Objective: Because dysregulated Epstein-Barr virus (EBV)-infected B cells may induce meningeal inflammation, which contributes to cortical pathology in multiple sclerosis (MS), we investigated associations between antibody responses to EBV and development of cortical pathology in MS. Methods: We included 539 patients with MS (369 with relapsing-remitting MS, 135 with secondary progressive MS, and 35 with primary progressive MS), 66 patients with clinically isolated syndrome (CIS), 63 patients with other neurologic diseases (OND), and 178 age- and sex-matched healthy controls (HC). All participants were scanned on 3T MRI. Serum samples were analyzed for IgG antibodies against EBV viral capsid antigen (VCA) and EBV nuclear antigen-1 (EBNA-1), and their quartiles were determined on the whole study sample. Differences between the study groups were assessed using analysis of covariance adjusted for multiple comparisons. Results: More than 30% of patients with MS and CIS presented with the highest quartile of anti-EBV-VCA and -EBNA-1 status compared to ≤10% of HC (p < 0.001). The figures were 9 (14.3%) and 7 (12.3%) for patients with OND. Patients with MS with the highest quartile of anti-EBV-VCA showed significantly increased T2 lesion volume (p = 0.001), T1 lesion number (p = 0.002), and T1 lesion volume (p = 0.04) and decreased gray matter (p = 0.041) and cortical (p = 0.043) volumes compared to patients with MS with lower quartiles. No significant differences of MRI outcomes in patients with CIS, patients with OND, and HC with lower or highest quartiles of anti-EBV-VCA and -EBNA-1 were detected. Conclusions: Humoral response to anti-EBV-VCA and -EBNA-1 is associated with more advanced cortical atrophy, accumulation of chronic T1 black holes, and focal white matter lesions in patients with MS.

Objectives: To investigate the associations between antibody responses to Epstein-Barr virus (EBV) and the development of cortical pathology in multiple sclerosis (MS). Background: Cortical pathology could be the net result of cortical subpial lesions. Dysregulated EBV-infected B cells may induce meningeal inflammation that could contribute to cortical pathology in MS. Methods: In this prospective study, we included 539 MS patients (342 relapsing-remitting, 135 secondary-progressive and 35 primary-progressive), 66 patients with clinically isolated syndrome (CIS) and 178 age- and sex- matched healthy controls (HC). All subjects were scanned on a 3T MRI scanner and serum samples were obtained and analyzed for IgG antibodies against EBV viral capsid antigen (VCA) and nuclear antigen-1 (EBNA-1). Participants were grouped based on disease status and disease course. MRI measures assessed brain and lesion volumes (LVs). Differences between the groups were assessed using analysis of covariance, adjusted for age. Results: More than 30[percnt] of MS and CIS patients presented with highest quartile of VCA and EBNA-1 status, compared to less than 10[percnt] of HCs (p<0.001). MS patients with the highest quartile of VCA showed significantly increased T2-LV (p=0.001), T1-LV (p=0.002) and decreased gray matter (GM) (p=0.041) and cortical (p=0.05) volumes, compared to MS patients with lower quartiles. These differences were significant in both relapsing-remitting and secondary-progressive MS patients. In addition, relasping-remitting patients with the highest quartile of EBNA-1 showed decreased GM (p=0.008) and cortical volumes (p=0.035) and increased T1-LV (p=0.035). No significant differences in MRI measures between HCs with lower or highest quartiles of VCA and EBNA were detected. Conclusions: Humoral response to anti-EBV VCA and EBNA-1 is associated with more advanced cortical pathology and accumulation of chronic T1 black hole lesions in patients with with relapsing-remitting and secondary-progressive MS. Humoral response to anti-EBV VCA is also associated with more severe WM focal pathology. Supported by:

Background Oxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers. Methods A method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated. Results Our method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis. Conclusion The method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.

To compare frequencies of autoreactive antibody responses to endogenous disease-associated antigens in healthy controls (HC), relapsing and progressive MS and to assess their associations with clinical and MRI measures of MS disease progression. Methods The study analyzed 969 serum samples from 315 HC, 411 relapsing remitting MS (RR-MS), 128 secondary progressive MS (SP-MS), 33 primary progressive MS (PP-MS) and 82 patients with other neurological diseases for autoantibodies against two putative MS antigens CSF114(Glc) and KIR4.1a and KIR4.1b and against 24 key endogenous antigens linked to diseases such as vasculitis, systemic sclerosis, rheumatoid arthritis, Sjogren’s syndrome, systemic lupus erythematosus, polymyositis, scleroderma, polymyositis, dermatomyositis, mixed connective tissue disease and primary biliary cirrhosis. Associations with disability and MRI measures of lesional injury and neurodegeneration were assessed. Results The frequencies of anti-KIR4.1a and anti-KIR4.1b peptide IgG positivity were 9.8% and 11.4% in HC compared to 4.9% and 7.5% in RR-MS, 8.6% for both peptides in SP-MS and 6.1% for both peptides in PP-MS (p = 0.13 for KIR4.1a and p = 0.34 for KIR4.1b), respectively. Antibodies against CSF114(Glc), KIR4.1a and KIR4.1b peptides were not associated with MS compared to HC, or with MS disease progression. HLA DRB1*15:01 positivity and anti-Epstein Barr virus antibodies, which are MS risk factors, were not associated with these putative MS antibodies. Conclusions Antibody responses to KIR4.1a and KIR4.1b peptides are not increased in MS compared to HC nor associated with MS disease progression. The frequencies of the diverse autoreactive antibodies investigated are similar in MS and HC.

OBJECTIVE:In this study, we survey the frequencies of auto-reactive antibody responses to endogenous disease-associated antigens in healthy controls (HC), relapsing and progressive MS and to assess their associations with clinical and MRI measures of MS disease progression. BACKGROUND:Neurodegeneration in Multiple Sclerosis (MS) is driven by complex molecular mechanisms involving cellular and humoral immune histotoxicity in combination with altered metabolic and intracellular processes. DESIGN/METHODS:969 serum samples from 315 healthy controls (HC), 411 relapsing remitting MS (RR-MS), 128 secondary progressive MS (SP-MS), 33 primary progressive MS (PP-MS) and 82 patients with other neurological diseases for autoantibodies against two putative MS antigens CSF114, KIR4.1 a and b peptides and 26 key auto-antigens linked to systemic and organ specific autoimmune diseases using ELISA or LIA (Line Immunoassay). Associations with disability and MRI measures of lesional injury and neurodegeneration were also assessed. RESULTS:The frequencies of anti-KIR4.1a and anti-KIR4.1b IgG positivity were 9.8[percnt] and 11.4[percnt] in HC compared to 4.9[percnt] and 7.5[percnt] in RR-MS, 8.6[percnt] for both peptides in SP-MS and 6.1[percnt] for both peptides in PP-MS (p = 0.13 for KIR4.1a and p = 0.34 for KIR4.1b), respectively. These putative MS antibodies were not associated with MS compared to HC, or with MS disease progression. HLA DRB1*1501 positivity and anti- Epstein Barr virus antibodies, which are MS risk factors, were not associated with these putative MS antibodies. CONCLUSIONS:Antibody responses to KIR4.1a and KIR4.1b peptides are not increased in MS compared to HC nor associated with MS disease progression. The frequencies of the diverse auto-antibodies investigated are similar in MS and HC. Study Supported by:Immco Diagnostics Inc.,Buffalo, NY 14228

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 There is increasing evidence that serum lipoprotein cholesterol biomarkers are associated with disease progression in clinically isolated syndromes (CIS). Apolipoproteins (Apo) are recognition ligands that mediate the physiological interactions of cholesterol-containing lipoproteins. The objective of this study was to investigate whether serum Apo levels are associated with CIS disease progression. Methods ApoB, ApoAI, ApoAII, ApoE and lipoprotein (a) (Lpa) levels were measured in serum samples obtained prior to the start of treatment from 181 CIS patients (123 women, 58 men, 68% women; mean age: 28.1±SD 8.1 years). All patients were treated with intramuscular interferon-β as part of the prospective study. Clinical and MRI assessments were obtained at baseline, 6, 12 and 24 months after start of interferon-β treatment. Results Greater ApoB levels were associated with increased number of new T2 lesions (p<0.001) and increased number of new or enlarging T2 lesions (p<0.001) over 2 years. Each 10 mg/dL of greater baseline ApoB is associated with a 16% increase in the number of new T2 lesions over 2 years. ApoAI, ApoAII, ApoE and Lpa were not associated with T2 lesions. Greater ApoE levels were associated with greater deep grey matter atrophy (partial correlation rp=−0.28, p<0.001). Each 1 mg/dL increment in ApoE levels was associated with a 1% increase in deep grey matter atrophy over 2 years. Conclusions Serum ApoB levels are associated with new lesion accumulation whereas ApoE levels are associated with deep grey matter atrophy in high risk CIS patients treated with interferon β-1a.

Context: High serum cholesterol is adversely associated with clinical and imaging disease progression outcomes in multiple sclerosis (MS) and in clinically isolated syndrome (CIS), the earliest stage of MS. Low vitamin D levels are associated with an increased risk of disease progression.Objectives: To investigate the mechanisms mediating the adverse effects of cholesterol in CIS and to determine the role of the nexus between the vitamin D-3 (D-3) and cholesterol pathways.Design: Multi-center, prospective, longitudinal prospective study.Setting: University hospital multiple sclerosis centers.Intervention: Serum samples were obtained prior to any treatment from study subjects.Methods: Serum obtained prior to any treatment from 172 CIS patients enrolled in a multi-center, prospective, longitudinal study (119 females: 53 males, age: 28.1 +/- SD 8.1 years) were analyzed for high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein AI (ApoAI), ApoAII, ApoB, ApoE, and lipoprotein-a. Levels of 25-hydroxy vitamin D-3 (25(OH)D-3), 1,25-dihydroxy D-3, and 24,25-dihydroxy D-3 were measured using liquid chromatography-mass spectrometry.Results: Greater levels of HDL-C biomarkers (e.g., HDL-C itself, ApoAI, ApoAII and paroxonase arylesterase activity) and LDL-C biomarkers (e.g., LDL-C itself, Apo B) were associated with greater 25(OH)D3. The effects of HDL-C biomarkers were stronger than those of LDL-C. Free cholesterol and cholesteryl ester levels were positively associated with higher 25(OH)D3 levels. Cholesterol palmitate was particularly potent. The nexus between the D3 and cholesterol pathways was proximal to, or in linkage disequilibrium with, 7-dehydrocholesterol reductase DHCR7 rs1790349, endothelial lipase LIPG rs4939883 and proprotein convertase subtilisin/kexin type 9 PCSK9 rs11206510.Conclusions: The associations between cholesterol biomarkers and vitamin D metabolite levels in CIS are consistent with the biochemical inter-dependence between the two pathways. Cholesterol biomarkers should be considered for inclusion as covariates when assessing vitamin D levels in CIS. (C) 2014 Elsevier Ltd. All rights reserved.

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<0.001) in patients with high LDL-C.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.

Objectives To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event. Methods We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months. Results The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies. Conclusions Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.

OBJECTIVES:To investigate the associations of serum lipid profile with disease progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event.METHODS:High density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) were obtained in pretreatment serum from 135 high risk patients with CIS (≥ 2 brain MRI lesions and ≥ 2 oligoclonal bands) enrolled in the Observational Study of Early Interferon β-1a Treatment in High Risk Subjects after CIS study (SET study), which prospectively evaluated the effect of intramuscular interferon β-1a treatment following the first demyelinating event. Thyroid stimulating hormone, free thyroxine, 25-hydroxy vitamin D3, active smoking status and body mass index were also obtained. Clinical and MRI assessments were obtained within 4 months of the initial demyelinating event and at 6, 12 and 24 months.RESULTS:The time to first relapse and number of relapses were not associated with any of the lipid profile variables. Higher LDL-C (p=0.006) and TC (p=0.001) levels were associated with increased cumulative number of new T2 lesions over 2 years. Higher free thyroxine levels were associated with lower cumulative number of contrast-enhancing lesions (p=0.008). Higher TC was associated as a trend with lower baseline whole brain volume (p=0.020). Higher high density lipoprotein was associated with higher deseasonalised 1,25-dihydroxy vitamin D3 (p=0.003) levels and a trend was found for deseasonalised 25-hydroxy vitamin D3 (p=0.014).CONCLUSIONS:In early multiple sclerosis, lipid profile variables particularly LDL-C and TC levels are associated with inflammatory MRI activity measures.

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<0.001). The EDSS, NGMV and NLVV exhibited a non-linear dependence on duration of alcohol consumption after MS onset: non-linear regression analyses indicated that EDSS and NLVV were lower and the NGMV was greater in MS patients who had consumed for a period of 15years or less after MS onset compared those who did not consume alcohol or consumed it for more than 15years.CONCLUSION:The duration of alcohol consumption is associated with disability and MRI measures in MS. Prospective, longitudinal studies of the role of alcohol in MS disease progression are warranted.

Background: Chronic cerebrospinal venous insufficiency (CCSVI) was described as a vascular condition characterized by anomalies of veins outside the skull was reported to be associated with multiple sclerosis (MS). The objective was to assess the associations between HLA DRB1*1501 status and the occurrence of CCSVI in MS patients.Methodology/Principal Findings: This study included 423 of 499 subjects enrolled in the Combined Transcranial and Extracranial Venous Doppler Evaluation (CTEVD) study. The HLA DRB1*1501 status was obtained in 268 MS patients and 155 controls by genotyping rs3135005, a SNP associated with DRB1*1501 status. All subjects underwent a clinical examination and Doppler scan of the head and neck. The frequency of CCSVI was higher (OR = 4.52, P < 0.001) in the MS group 56.0% vs. 21.9% in the controls group and also higher in the progressive MS group 69.8% vs. 49.5% in the non-progressive MS group. The 51.9% frequency of HLA DRB1*1501 positivity (HLA(+)) in MS was higher compared (OR = 2.33, P < 0.001) to 31.6% to controls. The HLA+ frequency in the non-progressive (51.6%) and progressive MS groups (52.3%) was similar. The frequency of HLA(+) CCSVI+ was 40.7% in progressive MS, 27.5% in non-progressive MS and 8.4% in controls. The presence of CCSVI was independent of HLA DRB1*1501 status in MS patients.Conclusions/Significance: The lack of strong associations of CCSVI with HLA DRB1*1501 suggests that the role of the underlying associations of CCSVI in MS should be interpreted with caution. Further longitudinal studies should determine whether interactions between these factors can contribute to disease progression in MS.

PURPOSE:To characterize gene expression in multiple sclerosis (MS) patients after the first dose and chronic dosing of 30 microg, once weekly, intramuscular interferon-beta1a (IFN-beta) and to delineate the pharmacogenomic differences between Good Responders and Partial Responders to IFN-beta therapy. METHODS:The treatment responses after the first IFN-beta dose and chronic IFN-beta 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-beta dose and at 1, 6 and 12 months after chronic dosing with once-weekly 30 microg IFN-beta-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-beta 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-beta effect showed differences in time profiles for the Good Responder and Partial Responders to IFN-beta therapy and the Jak-STAT, TNFRSF10B, IL6, TGFbeta, retinoic acid and CDC42 pathways were differentially modulated. The patients with side effects to therapy showed differences in the TGFbeta1, IFNG/STAT3 and TNF pathways. CONCLUSIONS:Gene expression is a valuable tool for understanding the molecular mechanisms of IFN-beta action in MS patients.

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, T I-, 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 (W13) parenchyma diffasivity 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 WNI 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. (c) 2007 Elsevier B.V. All rights reserved.

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.

PURPOSE:Multiple sclerosis (MS) patients have a high risk of low bone density. The purpose of this study was to examine the molecular mechanisms potentially capable of modulating bone homeostasis in response to interferon-beta-1a (IFN-beta-1a) treatment and the focus was the bone-modulating system comprised of receptor activator of nuclear factor-kappaB (RANK), its ligand RANKL and its decoy receptor, osteoprotegerin (OPG).METHODS:In this open-label pharmacodynamic study, peripheral blood was obtained from relapsing-remitting MS patients just prior to and at multiple time points after intramuscular injection of 30 microg IFN-beta-1a. Samples were analysed for RANKL, tumour necrosis factor related apoptosis-inducing ligand (TRAIL), OPG and macrophage inflammatory protein-1 alpha/beta expression. Osteoclast precursor differentiation from peripheral blood cells of MS patients in the presence of exogenously added IFN-beta-1a was also assessed. Additionally, the changes in plasma levels of osteocalcin and the C-telopeptides after 1 year of treatment were measured as surrogate markers of bone formation and degradation, respectively.RESULTS:IFN-beta-1a treatment modulated RANKL and OPG in a selective, time-dependent manner. The levels of OPG protein decreased 25% at the 8-h time point, then increased 43% at the 24-h time point. The levels of free RANKL reached a maximum at the 8-h time point. Increases in the levels of macrophage inflammatory protein-1beta (MIP-1beta), a chemokine that increases osteolysis, were observed. The levels of the bone formation marker, osteocalcin, were lower in MS patients compared to controls and increased after one year of treatment. Ex vivo treatment of peripheral blood lymphocytes with IFN-beta resulted in a marked reduction of osteoclast-like cells in the presence of RANKL and macrophage colony stimulating factor.CONCLUSIONS:IFN-beta treatment induces complex, specific and time-dependent changes in multiple proteins and mRNAs related to bone homeostasis in MS patients.