority of their T cell lines (TCL) (residues 16-38 and 86-99, respectively). TCL sharing the same restriction element were re-assayed in the presence of reciprocally mismatched antigen-presenting cells (APC). The TCL recognized both the whole MBP and the relevant peptide also in the presence of non-autologous APC, (compatibility for processing, despite a difference in the DM haplotype). The same protocol, performed in serum-free pulsing experiments or in the presence of 'fixed' APC, excluded extracellular processing or mutual T cell presentation, and confirmed the need for MBP processing in our system. The finding, that only TCL recognizing MBP peptide 16-38 (a region not previously related to the DR2 haplotype) used a novel Vbeta, supports the importance of the TCR repertoire over the processing-presentation machinery in the selection of MBP epitopes in MS.

Global immune disregulation in multiple sclerosis: from the adaptive response to the innate immunity.

Increasing evidences show a global immune disregulation in multiple sclerosis (MS). The possible involvement of myelin and non-myelin (auto-)antigens in the autoaggressive process as well as the disregulation of both adaptive and innate immunity challenge the concept of specific immunotherapy. T cells at the boundary between innate and adaptive immunity, whose immunoregulatory role is becoming increasingly clear, have recently been shown to bear relevance for MS pathogenesis. Global immune interventions (and type I interferons may be considered as such) aimed at interfering with both innate and acquired immune responses seem to be a most promising therapeutic option in MS.

T cell response to myelin basic protein before and after treatment with interferon beta in multiple sclerosis.

Studies on the in vivo effects of interferon-beta (IFNbeta) therapy on autoreactive T cells have never been carried out in multiple sclerosis (MS). We investigated the T cell response to myelin basic protein (MBP), before and after IFN-beta therapy, raising MBP-specific T cell lines (TCL) from the peripheral blood of six MS patients with a satisfactory response to the treatment. IFNbeta did not affect the relative frequency and epitope specificity of the TCL. After IFNbeta therapy, the production of interleukin-4 was decreased in MBP-stimulated TCL while the secretion of interferon-gamma was increased in unstimulated TCL. Interleukin-10 and tumor necrosis factor-alpha did not show significant variations. This finding supports recent suggestions about the complexity of the T helper 1/T helper 2 paradigm in MS and other organ-specific autoimmune diseases. In fact, the beneficial effects of IFNbeta do not exclude an immunostimulatory action that may involve potentially autoreactive T cells. This has implications for future treatment options, including combination therapies.

Evidence for a role of gammadelta T cells in demyelinating diseases as determined by activation states and responses to lipid antigens.

In this report we review current information on the phenotypic and functional properties of gammadelta T cells in demyelinating disorders. The results support the conclusion that although gammadelta T cells show evidence of activation in patients with either multiple sclerosis (MS) or Guillain Barrè syndrome (GBS), differences exist in the phenotypic and functional properties of these cells between the two diseases. In particular, our data indicate that in patients with MS the Vdelta2 subset is activated and that these cells can be induced to secrete high levels of proinflammatory cytokines. In contrast, in patients with GBS, the Vdelta1 subset is expanded and can be induced to secrete cytokines more associated with a humoral response.

Anatomical and functional correlates of cognitive deficit in multiple sclerosis.

This brief article reviews the present state of knowledge concerning the relationship between structural and functional cerebral abnormalities and the cognitive deficits associated with multiple sclerosis. Currently available neuroimaging techniques have substantially contributed to a better understanding of the latter, suggesting that cortical-subcortical disconnection is the most likely cause of the cognitive disturbance. Longitudinal studies are needed to determine the natural history of the cognitive deficit and its relationship to the cerebral changes detected by neuroimaging techniques.

Crossed quadrant homonymous hemianopsia in a case of multiple sclerosis.

We describe the first report of crossed quadrant hemianopsia (CQHH) occurring in a patient with a well documented clinical history of Multiple Sclerosis (MS). The visual field studied by a Humphrey Field Analyzer (Mod.630, 30-2 program) showed a CQHH, involving right superior quadrants and left inferior ones. Magnetic resonance imaging (MRI) showed two lesions located in both left and right trigone area, corresponding to the geniculocalcarine pathways. CQHH is a very rare visual field defect commonly caused by vertebro-basilar ischemia and attributed to bilateral injury of the calcarine fissure. In the absence of lesions in the primary visual cortex, a direct relationship between the quadrantonopic defect and two small demyelinating lesions in posterior periventricular white matter, corresponding to the geniculocalcarine pathways, is implied.

Serum elements and oxidative status in clinically isolated syndromes: imbalance and predictivity.

BACKGROUND: Metals are suspected of being involved in the pathogenesis of various neurologic diseases. We previously found a complex imbalance in serum chemical elements and oxidative status in patients with clinically definite multiple sclerosis (CDMS). OBJECTIVE: To understand whether this imbalance affects people with clinically isolated syndrome (CIS) and, if so, whether it predicts conversion to CDMS. METHODS: We studied 22 chemical elements and the oxidative status in 49 patients with CIS, 49 patients with CDMS, and 49 healthy donors (HD). Univariate and multivariate approaches were used to identify profiles for each group. A logistic regression analysis was used to identify the predictive potential of baseline data (elements, oxidative status, and MRI findings) for conversion to CDMS over 36 months. RESULTS: Several elements and oxidative status values differed significantly among the 3 groups. Discriminant analysis revealed a major contribution of Ca, Fe, Sn, Zn, serum antioxidant capacity, and serum oxidative status, which resulted in distinct profiles (the prediction of group membership was 96% [cross-validated 92%] for HD, 92% [cross-validated 92%] for CDMS, and 90% [cross-validated 86%] for CIS). A weighted combination of element concentrations and oxidative status values, adjusting for all other predictors, would predict a reduction in the risk of conversion to CDMS within 3 years (odds ratio 0.37; 95% confidence interval 0.18-0.76; p = 0.007), thereby proving more effective than MRI at baseline. CONCLUSIONS: The peculiar imbalance in serum elements and oxidative status that characterizes patients with CIS and may predict conversion to CDMS warrants studies on larger sample sizes.

Riluzole in cerebellar ataxia: a randomized, double-blind, placebo-controlled pilot trial.

BACKGROUND: The pleiotropic effects of riluzole may antagonize common mechanisms underlying chronic cerebellar ataxia, a debilitating and untreatable consequence of various diseases. METHODS: In a randomized, double-blind, placebo-controlled pilot trial, 40 patients presenting with cerebellar ataxias of different etiologies were randomly assigned to riluzole (100 mg/day) or placebo for 8 weeks. The following outcome measures were compared: proportion of patients with a decrease of at least 5 points in the International Cooperative Ataxia Rating Scale (ICARS) total score after 4 and 8 weeks compared with the baseline score; mean changes from the baseline to posttreatment ICARS (total score and subscores at 8 weeks); and occurrence of adverse events. RESULTS: Riluzole and placebo groups did not differ in baseline characteristics. The number of patients with a 5-point ICARS drop was significantly higher in the riluzole group than in the placebo group after 4 weeks (9/19 vs 1/19; odds ratio [OR] = 16.2; 95% confidence interval [CI ] 1.8-147.1) and 8 weeks (13/19 vs 1/19; OR = 39.0; 95% CI 4.2-364.2). The mean change in the riluzole group ICARS after treatment revealed a decrease (p < 0.001) in the total score (-7.05 [4.96] vs 0.16 [2.65]) and major subscores (-2.11 [2.75] vs 0.68 [1.94] for static function, -4.11 [2.96] vs 0.37 [2.0] for kinetic function, and -0.74 [0.81] vs 0.05 [0.40] for dysarthria). Sporadic, mild adverse events occurred. CONCLUSIONS: These findings indicate the potential effectiveness of riluzole as symptomatic therapy in diverse forms of cerebellar ataxia. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that riluzole reduces, by at least 5 points, the ICARS score in patients with a wide range of disorders that cause cerebellar ataxia (risk difference 63.2%, 95% CI 33.5%-79.9%).