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Objectives To investigate the associations between antibody responses to herpesviruses and the development of thalamic, total deep gray matter, cortical and central atrophy in high-risk clinically isolated syndromes (CIS) after the first demyelinating event. Methods We analyzed volumetric brain outcomes in 193 CIS patients enrolled in a multi-center study of high-risk CIS. All patients had 2 or more MRI brain lesions and two or more oligoclonal bands in cerebrospinal fluid. Serum samples obtained at the screening visit prior to any treatment were analyzed for IgG antibodies against cytomegalovirus (anti-CMV) and Epstein–Barr virus (EBV) viral capsid antigen (VCA). All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months. Results Anti-EBV VCA highest quartile status was associated with regional atrophy measures for percent decrease in thalamus. Anti-CMV positivity was associated with greater total deep gray matter atrophy and whole brain atrophy. Anti-EBV VCA highest quartile status was associated as trends with greater whole brain, gray matter atrophy and central atrophy. The associations of anti-EBV VCA antibodies with thalamic atrophy were mediated by its associations with T2 lesions whereas the associations of anti-CMV positivity with deep gray matter atrophy were relatively independent of T2 lesions. Conclusions Antibody responses to EBV and CMV are associated with global and regional brain atrophy in CIS patients treated with interferon-beta.

Objectives To investigate whether anti-herpesvirus antibodies are associated with serum cholesterol profiles in clinically isolated syndromes (CIS). Methods Pre-treatment serum samples from 118 high-risk CIS patients were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) viral capsid antigen (VCA) and EBV nuclear antigen-1 (EBNA-1). A lipid profile consisting of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) was obtained. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months after start of interferon-beta treatment. Results The study included 118 CIS patients (77 females, 41 males, 65.3% female; mean age: 28.1 ± SD 8.1 years). Anti-EBV EBNA-1 antibody levels were associated with LDL-C ( p = 0.009) and TC ( p = 0.008) levels. Anti-CMV positivity status was associated with reduced time to relapse ( p = 0.006) and the greater number of relapses ( p = 0.009) in patients with high HDL-C. Anti-EBV VCA antibody levels were associated with greater number of new T2 lesions ( p = 0.002) and with increased brain atrophy ( p Conclusions Our results indicate that higher levels of anti-EBV EBNA-1 antibodies are associated with higher LDL-C and TC levels. Anti-CMV positive individuals have greater disease progression in the presence of higher HDL-C levels. Individuals with higher levels of anti-EBV VCA antibodies have greater progression on MRI measures in the presence of higher LDL-C.

Anti-myelin basic protein (MBP) antibodies in pediatric-onset MS and controls were characterized. Serum samples were obtained from 94 children with MS and 106 controls. Paired CSF and serum were obtained from 25 children with MS at time of their initial episode of acute demyelinating syndrome (ADS). Complementary assays were applied across samples to evaluate the presence, and the physical binding properties, of anti-MBP antibodies. While the prevalence and titers of serum anti-MBP antibodies against both immature and mature forms of MBP were similar in children with MS and in controls, binding characteristics and formal Surface Plasmon Resonance (SPR) studies indicated surprisingly high binding affinities of all pediatric anti-MBP antibodies. Serum levels of anti-MBP antibodies correlated significantly with their CSF levels, and their presence in children with MS was associated with significantly increased risk of an acute disseminated encephalomyelitis-like initial clinical presentation. While antibodies to both immature and mature forms of MBP can be present as part of the normal pediatric humoral repertoire, these anti-myelin antibodies are of surprisingly high affinity, can access the CNS during inflammation, and have the capacity to modulate disease expression. Our findings identify an immune mechanism that could contribute to the observed heterogeneity in spectrum of clinical presentations in early-onset MS.

Purpose To determine the role of gene–environmental interactions between the Class I and Class II HLA alleles and the humoral anti-Epstein–Barr Virus (EBV) responses in the development of brain injury and clinical disability in multiple sclerosis (MS) patients. Methods A total of 93 MS patients (62 females; 31 males) and 122 healthy controls underwent HLA typing and testing for antibodies against EBV. The MS patients underwent brain MRI and quantitative measurements of T1- and T2-lesion volumes (LVs) and brain parenchymal fraction (BPF) were obtained. There were 54 MS cases that underwent MRI and EBV-antibody assessments at the 3-year follow-up. The anti-EBV panel included measurements of the levels of anti-EBV early antigen (EA) IgG, anti-EBV nuclear antigen (EBNA) IgG and anti-EBV viral capsid antigen (VCA) IgM and anti-EBV VCA IgG. The relationships between HLA alleles, anti-EBV antibody levels, MRI and clinical parameters were assessed in regression analysis. Results The presence of HLA B7 was associated with increased T1-LV and trends indicating increased anti-EBV VCA IgG levels, higher disability (EDSS) and more destructive MRI parameters (increased T2-LV and decreased BPF). The presence of HLA A2 was associated with lower EDSS and a trend toward decreased anti-EBV VCA IgG levels; the associations with MRI variables were not significant. The HLA B7–A2 haplotype was significantly associated with higher T2-LV and T1-LV and a trend toward lower BPF was observed. Conclusions Our data suggest that gene–environment interactions between specific HLA Class I loci and EBV exposure are associated with MRI markers of lesion injury and brain atrophy in MS patients.

Purpose To characterize gene expression in multiple sclerosis (MS) patients after the first dose and chronic dosing of 30 µg, once weekly, intramuscular interferon-β1a (IFN-β) and to delineate the pharmacogenomic differences between Good Responders and Partial Responders to IFN-β therapy. Methods The treatment responses after the first IFN-β dose and chronic IFN-β dosing were assessed in 22 relapsing MS patients (17 females, 5 males; average age: 41.5 ± SD 10.4 years). Gene expression profiles in peripheral blood mononuclear cells were obtained prior to treatment and at 1, 2, 4, 8, 24, 48, 120, 168 h after the first IFN-β dose and at 1, 6 and 12 months after chronic dosing with once-weekly 30 µg IFN-β-1a intramuscularly. Repeated measures statistics with false discovery rate control were used. The functional characteristics, biological pathways and transcription factor sites were analyzed. Results Of the 1000 genes modulated following the first dose and upon chronic dosing of IFN-β in MS patients, approximately 35% were up-regulated and 65% were down- regulated; the percentage of modulated genes in common was approximately 50%. The expression of the pharmacodynamic mRNA markers of IFN-β effect showed differences in time profiles for the Good Responder and Partial Responders to IFN-β therapy and the Jak-STAT, TNFRSF10B, IL6, TGFβ, retinoic acid and CDC42 pathways were differentially modulated. The patients with side effects to therapy showed differences in the TGFβ1, IFNG/STAT3 and TNF pathways. Conclusions Gene expression is a valuable tool for understanding the molecular mechanisms of IFN-β action in MS patients.

The myxovirus resistance A (MXA) mRNA has been extensively investigated for assessing the biologic responses of multiple sclerosis (MS) patients to interferon-beta (IFN-beta) therapy. The objective of this study was to evaluate the associations between two MXA promoter region single nucleotide polymorphisms (rs2071430 and rs17000900) and the gene expression responses, clinical and MRI phenotypes in IFN-beta treated MS patients. The rs2071430 and rs17000900 SNPs, which are located in or near an interferon-stimulated response element (ISRE), were genotyped in 179 relapsing MS patients. Quantitative MRI measurements were available for 101 patients on IFN-beta monotherapy. Gene expression was assessed in 22 anti-interferon-beta neutralizing antibody negative patients. No significant association was found between the MXA genotype at these two SNPs and clinical, MRI and MXA gene expression in MS patients treated with IFN-beta therapy.

The objective of this study was to evaluate multiple interferon (IFN) specific mRNA biomarkers in multiple sclerosis (MS) patients with anti-IFN-beta neutralizing antibodies (NAB) using a pharmacodynamic study design. Thirty patients were enrolled. Blood samples were drawn at pre-treatment, 4-, 8-h time points following the intramuscular dose of IFN-beta-1a. Total RNA was obtained from peripheral blood cells, processed to cDNA and analyzed using quantitative real-time polymerase chain reaction. Pre-treatment serum samples were analyzed for anti-IFN-beta binding and neutralizing antibodies: 22 patients were NAB negative; equal numbers of the eight remaining patients were either NAB positive or had borderline NAB status. The results showed that early assessment (at 4 h after IFN-beta injection) of mRNAs for Stat-1, MxA, MxB and TRAIL was more sensitive than the later measurements. Furthermore, the NAB positive patients had strongly attenuated gene expression responses on all the mRNAs. Patients with borderline NAB had average responses that appear to be lower than NAB negative patients on several genes, notably Stat-1, TRAIL and beta2 microglobulin.

Background and objective : This report provides results of CSF analyses done in a subset of relapsing remitting MS patients participating in a placebo-controlled, double-blind, phase III clinical trial of IFNβ-Studies supported by the National Multiple Sclerosis Society (grants RG2019, RG2827),a (Avonex®, Biogen). The clinical trial demonstrated that IFNβ-1a treatment resulted in significantly reduced disability progression, annual relapse rate, and new brain lesions visualized by cranial magnetic resonance imaging. The objectives of the current study were to determine: (a) whether CSF abnormalities in MS patients correlated with disease or MRI characteristics, and (b) effects of IFNβ-1a therapy on these CSF abnormalities. Methods : CSF was analyzed from 262 (87%) of the 301 study subjects at entry into the clinical trial, and a second CSF sample was analyzed from 137 of these 262 subjects after 2 years of therapy. CSF cell counts, oligoclonal bands (OCB), IgG index, and free kappa light chains were measured using standard assays. Baseline CSF results were compared with demographic, disease, and MRI parameters. Differences in on-study relapse rate, gadolinium enhancement, and EDSS change according to baseline CSF status was used to determine the predictive value of CSF for subsequent clinical and MRI disease activity. Change in CSF parameters after 104 weeks were used to determine the effects of treatment. Results : (1) At study baseline, 37% of the subjects had abnormal CSF WBC counts, 61% had abnormal levels of CSF free kappa light chains, 84% had abnormal IgG index values, and 90% were positive for OCB. (2) Baseline IgG index, kappa light chains, and OCB showed weakly positive, statistically significant correlations with Gd-enhanced lesion volume and T2 lesion volume. WBC showed a statistically significant correlation with Gd-enhancing lesion volume but was uncorrelated with T2 lesion volume. (3) There was an associated between baseline CSF WBC counts and on-study clinical and MRI disease activity in placebo recipients. (4) IFNβ-1a treatment resulted in significantly reduced CSF WBC counts, but there was no treatment-related change in CSF IgG index, kappa light chains, or OCB, which remained relatively stable over time in both patient groups. Conclusions : The current study documents significant reductions in CSF WBC counts in patients treated with IFNβ-1a for 104 weeks. This finding is considered relevant to the therapeutic response, since CSF WBC counts were found to be positively correlated with subsequent clinical and MRI disease activity in placebo-treated relapsing MS patients.

Objective To examine the associations of alcohol consumption patterns with disability and brain injury in multiple sclerosis (MS) patients. Design This study included 423 subjects (272 MS patients, 151 healthy controls) participating in a study of clinical, environmental and genetic risk factors in MS. Disability was assessed with the Expanded Disability Status Scale (EDSS) and the MS Severity Scale (MSSS). Brain injury was assessed using the quantitative MRI measures of T2-lesion volume (T2-LV), T1-LV, normalized volumes of brain parenchyma (NBV), gray matter (NGMV) and lateral ventricle (NLVV). Information related to alcohol-consumption patterns was obtained with standardized questionnaire during an in-person interview. The associations of alcohol consumption variables with disability and MRI measures were assessed in regression analyses. Results The frequency of MS patients who did not consume alcohol after MS (19.4%) was higher than the frequency before MS (p

The presence of MRI lesions at the first demyelinating event increases the risk of developing clinically definite multiple sclerosis (MS). The HLA DRB1*1501 genotype is linked to MS susceptibility but its relationship to quantitative MRI parameters at the first demyelinating event has not been assessed. The objectives were to assess the associations between HLA DRB1*1501 status and magnetic resonance imaging (MRI) measures in clinically isolated syndromes (CIS) at the first demyelinating event.We genotyped 205 CIS patients (age: 29.0±7.7 years) enrolled in the Observational Study of Early Interferon beta 1-a Treatment in High Risk Subjects after CIS (SET study), a multi-center, clinical study of CIS for rs3135005, a single nucleotide polymorphism associated with HLA DRB1*1501 status. The inclusion criteria required 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. Clinical and MRI assessments were obtained within 4 months of the initial demyelinating event.The frequency of HLA DRB1*1501 positivity was 102/205 (49.7%). HLA DRB1*1501 positivity was associated with higher contrast-enhancing (CE) lesion number (p=0.002), higher CE-lesion volume (LV) (p0.001) and exhibited a trend with higher T2-LV (p=0.012). There was no evidence for significant associations of HLA DRB1*1501 positivity with disability, symptoms at CIS presentation, whole brain, white and gray matter atrophy.HLA DRB1*1501 positivity is associated with increased brain inflammatory processes at first clinical onset. However, the effect sizes of the HLA DRB1*1501 associations with MRI are modest, which potentially limits the clinical usefulness.

The study is aimed to determine the role of quercetin (3,3'4',5,7-pentahydroxy flavone), alone and in combination with human interferon-beta (IFN-beta), in modulating the immune response(s) of peripheral blood mononuclear cells (PBMC) isolated from multiple sclerosis (MS) patients and from normal healthy subjects. PBMC proliferation in the presence or absence of these drugs was determined and the production of proinflammatory cytokines (IL-1beta, TNF-alpha), and the ratio of cell migration mediator MMP-9, and its inhibitor, TIMP-1 were assessed in the culture supernatants. Quercetin reduced, in a dose-dependent manner, the proliferation of PBMC and modulated the level of IL-1beta and TNF-alpha released by PBMC in the culture supernatants. Quercetin reduced the MMP-9/TIMP-1 ratio via lowering MMP-9 production. Quercetin, when combined with IFN-beta, had additive effects in modulating TNF-alpha and MMP-9. These immunomodulatory responses to quercetin were similar between MS patients and healthy control (HC) subjects.

Purpose To investigate the relationship between immune cell secretion of brain-derived neurotrophic factor (BDNF) with clinical and MRI variables in multiple sclerosis (MS) patients. Background BDNF exerts beneficial effects on neuronal growth and repair and is secreted by both neurons and immune cells. Consequently, it may mediate the crosstalk between the immune system and CNS in autoimmune diseases such as MS. Methods Fifty-two relapsing MS patients (41 females, age: 48.8 ± 6.6 years, disease duration: 12.7 ± 8.4 years) were enrolled. Clinical and MRI measurements (including, T1-, T2- and contrast-enhancing (CE) lesion volumes (LVs); normalized measures of whole brain, white matter (WM) and gray matter (GM) volumes; diffusion weighted imaging measure of mean whole brain (WB) parenchyma diffusivity and magnetization transfer ratio (MTR) measures were obtained. Results Immune cell BDNF secretion after anti-CD3 plus anti-CD28 stimulation was positively associated with increased CE–LV (p = 0.026). The MTR of CE–LV and normal-appearing (NA) WM (NAWM) were negatively associated with immune cell BDNF secretion after anti-CD3 plus anti-CD28 stimulation. Immune cell BDNF secretion after anti-CD3 plus anti-CD28 was positively associated with higher WM volume (p = 0.027). Immune cell BDNF secretion after anti-CD3 plus anti-CD28 stimulation was decreased with increasing disease duration (p = 0.031). The BDNF secretion was independent of the BDNF Val66Met (dBSNP ID: rs6265) SNP genotype. Conclusions Immune cell BDNF secretion is associated with the sites of higher inflammatory activity as evidenced by CE lesions and may represent an important factor associated with the WM volume of patients with MS.

Background: Autoreactive antibodies (ARAB) occur more frequently in patients with multiple sclerosis (MS) than in general population and the presence of these antibodies often causes uncertainty regarding the disease course, response to therapy and the diagnosis of MS. Methods: Retrospective analyses of the ARAB, clinical and MRI data of a consecutive patient cohort of MS and clinically isolated syndrome (CIS) patients were conducted. The patients were evaluated for an extensive panel that included various subtypes of antiphospholipid antibody (APLA) including anti-phosphatidylethanolamine (APE), anti-phosphatidylserine (APS), anti-beta-2-glycoprotein-1 (ABGP), anti-cardiolipin (ACA), and several other ARAB such as antinuclear antibody (ANA), anti-neutrophilic cytoplasmic antibodies (ANCA), anti-thyroid peroxidase antibodies (ATA), anti-SS-A, and anti-SS-B antibodies. Quantitative MRI analysis was performed in a subgroup of MS patients measuring T2-lesion volume (LV), T1 black hole LV and brain parenchymal fraction (BPF). Results: A total of 137 patients (mean age 44.7, 84% female) with either MS ( n = 111; age: mean 46.5 ± S.D. 10.3 years; disease duration: mean 13.0 ± S.D. 10.4 years; EDSS: mean 3.2 ± S.D. 1.9) or CIS ( n = 26; age: mean 37.7 ± S.D. 7.8 years; disease duration: mean 1.3 ± S.D. 1.1 years; EDSS: mean 1.0 ± S.D. 0.7) were enrolled. Among MS patients, 82 were RRMS, 26 SPMS, and 3 had PPMS. Seventy-seven (69%) of MS patients showed presence of one or more ARAB. The proportion of MS patients with APLA was 55% (61 patients); IgM subtype was most frequent. Co-occurrence of ACA and APE was more frequent in SPMS as compared to RRMS (15.4% vs. 1.2%, p = 0.012). The proportion of CIS patients with ARAB was 75% with IgM subtype being the most frequent. However, the ARAB in majority of CIS patients (9 out of 14, 64%) were transient on repeated testing. In a subgroup of 62 MS patients, quantitative MRI analysis showed significantly higher T2-LV in patients with positive APLA (15.1 ml for APLA positive vs. 6.75 ml for APLA negative) after correcting for the disease duration ( p = 0.048). The patients with ATA also had significantly higher T2-LV after correction for disease duration (19.0 ml vs.8.5, p = 0.044). Conclusions: ARAB were present in more than two thirds of MS and CIS patients although most of APLA in CIS were transient. The presence of APLA in MS patients was associated with higher T2-LV.

Purpose : To investigate the effects of interferon-β-1a (IFN-β-1a) on the trafficking of cell populations in peripheral blood cells of multiple sclerosis (MS) patients. Methods : In this open-label pharmacodynamic study, peripheral blood was obtained from 10 relapsing–remitting (RR) MS patients just prior to and at 1, 2, 4, 8, 24, 48, 120, and 168 h after intramuscular injection of 30-μg IFN-β-1a. Timed samples were also obtained from five controls at 0, 8, 24, 48 and 168 h. The blood cells were analyzed using four-color flow cytometry with antibody conjugates directed against cell surface proteins specific for T cells, B cells, NK cells, and the activation marker, CD69. Results : IFN-β-1a treatment resulted in selective, time-dependent effects on many cell populations in peripheral blood. The trafficking of T-helper and T-suppressor/cytotoxic subsets of T cells were qualitatively different. The most prominent effects were on the trafficking of natural killer cells, the levels of which decreased to 23.5% of pretreatment values at 8 h after treatment. The levels of CD69-positive NK cells increased to a peak value of 606% of pretreatment levels at the 24-h time point. In untreated controls, these characteristic trafficking effects were not observed. There was inter-patient heterogeneity in the levels of activated NK cells at the 6-month time point that may potentially be relevant for individualizing IFN-β therapy. Conclusions : IFN-β treatment can induce specific, selective, and time-dependent trafficking of cells and its effects on different subsets of a given cell type are not qualitatively similar. The dynamics indicate that the activation of NK cells by IFN-β is possibly dependent on the trafficking of NK cells. The activated NK cell levels after prolonged therapy may potentially provide a surrogate marker for IFN-β exposure.

Objectives: To use DNA arrays to identify differences in gene expression associated with relapsing–remitting (RR) MS. Methods: Total RNA was isolated from monocyte depleted peripheral blood mononuclear cells of 15 RR MS patients and 15 age- and sex-matched controls. The RNA was reverse transcribed to radiolabeled cDNA and the resultant cDNA was used to probe a DNA array containing over 4000 named human genes. The binding of radiolabeled cDNA to the probes on the array was measured by phosphorimager. Results: Of more than 4000 genes tested, only 34 were significantly different in RR-MS patients from controls. Of these, 25 were significantly increased and 9 significantly decreased in the RR MS patients. Twelve of these genes have inflammatory and/or immunological functions that could be relevant to the MS disease process. The potentially relevant genes that were elevated (15% to 28%) were P protein, LCK, cAMP responsive element modulator, IL-7 receptor, matrix metalloproteinase-19, M130 antigen, and peptidyl–prolyl isomerase. Those that were significantly decreased (15% to 35%) were SAS transmembrane 4 superfamily protein, STRL22 (C–C chemokine receptor 6), AFX protein, DNA fragmentation factor-45 and immunoglobulin gamma 3 (Gm marker). Conclusions: The RR-MS disease effect was relatively restricted and most of the mRNAs tested were not different from the normal controls. However, there were significant differences identified in the expression of a subset of mRNAs, including 13 with inflammatory/immune functions that could be relevant to MS. The systematic use of DNA arrays can provide insight into the dynamic cellular pathways involved in MS pathogenesis and its phenotypic heterogeneity.