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Background: Results from previous studies on a possible interaction between smoking and Epstein-Barr virus (EBV) in the risk of multiple sclerosis (MS) are conflicting., Objectives: To examine the interaction between smoking and infectious mononucleosis (IM) in the risk of MS., Methods: Within the case-control study on Environmental Factors In Multiple Sclerosis (EnvIMS), 1904 MS patients and 3694 population-based frequency-matched healthy controls from Norway, Italy, and Sweden reported on prior exposure to smoking and history of IM. We examined the interaction between the two exposures on the additive and multiplicative scale., Results: Smoking and IM were each found to be associated with an increased MS risk in all three countries, and there was a negative multiplicative interaction between the two exposures in each country separately as well as in the pooled analysis ( p = 0.001). Among those who reported IM, there was no increased risk associated with smoking (odds ratio (OR): 0.95, 95% confidence interval (CI): 0.66-1.37). The direction of the estimated interactions on the additive scale was consistent with a negative interaction in all three countries (relative excess risk due to interaction (RERI): -0.98, 95% CI: -2.05-0.15, p = 0.09)., Conclusion: Our findings indicate competing antagonism, where the two exposures compete to affect the outcome.

Background: The relevance of human leukocyte antigen (HLA)-G in dimeric form in multiple sclerosis (MS) is still unknown., Objective: To investigate the contribution of cerebrospinal fluid (CSF) HLA-G dimers in MS pathogenesis., Methods: CSF amounts of 78-kDa HLA-G dimers were measured by western blot analysis in 80 MS relapsing-remitting MS (RRMS) patients and in 81 inflammatory and 70 non-inflammatory controls., Results: CSF amounts of 78 kDa HLA-G dimers were more frequent in RRMS than in inflammatory (p<0.01) and non-inflammatory controls (p<0.001) and in magnetic resonance imaging (MRI) inactive than in MRI active RRMS (p<0.00001)., Conclusion: Our findings suggest that HLA-G dimers may be implicated in termination of inflammatory response occurring in MS., (© The Author(s), 2015.)

Background: The activity of matrix metalloproteinase-9 (MMP-9) depends on two isoforms, an 82 kDa active MMP-9 modulated by its specific tissue inhibitor (TIMP-1), and a 65 kDa TIMP-1 resistant active MMP-9. The relevance of these two enzymatic isoforms in multiple sclerosis (MS) is still unknown., Objective: To investigate the contribution of the TIMP-1 modulated and resistant active MMP-9 isoforms to MS pathogenesis., Methods: We measured the serum levels of the 82 kDa and TIMP-1 resistant active MMP-9 isoforms by activity assay systems in 86 relapsing-remitting MS (RRMS) patients, categorized according to clinical and magnetic resonance imaging (MRI) evidence of disease activity, and in 70 inflammatory (OIND) and 69 non-inflammatory (NIND) controls., Results: Serum levels of TIMP-1 resistant MMP-9 were more elevated in MS patients than in OIND and NIND (p < 0.05, p < 0.02, respectively). Conversely, 82 kDa active MMP-9 was higher in NIND than in the OIND and MS patients (p < 0.01 and p < 0.00001, respectively). MRI-active patients had higher levels of TIMP-1 resistant MMP-9 and 82 kDa active MMP-9, than did those with MRI inactive MS (p < 0.01 and p < 0.05, respectively)., Conclusion: Our findings suggested that the TIMP-1 resistant MMP-9 seem to be the predominantly active isoform contributing to MS disease activity., (© The Author(s), 2015.)

Background: Obesity may be a risk factor for developing multiple sclerosis (MS)., Objective: We examined if body size influences the risk of MS in a population-based, case control study., Methods: A total of 953 cases and 1717 controls from Norway and 707 cases and 1333 controls from Italy reported their body size by choosing a silhouette 1 to 9 (largest) every fifth year from age 5 to 30 and at time of study. The body size-related MS risk was defined by odds ratios (ORs) in logistic regression analyses adjusting for age, smoking and outdoor activity., Results: In Norway a large body size (silhouettes 6-9) compared to silhouette 3 increased the risk of MS, especially at age 25 (OR 2.21; 95% CI 1.09-4.46 for men and OR 1.43; 95% CI 0.90-2.27 for women). When comparing silhouette 9 to 1, we found a significant dose-response from age 10 until age 30 peaking at age 25 (sex-adjusted OR 2.83; 95% CI 1.68-4.78). The association was present for at least 15 years prior to disease onset. No significant associations were found in Italy., Conclusions: Obesity from childhood until young adulthood is a likely risk factor for MS with a seemingly stronger effect in Norway than in Italy., (© The Author(s), 2014.)

Objectives: The objective of this paper is to estimate the association between multiple sclerosis (MS) and measures of sun exposure in specific age periods in Norway and Italy., Methods: A total of 1660 MS patients and 3050 controls from Italy and Norway who participated in a multinational case-control study (EnvIMS) reported sun habits during childhood and adolescence., Results: A significant association between infrequent summer outdoor activity and increased MS risk was found in Norway and in Italy. The association was strongest between the ages of 16 and 18 years in Norway (odds ratio (OR) 1.83, 95% confidence interval (CI) 1.30-2.59), and between birth and age 5 years in Italy (OR 1.56, 95% CI 1.16-2.10). In Italy a significant association was also found during winter (OR 1.42, 95% CI 1.03-1.97). Frequent sunscreen use between birth and the age of 6 years was associated with MS in Norway (OR 1.44, 95% CI 1.08-1.93) after adjusting for outdoor activity during the same period. Red hair (OR 1.67, 95% CI 1.06-2.63) and blonde hair (OR 1.36, 95% CI 1.09-1.70) were associated with MS after adjusting for outdoor activity and sunscreen use., Conclusion: Converging evidence from different measures underlines the beneficial effect of sun exposure on MS risk., (© The Author(s) 2014.)

Background: Seasonal fluctuations in solar radiation and vitamin D levels could modulate the immune response against Epstein-Barr virus (EBV) infection and influence the subsequent risk of multiple sclerosis (MS)., Methods: Altogether 1660 MS patients and 3050 controls from Norway and Italy participating in the multinational case-control study of Environmental Factors In Multiple Sclerosis (EnvIMS) reported season of past infectious mononucleosis (IM)., Results: IM was generally reported more frequently in Norway (p=0.002), but was associated with MS to a similar degree in Norway (odds ratio (OR) 2.12, 95% confidence interval (CI) 1.64-2.73) and Italy (OR 1.72, 95% CI 1.17-2.52). For all participants, there was a higher reported frequency of IM during spring compared to fall (p<0.0005). Stratified by season of IM, the ORs for MS were 1.58 in spring (95% CI 1.08-2.31), 2.26 in summer (95% CI 1.46-3.51), 2.86 in fall (95% CI 1.69-4.85) and 2.30 in winter (95% CI 1.45-3.66)., Conclusions: IM is associated with MS independently of season, and the association is not stronger for IM during spring, when vitamin D levels reach nadir. The distribution of IM may point towards a correlation with solar radiation or other factors with a similar latitudinal and seasonal variation.

Cerebrospinal fluid and serum levels and intrathecal synthesis of anti-Epstein-Barr virus (EBV) IgG were measured by enzyme-linked immunosorbent assay in 80 relapsing-remitting multiple sclerosis patients grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. Eighty patients with other inflammatory neurological disorders (OIND) and 80 patients with non-inflammatory neurological disorders (NIND) served as neurological controls. Cerebrospinal fluid concentrations were higher in OIND than in multiple sclerosis (p < 0.0001) and NIND (p < 0.01) for anti-viral-capsid-antigen (anti-VCA) IgG, in multiple sclerosis than in NIND (p < 0.01) and in OIND than in NIND (p < 0.05) for anti-EBV nuclear antigen-1 (EBNA-1) IgG. Serum levels were more elevated in OIND than in multiple sclerosis (p < 0.05) and in MRI inactive than in MRI active multiple sclerosis (p < 0.0001) for anti-VCA IgG, and in multiple sclerosis than in OIND and NIND (p < 0.01) for anti-EBNA-1 IgG. Serum titres of anti-VCA and anti-EBNA-1 IgG were also positively (p < 0.05) and inversely (p < 0.001) correlated, respectively, with the Expanded Disability Status Scale. An intrathecal IgG production of anti-VCA and anti-EBNA-1 IgG, as indicated by Antibody Index, was present only in a limited number of multiple sclerosis patients and controls (range from 1.3 to 6.3%). These findings do not support a direct pathogenetic role of EBV-targeted humoral immune response in multiple sclerosis.

Women have about twice the risk of developing multiple sclerosis (MS) compared with men, a ratio that seems to be increasing. Most studies show that female patients seem to have a more favourable outcome of the disease. We studied the gender-specific impact of MS on health-related quality of life. We surveyed the population prevalence of MS patients in Ferrara, Italy. Data were extracted from the MS registry of the study area. Health-related quality of life was assessed using the MSQOL54 questionnaire. We analysed 370 patients (105 men and 265 women). They had worse scores than the general population in all health-related quality of life dimensions, ranging from 2.5 standard deviations (SD) lower for physical functioning to less than 0.5 standard deviations for mental health. Health-related quality of life scores were inversely correlated with disability scores. The impact of disability on health-related quality of life was higher for men than women regarding physical functioning (p < 0.01), vitality (p < 0.001), social functioning (p < 0.001), emotional wellbeing (p < 0.05) and mental health (p < 0.01). For scales reflecting mental health, a marked reduction with increasing disability was seen for men, while a linear reduction in the range of Expanded Disability Status Scale score 0-5 was reported for women, followed by no clear decrease for higher scores. We conclude that MS affects health-related quality of life in all of its dimensions. The impact of disability seems to be stronger among men, in particular for scales related to mental well-being. This could indicate that interventions should to be gender specific in order to better meet patients' needs.

Background: Little is known about the involvement of matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor TIMP-2 in multiple sclerosis (MS)., Objective: To elucidate the actual implication of MMP-2 and TIMP-2 in MS., Methods: Cerebrospinal fluid (CSF) and serum levels of active MMP-2 and TIMP-2 were measured by activity assay system and ELISA, respectively, in 67 patients with relapsing-remitting MS (RRMS), categorized according clinical and magnetic resonance imaging (MRI), and in 129 controls., Results: Cerebrospinal fluid and serum active MMP-2/TIMP-2 ratio mean values and an intrathecal active MMP-2 production were more increased in RRMS than in non-inflammatory conditions (P < 0.001, P < 0.05, and P < 0.0001, respectively) and in MRI inactive than in MRI active RRMS (P < 0.02, P < 0.01 and P < 0.001, respectively). An intrathecal synthesis of active MMP-2 was more frequent in RRMS than in inflammatory disorders (P < 0.01). Serum active MMP-2/TIMP-2 ratio and MS disease duration were positively correlated (P < 0.02)., Conclusion: These findings suggest a potential role for MMP-2 activity in the termination of MS neuroinflammation related to remission of the disease and seem to indicate that serum MMP-2/TIMP-2 ratio may represent a useful biomarker for monitoring MS recovery phase.

Cerebrospinal fluid (CSF) concentrations of soluble human leukocyte antigen class I (HLA-I) (sHLA-I), HLA-G (sHLA-G) and anti-apoptotic Fas (sFas) molecules were measured by enzyme linked immunosorbent assay technique in 65 relapsing-remitting (RR) MS patients classified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. Sixty-four patients with other inflammatory neurological disorders (OIND) and 64 subjects with noninflammatory neurological disorders (NIND) served as controls. CSF concentrations were higher in RRMS and in OIND than in NIND patients for sHLA-I (P < 0.02), greater in RRMS than in OIND and in NIND for sHLA-G (P < 0.001 and P < 0.01, respectively) and lower in RRMS than in OIND and in NIND for sFas (P < 0.001 and P < 0.02, respectively). An increase in CSF levels was identified in MRI active RRMS for sHLA-I (P < 0.01) and in MRI stable RRMS for sHLA-G (P < 0.01), whereas CSF values of sFas were decreased in RRMS without Gd-enhancing lesions (P < 0.02). In MS patients with no evidence of MRI disease activity, a trend towards an inverse correlation was found between CSF concentrations of sHLA-G and sHLA-I and between CSF levels of sHLA-G and sFas. Our results indicate that enhanced CSF levels of sHLA-I antigens most likely represent an indirect manifestation of intrathecal immune activation taking place in neuroinflammation. Conversely, reciprocal fluctuations in CSF sHLA-G and sFas levels observed when MRI disease activity resolved suggest that sHLA-G could play an immunomodulatory role in MS through Fas/FasL-mediated mechanisms.

The presence of Chlamydia-like organism DNA was investigated by polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) samples from 27 patients previously found positive for Chlamydia pneumoniae DNA: 12 with multiple sclerosis (MS), grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity, 8 with other inflammatory neurological disorders and 7 with non-inflammatory neurological disorders. PCR evidence of Chlamydia-like organisms in CSF was observed only in two relapsing-remitting MS patients with clinical and MRI disease activity. These findings suggest a possible association between C. pneumoniae and Chlamydia-like organism brain infections as a cofactor in MS development.

Studies on the distribution of multiple sclerosis (MS) carried out in Southern Europe in the last years have shown a significant increase in the frequency of the disease. A previous descriptive survey in the Republic of San Marino, northern Italian peninsula, published in 1984 established that this area is at high risk for MS. We updated the frequency estimates of the disease by adopting a complete enumeration approach. On 31 December 2005, 50 MS patients (36 women and 14 men) yielded a crude prevalence rate of 166.7 per 100, 000 (95% CI 123.7-220), 235.3 (95% CI 165-327.4) for women and 95.2 (95% CI 52-160) for men. The average incidence from 1990 to 2005 was 7.9 (95% CI 5.3-11.1) per 100,000, 11.7 (95% CI 7.6-17.3) for women and 3.9 (95% CI 1.7-7.7) for men. We did not detect any significant temporal trend over the study period. These results confirm that in San Marino the disease occurs more frequently than that suggested in the past and support the data on MS frequency in continental Italy. The marked increase in MS prevalence ratio is partly due to the increasing survival of patients and the accumulation of new incidence cases owing to the reduction in diagnostic latency for better quality of neurological diagnostic procedures. However, an increased incidence of the disease could be considered.

We evaluated the risk of worsening according to the length of exposure to interferon beta (IFNbeta) in a large cohort of 2090 multiple sclerosis patients collected by the Italian MS Database Network. Overall 44,140 patient-visits with a follow-up of 22,143 patient-years were evaluated. Forty-one per cent of patients were exposed to IFNbeta for up to 2 years, 39% for 2-4 years and 20% for more than 4 years. A Cox regression model was used to analyse two clinical outcomes: disability progression and worsening of relapse rate. The technique of propensity score was applied to reduce bias in the comparison of non-randomized groups. The risks of disability progression (HR =0.23; 95% CI: 0.17-0.30) and worsening of relapse rate (HR =0.19; 95% CI: 0.14-0.27) were reduced by about 4-5-fold in patients exposed to IFNbeta for more than four years, compared with patients exposed for up to two years. The propensity score technique confirmed the findings. The proportion of days covered by IFNbeta treatment was lower (P <0.0001) in patients exposed to IFNbeta for up to two years than in other groups. A clinical stabilization over two years of IFNbeta exposure may predict a subsequent good clinical response to treatment.

In this study, we employed a sensitive activity assay system to measure cerebrospinal fluid (CSF) and serum levels of active matrix metalloproteinase-9 (MMP-9) in 37 relapsing-remitting (RR), 15 secondary progressive (SP) and nine primary progressive (PP) multiple sclerosis (MS) patients, grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also studied, as neurological controls, 48 patients with other inflammatory neurological disorders (OIND) and 48 with non-inflammatory neurological disorders (NIND). To assess active MMP-9/TIMP-1 circuit, CSF and serum levels of MMP-9 tissue inhibitor TIMP-1 were quantified by ELISA in the same patient population. CSF mean levels of active MMP-9, CSF active MMP-9/TIMP-1 ratios and intrathecal active MMP-9 synthesis, as indicated by specific index, were more elevated in MS than in NIND (P < 0.05, < 0.02 and < 0.02, respectively), serum active MMP-9/TIMP-1 ratio was higher in MS (P < 0.01) and OIND (P < 0.02) than in NIND, and serum TIMP-1 concentrations were lower in MS than in NIND (P<0.05). More importantly, serum active MMP-9 mean levels, serum active MMP-9/TIMP-1 ratio and intrathecal production of active MMP-9 were increased in MS patients with clinical (P < 0.001, < 0.001 and < 0.05, respectively) and MRI (P < 0.001, < 0.001 and < 0.02, respectively) disease activity, whereas CSF mean concentrations of active MMP-9 and CSF active MMP-9/TIMP-1 ratio were enhanced only in MS patients with MRI evidence of disease activity (P < 0.02 and < 0.01, respectively). Altogether, these findings suggest that a shift in MMP-9/TIMP-1 balance towards proteolytic activity of MMP-9 could be relevant in MS immune dysregulation. In addition, our results indicate that CSF and serum levels of active MMP-9 may represent a potential surrogate biomarker for monitoring MS disease activity. In particular, serum active MMP-9/TIMP-1 ratio seems to be a very appropriate indicator of ongoing MS inflammation, since it is easily measurable.

The aim of this study was to provide further insight into the effective contribution of classical soluble HLA-A, B and C class Ia (sHLA-I) and non-classical soluble HLA-G class Ib (sHLA-G) molecules in immune dysregulation occurring in multiple sclerosis (MS). We evaluated by enzyme-linked immunosorbent assay (ELISA) technique intrathecal synthesis and cerebrospinal fluid (CSF) and serum levels of sHLA-I and sHLA-G in 69 relapsing-remitting (RR), 21 secondary progressive (SP) and 13 primary progressive (PP) MS patients stratified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also tested, as neurological controls, 91 patients with other inflammatory neurological disorders (OIND) and 92 with non-inflammatory neurological disorders (NIND). Eighty-two healthy volunteers served as further controls for sHLA-I and sHLA-G determinations. An intrathecal production of sHLA-I and sHLA-G detected by specific indexes was significantly more frequent in MS patients than in controls (P<0.01). An intrathecal synthesis of sHLA-I was prevalent in clinically (P<0.02) and MRI active (P<0.001) MS, whereas a CSF-restricted release of sHLA-G predominated in clinically (P<0.01) and MRI stable (P<0.001) MS. sHLA-I levels were low in the serum of clinically active (P<0.001) and high in the CSF of MRI active (P<0.01) MS. Conversely, sHLA-G concentrations were decreased in the serum of clinically stable MS (P<0.01) and increased in the CSF of MRI inactive MS (P<0.001). The trends towards a negative correlation observed between CSF and serum concentrations and intrathecal synthesis of sHLA-I and sHLA-G in patients without evidence of clinical and MRI activity confirmed that intrathecal production and fluctuations in CSF and serum concentrations of sHLA-I and sHLA-G were reciprocal in MS. Our results suggest that, in MS, a balance between classical sHLA-I and non-classical sHLA-G products modulating both MRI and clinical disease activity in opposite directions may exist.

The presence and the replicative state of human herpesvirus 6 (HHV-6) were evaluated in clinical samples from multiple sclerosis (MS) patients at the first time of MS diagnosis. HHV-6 variant B was present in peripheral blood mononuclear cells of 5/32 (15%) patients, but persisted with a latent infection. Viral sequences were present also in cerebrospinal fluid (CSF), both free in the liquid (7/32, 22%) and latent in the cellular fraction (3/32, 9%), as shown by analysis of viral transcription. In these cases, variant A was detected. HHV-6 DNA sequences present in the CSF were associated to mature viral particles. In fact, in vitro infectious assays of CSF showed the presence of replication-competent virions. These results show that about 20% of MS patients have active foci of HHV-6 variant A infection in the early stages of the disease and suggest that viral replication takes place within the central nervous system.

To further explore the link between Chlamydia pneumoniae and multiple sclerosis (MS), we examined cerebrospinal fluid (CSF) samples from 71 patients with MS and from 72 patients suffering from other inflammatory neurological disorders (OIND) or noninflammatory neurological disorders (NIND). All samples were analysed by a touchdown nested polymerase chain reaction (n-PCR) for C. pneumoniae with primer sets which amplify target sequence genes encoding the major outer membrane protein (MOMP), the 16S rRNA and the Hsp-70 protein. A molecular study was also performed to evaluate genetic diversity among isolates of C. pneumoniae and to compare chlamydial sequences. PCR was found positive in 36.6% of total MS, in 28.1% of OIND and in 37.5% of NIND patients, without any statistical differences among the various groups examined. CSF PCR evidence of C. pneumoniae was significantly more frequent in relapsing-remitting (RR) than in secondary progressive (SP) (P < 0.001) and in primary progressive (PP)MS (P < 0.05), in clinically active than in clinically stable MS (P < 0.05) and in MRI active than in MRI inactive MS (P < 0.001). The analysis of CSF expression of each single C. pneumoniae-specific gene revealed that detectable levels of MOMP were significantly more frequent in MS patients with relapse (P < 0.05), whereas PCR positivity for MOMP and 16S rRNA genes were more represented in MS patients with clinical and MRI evidence of disease activity (P < 0.05). Similar rates for MOMP and 16S rRNA genes were detected in CSF of both MS patients and controls, whereas CSF PCR positivity for Hsp-70 gene was observed in only three active RR MS patients. Sequence analysis revealed significant homologies with C. pneumoniae compared to other Chlamydial spp. These findings confirm that the C. pneumoniae detection within the central nervous system (CNS) is not selectively restricted to MS, but accounts in a variety of neurological diseases. In addition, our results suggest that CSF C. pneumoniae-specific DNA detection can occur in a subset of MS patients with clinical and MRI active RR form in whom a C. pneumoniae brain chronic persistent infection may play a significant role in the development of disease.

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), has been proposed as a possible aetiopathogenic factor in multiple sclerosis (MS). We performed a study to search the LT region of JCV genome by nested PCR in cerebrospinal fluid (CSF), peripheral blood mononuclear cell (PBMC) and urine samples collected from 121 MS patients, 24 patients with other neurological disorders (OND), 30 non neurological patients (NND) and in PBMCs and urine of 40 healthy subjects. JCV DNA has been found in the CSF of 11 MS patients (9%) while all the CSFs from the 24 OND and the 30 NND cases were negative. No significant differences have been observed as regard to the frequency of JCV DNA detection in PBMCs and urine between the MS patients and the control groups. Nucleotide sequences analysis of seven JCV CSF isolates showed that five strains were identical the prototypal strain, while the other two had a base mutation (T-->C) in 4286 nucleotide (nt). The finding of JCV DNA in the CSF of MS patients suggest that JCV could play a role in the triggering and/or in the maintenance of MS aetiopathogenic process, and therefore it should be taken in consideration when monitoring this disease.