Effects of Bacille Calmette-Guerin after the first demyelinating event in the CNS.

OBJECTIVE: To evaluate Bacille Calmette-Guérin (BCG) effects after clinically isolated syndromes (CIS). METHODS: In a double-blind, placebo-controlled trial, participants were randomly assigned to receive BCG or placebo and monitored monthly with brain MRI (6 scans). Both groups then entered a preplanned phase with IM interferon-ß-1a for 12 months. From month 18 onward, the patients took the disease-modifying therapies (DMTs) that their neurologist considered indicated in an open-label extension phase lasting up to 60 months. RESULTS: Of 82 randomized subjects, 73 completed the study (33 vaccinated and 40 placebo). During the initial 6 months, the number of cumulative lesions was significantly lower in vaccinated people. The relative risks were 0.541 (95% confidence interval [CI] 0.308-0.956; p = 0.03) for gadolinium-enhancing lesions (the primary endpoint), 0.364 (95% CI 0.207-0.639; p = 0.001) for new and enlarging T2-hyperintense lesions, and 0.149 (95% CI 0.046-0.416; p = 0.001) for new T1-hypointense lesions. The number of total T1-hypointense lesions was lower in the BCG group at months 6, 12, and 18: mean changes from baseline were -0.09 ± 0.72 vs 0.75 ± 1.81 (p = 0.01), 0.0 ± 0.83 vs 0.88 ± 2.21 (p = 0.08), and -0.21 ± 1.03 vs 1.00 ± 2.49 (p = 0.02). After 60 months, the cumulative probability of clinically definite multiple sclerosis was lower in the BCG + DMT arm (hazard ratio = 0.52, 95% CI 0.27-0.99; p < 0.05), and more vaccinated people remained DMT-free (odds ratio = 0.20, 95% CI 0.04-0.93; p = 0.04). CONCLUSIONS: Early BCG may benefit CIS and affect its long-term course. CLASSIFICATION OF EVIDENCE: BCG, as compared to placebo, was associated with significantly reduced development of gadolinium-enhancing lesions in people with CIS for a 6-month period before starting immunomodulating therapy (Class I evidence).

CD161(high)CD8+T cells bear pathogenetic potential in multiple sclerosis.

To identify differentially expressed genes in multiple sclerosis, microarrays were used in a stringent experimental setting-leukapheresis from disease-discordant monozygotic twins and gene expression profiling in CD4(+) and CD8(+) T-cell subsets. Disease-related differences emerged only in the CD8(+) T-cell subset. The five differentially expressed genes identified included killer cell lectin-like receptor subfamily B, member 1, also known as natural killer receptor protein 1a/CD161, presented by the International Multiple Sclerosis Genetics Consortium as one of the non-MHC candidate loci. Flow cytometric analysis on peripheral blood of healthy donors and patients with multiple sclerosis and rheumatoid arthritis confirmed an upregulation of CD161 at the protein level, showing also a significant excess of CD161(high)CD8(+) T cells in multiple sclerosis. This subset prevalently included chemokine (C-C motif) receptor 6(+), cytokine-producing, effector-memory T cells with proinflammatory profiles. It also included all circulating interleukin-17(+)CD8(+) T cells. In the CD161(high)CD8(+) subset, interleukin-12 facilitated proliferation and interferon-γ production, with CD161 acting as a co-stimulatory receptor. CD161(+)CD8(+)CD3(+) T cells producing interferon-γ were part of intralesional immune infiltrates and ectopic B cell follicles in autopsy multiple sclerosis brains. Variations of CD161 expression on CD8(+) T cells identify a subset of lymphocytes with proinflammatory characteristics that have not been previously reported in multiple sclerosis and are likely to contribute to disease immunopathology.

Gene expression profiles reveal homeostatic dynamics during interferon-beta therapy in multiple sclerosis.

Understanding the mechanisms that sustain the effects of disease modifying drugs in multiple sclerosis (MS) may help refine current therapies and improve our knowledge of disease pathogenesis. By using cDNA microarrays, we investigated gene expression in the peripheral blood mononuclear cells (PBMC) of 7 MS patients, at baseline (T0) as well as after 1 (T1) and 3 months (T3) of interferon beta-1a (IFN-beta-1a; Rebif 44 microg) therapy. Gene expression changes involved genes of both immunological and non-immunological significance. We validated IL-10 up-regulation, which is in accordance with previous reports, and other novel changes that underscore the capacity of IFN-beta to impair antigen presentation and migration of inflammatory elements into the central nervous system (up-regulation of filamin B and down-regulation of IL-16 and rab7). Overall, gene expression changes became less pronounced after 3 months of therapy, suggesting a homeostatic response to IFN-beta. This may be of use for the design of new treatment schedules.

Characterization of CD8+ T cell repertoire in identical twins discordant and concordant for multiple sclerosis.

Autoreactive CD4+ and CD8+ T cells directed against CNS autoantigens may play a role in the development of multiple sclerosis (MS). Identical twins share the same genetic background but not the TCR repertoire that is shaped by the encounter with self or foreign antigens. To gain insights into the interplay between MS and T cell repertoire, peripheral blood CD4+ and CD8+ T lymphocytes and their CCR7+/CCR7- subsets from five pairs of identical twins (four discordant and one concordant for MS; none of which had taken disease-modifying therapy) were compared by TCR beta-chain (TCRB) complementary-determining region 3 (CDR3) spectratyping. CD4+ T cells generally showed a Gaussian distribution, whereas CD8+ T cells exhibited subject-specific, widely skewed TCR spectratypes. There was no correlation between CD8+ T cell oligoclonality and disease. Sequencing of predominant spectratype expansions revealed shared TCRB-CDR3 motifs when comparing inter- and/or intrapair twin members. In many cases, these sequences were homologous to published TCRs, specific for viruses implicated in MS pathogenesis, CNS autoantigens, or copaxone [glatiramer acetate (GA)], implying the occurrence of naturally GA-responding CD8+ T cells. It is notable that these expanded T cell clones with putative pathogenic or regulatory properties were present in the affected as well as in the healthy subject, thus suggesting the existence of a "MS predisposing trait" shared by co-twins discordant for MS.

Multiple sclerosis in twins from continental Italy and Sardinia: a nationwide study.

Knowledge about the balance between heritable and nonheritable risk in multiple sclerosis (MS) is based on twin studies in high-prevalence areas. In a study that avoided ascertainment limitations and directly compared continental Italy (medium-prevalence) and Sardinia (high-prevalence), we ascertained 216 pairs from 34,549 patients. This gives a twinning rate of 0.62% among MS patients, significantly less than that of the general population. In continental Italy, probandwise concordance was 14.5% (95% confidence interval, 5.1-23.8) for monozygotic and 4.0% (95% confidence interval, 0.8-7.1) for dizygotic twins. Results in Sardinia resemble those in northern populations but in limited numbers. Monozygotic concordance was 22.2% (95% confidence interval, 0-49.3) probandwise, but no concordant dizygotic pairs were identified. A questionnaire on 80 items possibly related to disease cause was administered to 70 twin pairs, 135 sporadic patients, and 135 healthy volunteers. Variables positively (7) or negatively (2) associated with predisposition and concordance in twins largely overlapped and were mainly linked to infection. If compared with previous studies, our data demonstrate that penetrance in twins appears to correlate with MS prevalence. They highlight the relevance of nonheritable variables in Mediterranean areas. The apparent underrepresentation of MS among Italian twins draws attention to protective factors, shared by twins, that may influence susceptibility.

Quantification of chemical elements in blood of patients affected by multiple sclerosis.

Although some studies suggested a link between exposure to trace elements and development of multiple sclerosis (MS), clear information on their role in the aetiology of MS is still lacking. In this study the concentrations of Al, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Ni, Pb, Sb, Si, Sn, Sr, Tl, V, W, Zn and Zr were determined in the blood of 60 patients with MS and 60 controls. Quantifications were performed by inductively coupled plasma (ICP) atomic emission spectrometry and sector field ICP mass spectrometry. When the two groups were compared, an increased level of Co, Cu and Ni and a decrement of Be, Fe, Hg, Mg, Mo, Pb and Zn in blood of patients were observed. In addition, the discriminant analysis pointed out that Cu, Be, Hg, Co and Mo were able to discriminate between MS patients and controls (92.5% of cases correctly classified).

Concentration of elements in serum of patients affected by multiple sclerosis with first demyelinating episode: a six-month longitudinal follow-up study.

Twenty-six chemical elements and oxidative status were determined in serum of 12 patients with first demyelinating episode and brain magnetic resonance imaging compatible with the disease at different time points. Quantifications of Al, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Ni, Pb, Sb, Si, Sn, Sr, V, Tl, W, Zn and Zr, as well as of serum oxidative status and antioxidant capacity were carried out. The results were compared with values obtained from healthy subjects living in the same geographic area. Concentration variability, expressed as coefficient of variation (CV), was evaluated over a six months longitudinal follow-up. The CV was higher for Li and Pb, while showed minimal variation for Ca, Cu, Mg and Zn--elements strictly body regulated. Significant difference (p < or = 0.05) in mean concentrations of Ba, Ca, Cd, Cr, Li, Mn, Mo, Ni, Sb, Si, Sn and Zr between patients at time 0 and controls was also found.

Cerebrospinal fluid isoprostanes are not related to inflammatory activity in relapsing-remitting multiple sclerosis.

Oxidative stress leads to lipid peroxidation and may contribute to the pathogenesis of lesions in multiple sclerosis (MS), an autoimmune disease characterised by inflammatory as well as degenerative phenomena. We previously found that cerebrospinal fluid (CSF) levels of isoprostane 8-epi-PGF2alpha, a marker of free radical damage and lipid peroxidation in vivo, were elevated in MS patients. Such levels were correlated with the degree of disability and reduced in subjects under steroid therapy. Here we investigated weather the CSF isoprostane levels correlated with disease inflammatory activity. To this aim, we enrolled 41 relapsing-remitting (RR) MS patients who underwent at the same time full neurological examination, NMR-imaging brain scan and diagnostic CSF test. No evidence of correlation was found between 8-epi-PGF2alpha levels and the presence of gadolinium (Gd)-enhancing NMR lesions or the time elapsed since the last relapse. We suggest that isoprostanes are not useful as surrogate inflammatory markers in MS. However, they may represent a sensitive index of degenerative phenomena, which can persist also in the absence of inflammatory activity.

A whole genome screen for linkage disequilibrium in multiple sclerosis performed in a continental Italian population.

We have systematically screened the genome for evidence of linkage disequilibrium (LD) with multiple sclerosis (MS) by typing 6000 microsatellite markers in case-control and family based (AFBAC) cohorts from the Italian population. DNA pooling was used to reduce the genotyping effort involved. Four DNA pools were considered: cases (224 Italian MS patients), controls (231 healthy Italians), index (185 index cases from trio families) and parents (the 370 parents of the patient included in the Index pool), respectively. After refining analysis of the most promising 14 markers to emerge from this screening process, only marker D2S367 retained evidence for association.

T cell response to amyloid-beta and to mitochondrial antigens in Alzheimer's disease.

Despite the vast amount of literature on non-specific immune mechanisms in Alzheimer's disease (AD), little is known about the role of antigen-specific immune responses. We investigated T cell reactivity to fragment 1-42 of amyloid-beta (Abeta) and to N-terminal peptides of human mitochondrial and control microbial proteins. Thirty subjects with a diagnosis of probable AD according to NINCDS-ADRDA criteria and 30 sex- and age-matched healthy controls were enrolled. T cell responses to Abeta fragment showed no significant differences between AD patients and controls. By contrast, the mean number of positive T cell responses to both human mitochondrial and microbial peptides was significantly decreased in AD patients compared to control subjects. No significant correlation was found between T cell responses and both the severity of cognitive impairment and duration of the disease. Our results suggest that antigen-specific immune responses are impaired in AD. Protective immune responses to harmful amyloidogenic substances may also be impaired, thus favoring their accumulation in the brain.

Prolactin and prolactin receptor gene polymorphisms in multiple sclerosis and systemic lupus erythematosus.

Genes encoding for prolactin (PRL) and its receptor (PRLR) are possible candidates for multiple sclerosis (MS) and systemic lupus erythematosus (SLE) susceptibility. In fact: (1) a prolactin secretion dysfunction has been described in several autoimmune diseases including SLE and MS and their animal models; (2) both PRL and PRLR are structurally related to members of the cytokine/hematopoietin family and have a role in the regulation of the immune response; and (3) both PRL and PRLR genes map in genomic regions that showed linkage with autoimmunity. Prolactin maps on chromosome 6p, about 11-kb telomeric to HLA-DRB1 and PRLR in 5p12-13, which revealed evidence of linkage with MS in different populations. To evaluate a possible role of these two genes in SLE and MS we performed an association study of 19 PRL and PRLR single nucleotide polymorphisms (SNPs). These were directly searched by DHPLC in a panel of SLE and MS patients and selected from databases and the literature. The SNP allele frequencies were determined on patient and control DNA pools by primer-extension genotyping and HPLC analysis. Moreover a panel of HLA typed SLE and control individuals were individually genotyped for the PRL G-1149T polymorphism previously described to be associated with SLE. No statistically significant difference in the allele distribution was observed for any of the tested variations.

[Twins in biomedical research and the creation of the "National Twin Registry"]. / I gemelli nella ricerca biomedica e l'istituzione del "Registro Nazionale Gemelli".

Twins are a valuable resource for the study of complex traits. The twin method is substantially based on the comparison between correlations and concordance in monozygotic (MZ) and dizygotic (DZ) twins and allows several applications in biomedical and molecular genetic research. It allows either the qualitative and quantitative evaluation of the influences that genetic and environmental factors exert on phenotypes or the estimation of trait variability. Moreover, classical genetic linkage analysis is more powerful if performed in DZ twins. However, the twin method has some pitfalls, such as the necessity that collected samples be representative of both twin and general population. For this reason, over the last few years, a number of Countries have established population-based twin registers, which guarantee the maximum level of representation and, consequently, are of extreme value for epidemiological studies. Italy is also implementing a national twin register. The following is the description of the procedure that led to the establishment of the Italian Twin Registry.

Protein tyrosine phosphatase receptor-type C exon 4 gene mutation distribution in an Italian multiple sclerosis population.

In this study, we investigate the role of the C-->G mutation in position 77 of exon 4 of the protein tyrosine phosphatase receptor-type C (PTPRC) gene, coding for the CD45 molecule, for the development of multiple sclerosis (MS) in an Italian continental population. The PTPRC mutated genotype has been recently described as associated with MS in three different case-control studies carried out in German MS patients, whereas similar studies performed in the US and Swedish populations failed to demonstrate such an association. The C-->G transition in position 77 was found in a small number of Italian MS patients and in none of the matched group of healthy controls (Fisher exact test, P value=0.02). This finding suggests a role, in at least a group of patients, for the PTPRC mutation in genetic susceptibility to MS.

Epidemiology and current treatment of multiple sclerosis in Europe today.

Multiple sclerosis (MS) is a chronic disease affecting the central nervous system, usually leading to early disablement in young adults. At least 350,000 persons in Europe have the disease. Wide variations exist both between and within European countries in the incidence and prevalence of the disease as well as in the general standard of care for MS patients. The needs, well-being, and social participation of people with MS are systematically influenced by their physical and cultural environment and the nature of the community services. Moreover, the rate of introduction of the new disease-modifying therapy also widely differs from country to country. This article helps clinical researchers to understand better the differences in epidemiology and in the current treatment of MS in Europe.

Identification of single nucleotide variations in the coding and regulatory regions of the myelin-associated glycoprotein gene and study of their association with multiple sclerosis.

The myelin-associated glycoprotein (MAG) gene is an appealing candidate in the 19q13 Multiple Sclerosis (MS) candidate region. Using denaturing high performance liquid chromatography (DHPLC), we identified 14 single nucleotide polymorphisms (SNPs) in MAG coding and regulatory regions, and we tested their possible association with MS in Italian patient and control DNA pools. Eight variations had a frequency <0.05, i.e. below the detection limit in the pools. Of these, Arg537Cys was further studied with individually genotyped individuals and was detected in 1/189 patients and 0/85 controls. The frequency of the six remaining SNPs were not significantly different in pools including a total of 1266 patient and 1612 control chromosomes. Considering the statistical power of the experimental design, these results exclude the MAG gene as an MS susceptibility factor with an odds ratio (OR) equal or higher than 1.3.

The Italian Twin Project: from the personal identification number to a national twin registry.

The unique opportunity given by the "fiscal code", an alphanumeric identification with demographic information on any single person residing in Italy, introduced in 1976 by the Ministry of Finance, allowed a database of all potential Italian twins to be created. This database contains up to now name, surname, date and place of birth and home address of about 1,300,000 "possible twins". Even though we estimated an excess of 40% of pseudo-twins, this still is the world's largest twin population ever collected. The database of possible twins is currently used in population-based studies on multiple sclerosis, Alzheimer's disease, celiac disease, and type 1 diabetes. A system is currently being developed for linking the database with data from mortality and cancer registries. In 2001, the Italian Government, through the Ministry of Health, financed a broad national research program on twin studies, including the establishment of a national twin registry. Among all the possible twins, a sample of 500,000 individuals are going to be contacted and we expect to enrol around 120,000 real twin pairs in a formal Twin Registry. According to available financial resources, a sub sample of the enrolled population will be asked to donate DNA. A biological bank from twins will be then implemented, guaranteeing information on future etiological questions regarding genetic and modifiable factors for physical impairment and disability, cancers, cardiovascular diseases and other age related chronic illnesses.