Article Text

Download PDFPDF

Research paper
Apolipoproteins are associated with new MRI lesions and deep grey matter atrophy in clinically isolated syndromes
  1. Richard W Browne1,
  2. Bianca Weinstock-Guttman2,
  3. Dana Horakova3,
  4. Robert Zivadinov2,4,
  5. Mary Lou Bodziak1,
  6. Miriam Tamaño-Blanco5,
  7. Darlene Badgett5,
  8. Michaela Tyblova3,
  9. Manuela Vaneckova6,
  10. Zdenek Seidl6,
  11. Jan Krasensky6,
  12. Niels Bergsland4,
  13. Deepa P Ramasamy4,
  14. Jesper Hagemeier4,
  15. Eva Havrdova3,
  16. Murali Ramanathan2,5
  1. 1Department of Biotechnical and Clinical Laboratory Sciences, State University of New York, Buffalo, New York, USA
  2. 2Department of Neurology, State University of New York, Buffalo, New York, USA
  3. 3Department of Neurology and Center of Clinical Neuroscience, Charles University in Prague, 1st Faculty of Medicine and General University Hospital, Charles University, Prague, Czech Republic
  4. 4Buffalo Neuroimaging Analysis Center, Department of Neurology, State University of New York, Buffalo, New York, USA
  5. 5Department of Pharmaceutical Sciences, State University of New York, Buffalo, New York, USA
  6. 6Department of Radiology, 1st Faculty of Medicine and General University Hospital, Charles University, Prague, Czech Republic
  1. Correspondence to Professor Murali Ramanathan, 355 Kapoor Hall, Department of Pharmaceutical Sciences, State University of New York, Buffalo, Buffalo, NY 14214-8033, USA; Murali{at}Buffalo.Edu

Abstract

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.

  • Apolipoproteins
  • Cholesterol
  • Interferon
  • MRI
  • Multiple Sclerosis

Statistics from Altmetric.com

Introduction

The mechanisms by which the environmental and genetic factors interact with each other and with other comorbidities to affect disease susceptibility and disease progression in multiple sclerosis (MS) are not well understood.1

In the context of MS disease progression, the pleiotropic roles of cholesterol in myelin integrity, neurodegeneration and immune processes are receiving renewed research interest.2–4 Chronic hypercholesterolaemia, a common comorbidity that is also found in MS patients, is often associated with abnormal metabolic, cardiovascular and inflammatory processes that could affect MS progression.

An epidemiological analysis of nearly 9000 subjects found that the category of vascular comorbidities, which included hypercholesterolaemia, was associated with increased disability progression.5 Serum low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) levels were associated with disability changes in a study of nearly 500 MS patients.4 There is also evidence for a role for cholesterol in disease progression in clinically isolated syndromes (CIS), which represent the earliest stages of MS. In a small study of 18 CIS patients followed with monthly MRI for 6 months, LDL-C and TC were associated with the number of contrast-enhancing lesions (CEL).2 In a study of 135 CIS patients at high risk of conversion to MS and treated with interferon β-1a, LDL-C and TC were associated with the number of new T2 lesions after 2 years.3 Key suspected risk factors for MS progression including vitamin D deficiency and humoral responses to Epstein–Barr virus exhibit inter-dependence with serum cholesterol.6 ,7

These results however, were obtained with TC, high-density lipoprotein cholesterol (HDL-C) and LDL-C from a routine clinical lipid profile, which is now considered a crude assessment because it only reflects the relative cholesterol content of the lipoprotein particles that transport cholesterol. Cholesterol homeostasis is dependent on the dynamic remodelling of lipoprotein particles and complex interactions among cells, lipoprotein particles, their lipid cargo and associated apolipoproteins, lipid transfer proteins and enzyme activities. In cardiovascular disease, a broader range of cholesterol-related biomarkers is being advocated to complement rudimentary HDL-C, LDL-C and TC measurements.8–11

Apolipoproteins, which are critical for proper cholesterol homeostasis because they mediate the interaction of lipoproteins with cells and other lipoproteins, are the specific focus of this study. Apolipoprotein B (ApoB) is the primary protein of LDL, intermediate-density lipoprotein (IDL) and very low-density lipoprotein (VLDL) and mediates their interactions with the LDL receptor on cells including the vascular endothelium of the blood–brain barrier. Lipoprotein(a) (Lpa) is an LDL-like lipoprotein whose characteristic apolipoprotein is apolipoprotein(a) covalently linked to ApoB. Apolipoprotein AI (ApoAI) is the primary protein of HDL and the recognition ligand for all HDL-interacting proteins.12 The HDL particle also contains other apolipoproteins, for example, ApoAII, ApoAIV and ApoC-III.13 Serum apolipoprotein E (ApoE) is found on chylomicrons, VLDL and IDL, and is a minor component of HDL.14 ApoE is the predominant apolipoprotein of the central nervous system15 where it is produced by glial cells, particularly astrocytes. ApoE is the recognition ligand component of the HDL-like particle that transports cholesterol from astrocytes to neurons by binding ApoE receptors.16

The goal of this study was to substantively extend the mechanistic reach of our earlier studies3 ,4 ,6 and to determine whether serum apolipoproteins measured prior to the start of treatment are associated with disease progression in high-risk CIS patients.

Methods

Study population

Study setting

A multi-centre, prospective, longitudinal observational study.

Informed consent

All patients provided written informed consent.

Clinical study design

The observational Study of Early Interferon beta 1-a Treatment in High Risk Subjects after CIS (SET study) design has been previously described.17 This prospective observational clinical study to determine clinical, MRI, environmental and genetic predictors of response to interferon β-1a therapy in CIS was coordinated by Charles University in Prague, Czech Republic. It screened 259 patients and enrolled 220 patients from eight Czech Republic MS centres.

Inclusion criteria

CIS patients with the following characteristics were included: 18–55 years of age, enrolled within 4 months from the clinical event, presence of ≥2 T2-hyperintense lesions on diagnostic MRI, presence of ≥2 oligoclonal bands in cerebrospinal fluid (CSF) obtained prior to steroid treatment and Expanded Disability Status Scale (EDSS)≤3.5.

Treatments

All patients were treated with 3–5 g of methylprednisolone for the first symptom and baseline MRI was performed ≥30 days after steroid administration.

All patients were started on 30 µg, once-weekly, intramuscular interferon β 1-a (AVONEX) treatment at baseline.

Visits and assessments

Clinical visits occurred every 3 months for 4 years and subsequent long-term follow-up in routine clinical practice. This report is based on the data obtained until the 24-month (2-year) follow-up.

Clinical and MRI outcomes (including time to second clinical demyelinating event, disability progression measures and volumetric MRI scans) were obtained longitudinally. MRI assessments were obtained at baseline, 6, 12 and 24 months. Disability was assessed using the EDSS.

Serum lipids and apolipoproteins

Serum was obtained from CIS patients at the screening visit prior to the start of corticosteroid or interferon treatment for analysis of apolipoproteins and lipid profile.

Apolipoprotein levels (AI, AII, B and E), Lpa and high sensitivity C reactive protein (CRP) were analysed by immunoturbidometric methodology using diagnostic kits from Kamiya Biomedical (Thousand Oaks, California, USA). TC, phospholipids and TG were measured using diagnostic reagent kits (Sekisui Diagnostics, Lexington, Massachusetts, USA). These assays require <10 µL per test and were adapted to the ABX Pentra 400 automated chemistry analyser (Horiba Instruments, Irvine, California, USA). The coefficient of variation of these assays is <5%.

The ε2, ε3 and ε4 variants of the apolipoprotein E gene (APOE) were genotyped using specific probes for two single nucleotide polymorphisms, rs7412 and rs429358 (OpenArray, Applied Biosystems, Life Technologies, Foster City, California, USA).

Clinical data collected included height and weight for body mass index (BMI) calculations, and history of statin use.

Serum 25-hydroxy vitamin D3 and cotinine were measured by liquid chromatography-mass spectrometry as described in.18 Active smoking status was determined from cotinine measurements.18

MRI acquisition and analysis

All MRI examinations were done in the same centre on a 1.5 T magnet (Philips Gyroscan NT 15, Best, the Netherlands) as previously described.18 Scans were analysed by the Buffalo Neuroimaging Analysis Center (Buffalo, New York, USA).

Lesion measures

T2 and CEL number and lesion volumes were measured as previously described.18 Subtraction image methodology was used to identify new or enlarging T2 lesions.18 A new or enlarging lesion on T2-weighted images was defined as a rounded or oval lesion (3 mm of size) arising from an area previously considered normal appearing brain tissue and/or showing an identifiable increase in size from a previously stable-appearing lesion.

Global and tissue-specific atrophy measures

Normalised measures for the whole brain volume, grey matter volume and white matter volume were obtained as previously described.18 ,19 The SIENA method20 was used to calculate the percent brain volume change (PBVC).18

DGM measures

The volumes of total subcortical deep grey matter (DGM) and thalamus were estimated from inpainted T1-weighted 3D SPGR images with FMRIB's Integrated Registration and Segmentation Tool (V.1.2).21 Normalised volumes were obtained by multiplying the volumes from this tool by the volumetric scaling factor from SIENAX and percent volume changes were obtained.19

Data analysis

SPSS (IBM Inc., Armonk, New York, USA, V.19.0) statistical programme was used. The statistical analysis methods were based on strategies previously described.3 ,7

A conservative p value ≤ 0.01 was used for significance assessment given the multiple testing involved. A p value ≤0.05 was considered a trend.

Time to second clinical demyelinating event, defined as the time interval from first to second relapse, was analysed with Cox regression. The observations were right censored at the 24-month (2-year) follow-up visit; the proportional hazards assumption was assessed with log-log plots. The apolipoprotein variable of interest (ApoB, Lpa, ApoAI, ApoAII, or ApoE), age, gender and BMI were used as main effects predictors. The total number of relapses over the 2-year period was analysed using negative binomial regression with the predictors described for Cox regression.

Linear regression analysis was used for the following dependent variables, which are percent changes in volume over 2 years: PBVC, grey matter, lateral ventricle, DGM and thalamus volumes. The following count variables (all of which are total number of lesions over 2 years) were analysed with negative binomial regression: CEL, new T2 lesions, enlarging T2 lesions, new or enlarging T2 lesions. Age at baseline, gender, BMI, the baseline value of the MRI variable of interest and the apolipoprotein variable of interest were used as main effects predictors.

The differences in ApoE and CRP levels among the ε2, ε3 and ε4 variants of APOE were assessed with analysis of variance (ANOVA) followed by post hoc Bonferroni-corrected t tests.

Results

Demographic and clinical characteristics

The demographic and apolipoprotein and lipid profile variables obtained prior to the start of corticosteroid or interferon β-1a treatment are shown in table 1. Table 2 summarises the progression on MRI and clinical measures over 2 years. None of the patients was on statins.

Table 1

Demographic, apolipoprotein and lipid profile characteristics (mean±SD)

Table 2

Progression on MRI and clinical characteristics at baseline and 2 years

Clinical associations

The time to second clinical demyelinating event was not associated with ApoB (p=0.73), Lpa (p=0.64), ApoAI (p=0.74), ApoAII (p=0.89) or ApoE (p=0.83). Likewise, the number of relapses during the 2-year period was also not associated with the ApoB (p=0.73), Lpa (p=0.83), ApoAI (p=0.93), ApoAII (p=0.94) or ApoE (p=0.68) variables.

MRI associations

High 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). The number of enlarging T2 lesions was not associated with ApoB levels. Each 10 mg/dL of greater baseline ApoB was associated with a 16% increase in the number of new T2 lesions. Figure 1A shows the dependence of the number of new or enlarging T2 lesions over 2 years for the lower quartiles and highest quartile of ApoB levels.

Figure 1

(A) Dependence of the number of new or enlarging T2 lesions over 2 years for the lower quartiles (green bars) and highest quartile of apolipoprotein B levels (red bars). (B) Changes in per cent deep grey matter volume over 2 years for the lower quartiles (green bars) and highest quartile of apolipoprotein B levels (red bars). The error bars represent standard errors.

We conducted regression analysis with CRP as an additional predictor to assess the contributions from non-specific inflammatory processes. The inclusion of CRP did not reduce the significance and strength of ApoB associations with the number of new T2 lesions and the number of new or enlarging T2 lesions over 2 years (p<0.001 for both).

ApoAII, ApoE and Lpa did not show evidence for associations with the number of new T2 lesions or the number of new or enlarging T2 lesions-dependent variables. A trend for increased number of new T2 lesions was found for ApoAI (p=0.025). Each 10 mg/dL of greater baseline ApoAI was associated with a 5% increase in the number of new T2 lesions.

Concordant with our earlier report,3 higher LDL-C (p=0.002) and TC (p=0.001) levels were associated with increased number of new T2 lesions. Each 10 mg/dL of greater baseline LDL-C and TC was associated with a 7.4% and 5.9%, increase in the number of new T2 lesions, respectively.

There were no significant associations of any of the apolipoproteins with the cumulative number of CEL, PBVC or percent change in gray matter volume over 2 years.

DGM and thalamic atrophy measures of neurodegeneration are more sensitive than global atrophy measures in CIS.22 Interestingly, greater ApoE levels were associated with greater DGM atrophy (partial correlation rp=−0.28, p<0.001). None of the other apolipoproteins were associated with DGM atrophy. Each 1 mg/dL increase in ApoE levels was associated with a 0.99% (or approximately 1%) increase in DGM atrophy as assessed by decreases in per cent DGM volume over 2 years. Figure 1B shows the decrease in per cent DGM volume for the lower quartiles and highest quartile of ApoE levels. The significance and strength of the associations of DGM atrophy with ApoE levels were not decreased substantially by the inclusion of CRP as a predictor (rp=−0.27, p=0.001) and to the inclusion of new T2 lesions as a predictor (rp=−0.27, p<0.001). Greater ApoE levels were also associated as a trend with increased thalamic atrophy (rp=−0.15, p=0.047).

We obtained genotyping calls on 172 and 176 of 181 patients for the APOE rs7412 and rs429358 SNPs, respectively. APOE ε2, ε3 and ε4 status was inferred for 166 patients and the frequencies of the variants were: ε2/ε2 (1.8%), ε2/ε3 (12%), ε2/ε4 (1.8%), ε3/ε3 (62%), ε3/ε4 (21.7%) and ε4/ε4 (0.6%).

ApoE levels (figure 2, ANOVA, p=0.001) in the ε2/ε3 group were modestly higher compared with the ε3/ε3 (p=0.001) and the  e3/ε4 groups (p=0.008); the other APOE groups had three or fewer patients and were not included in the analysis. The associations of increased DGM atrophy with greater ApoE levels remained significant (rp=−0.24, p=0.004) after adjusting for APOE ε2/ε3, ε3/ε3 and ε3/ε4 variant status. The mean CRP levels of the three APOE variants were not different (p=0.32).

Figure 2

(A) Dependence of the serum ApoE levels (mg/dL) with the apolipoprotein E gene (APOE)  e2/ε3, ε3/ε3 and ε3/ε4 variants. (B) Dependence of the serum C reactive protein levels (mg/dL) with the APOE  e2/ε3, ε3/ε3 and ε3/ε4 variants. The error bars represent standard errors.

Discussion

The goals of this study were to assess the associations of apolipoproteins, which mediate the interactions of lipoproteins with cells and other lipoproteins, with progression on MRI measures in high-risk CIS patients. Greater ApoB and ApoE levels were associated with greater number of new T2 lesions and greater DGM atrophy, respectively.

Much of our knowledge of lipoproteins as risk factors of disease comes from cardiovascular disease. LDL is widely recognised as the major atherogenic lipoprotein but LDL-C has known limitations as a marker of cardiovascular risk. LDL-C is not a true estimate of LDL particle concentration but rather of LDL cholesterol along with variable, usually smaller, amounts of VLDL, IDL and Lpa cholesterol, which are also atherogenic. As each particle carries a single ApoB, total ApoB may better reflect the totality of atherogenic proteins.8 ,9 A similar argument has been made for ApoAI relative to HDL-C.10 Additionally, fasting is not needed for interpreting ApoB and ApoAI values.23

Our ApoB findings are consistent with the earlier study from our group, which found associations of TC and LDL-C with new T2 lesions.3 This is not surprising given that ApoB is the marker for LDL-C, which contains the majority of TC. Giubilei et al found that LDL-C and TC levels were associated with CEL number;2 the number of T2 lesions was not reported. The p values for ApoB association with cumulative number of CEL in our study were not significant but low (p=0.12). Giubilei et al had six MRI at monthly intervals,2 which provided greater temporal resolution to detect transient CEL compared with our study, which used four MRI obtained at baseline, 6, 12 and 24 months. These findings suggest that higher ApoB, LDL-C and TC are associated with greater inflammatory lesion activity in CIS.

ApoE transports cholesterol from astrocytes to neurons and plays a central role in remyelination.24 ApoE also assists in presentation of lipid antigens bound to CD1 on antigen presenting cells to NKT cells.15 In animal models, higher serum ApoE levels adversely affect immune response to pathogens25 and increase levels of pathogenic amyloid-β in older mice.26 Further, high levels of serum ApoE are associated with increased cardiovascular mortality independent of APOE genotype.27 The APOE ɛ4 and APOE ɛ2 alleles are not associated with MS risk based on a definitive study with 29 000 subjects.28 The APOE ɛ4 allele may be associated with increased MS disease progression but the evidence is mixed.29 Much less work has been done examining serum ApoE in MS. Giubilei et al also examined ApoE levels in 18 CIS patients.2 Concordant with our findings, they did not find any evidence for associations of CEL with ApoE.2 Unfortunately, their 6-month study was not designed to address regional brain volume changes. A limitation of our study was the absence of a healthy control group. Three studies examining ApoE levels in MS versus control groups have reported decreased ApoE levels while two found no difference. Serum ApoE levels were approximately 40% lower in MS patients in remission compared with controls or MS patients in exacerbation; CSF ApoE levels were unchanged.30 ,31 A study comparing MS, Guillain–Barre syndrome and amyotropic lateral sclerosis reported decreased CSF ApoE, but not decreased serum ApoE, in MS.32 Serum ApoE levels were similar in MS compared with acute herpes zoster patients; however, this study pooled MS patients in remission and those with recent exacerbation, which may have obscured differences.33 Based on the known functions of ApoE, it is reasonable to posit that its effects on modulating inflammation in the periphery and in neuronal cholesterol homeostasis particularly during demyelination24 could be important in mediating its adverse effects on disease progression in CIS.15 ,16 Paradoxical effects on clinical progression and cognition were reported in ApoE knockout mice challenged with experimental autoimmune encephalomyelitis.34 The pathogenic mechanisms underlying our findings of increased DGM atrophy with ApoE levels are uncertain.

There are strengths and limitations of our study that merit discussion. We did not find evidence that ApoB, Lpa, ApoAI, ApoAII or ApoE were associated with time to relapse or with relapse rate. This may reflect the 5–10-fold greater sensitivity of MRI measures for MS disease activity compared with clinical measures. Our study was conducted on patients treated with interferon β-1a. This could potentially affect the generalisability of the findings to untreated CIS patients and to those on other FDA-approved drugs such as glatiramer acetate, natalizumab, fingolimod, teriflunomide and dimethyl fumarate. The implications for fingolimod treatment are beset with greater uncertainty because fingolimod binds ApoM and is HDL-associated.35 Interestingly, fingolimod causes hypercholesterolaemia in ApoE-deficient mice36 and inhibits atherosclerosis in LDL receptor-deficient mice.37

We did not find associations of baseline cholesterol biomarkers with clinical measures but our study was not designed to address whether decreases in cholesterol biomarkers might be associated with better clinical and MRI outcomes. Our findings might be relevant for interpreting clinical trials of statins, which were also negative on their primary clinical outcome measures. The SIMCOMBIN trial of once-daily 80 mg simvastatin added to intramuscular, once-weekly interferon β-1a in treatment-naive RR-MS did not show any additional benefit on relapse rate, time to relapse or T2 lesions.38 Likewise, the smaller placebo-controlled STAYCIS trial of once-daily 80 mg atorvastatin in CIS was negative on its primary endpoint (≥3 T2 lesions or one clinical relapse in 12 months). However, the atorvastatin-treated group was more likely to remain free of new T2 lesions.39 Surprisingly, both trials did not report cholesterol biomarker changes.

In conclusion, we have demonstrated that ApoB and ApoE are associated with lesions and with DGM atrophy in CIS. These findings add to the growing body of evidence for possible roles of cholesterol in MS disease progression.

Acknowledgments

The authors thank the patients who participated in this study. We thank the other clinical centres and investigators who participated in the SET study: (i) M Vachova, S Machalicka and J Kotalova from KZ a.s. Hospital, Teplice; (ii) Y Benesova, P Praksova and P Stourac from University Hospital, Brno, Bohunice; (iii) M Dufek from St Anne's University Hospital, Brno; (iv) E Meluzinova, J Pikova and E Houzvickova from Charles University in Prague, 2nd Faculty of Medicine, Motol; (v) D Zimova from Charles University in Prague, 3rd Faculty of Medicine, Kralovske Vinohrady; (vi) J Sucha from University Hospital, Plzen; (vii) V Sladkova and J Mares from University Hospital, Olomouc.

References

Footnotes

  • BW-G, DH and RZ contributed equally to this study.

  • Contributors RWB, DH and EH: Study concept and design, data analysis, manuscript preparation. JH: Data analysis. RZ: MRI data analysis, manuscript preparation. BW-G: Data interpretation, manuscript preparation. MLB, MT-B, DB, DPR, MT, NB, JK, MV, ZS: Data acquisition. MR: Study concept and design, data analysis, manuscript preparation.

  • Funding The SET study was supported by Czech Ministries of Education and Health (NT13237-4/2012, MSM 0021620849, PRVOUK-P26/LF1/4, RVO-VFN64165/2012) and Biogen Idec. The work was funded by a grant from the National Multiple Sclerosis Society (RG4836-A-5). Support from Department of Defense Multiple Sclerosis Program (MS090122) and the National MS Society (RR 2007-A-2) to the Ramanathan laboratory is also gratefully acknowledged. The funding agencies had no role in design and conduct of the study; collection, management, analysis and interpretation of the data; and preparation, review or approval of the manuscript; and decision to submit the manuscript for publication.

  • Competing interests DH received speaker honoraria and consultant fees from Biogen Idec, Novartis, Merck Serono, Teva and Bayer Schering and financial support for research activities from Biogen Idec. RZ has received speaker honoraria and consultant fees from Teva Pharmaceuticals, Biogen Idec, Genzyme-Sanofi, Novartis, Bayer and EMD Serono. He has received research support from the National Multiple Sclerosis Society, Department of Defense, Biogen Idec, Teva Neuroscience, Teva Pharmaceuticals, EMD Serono, Genzyme-Sanofi, Novartis and Greatbatch. BW-G received honoraria for serving in advisory boards and educational programmes from Teva Pharmaceuticals, Biogen Idec, Novartis, Accorda EMD Serono, Pfizer, Novartis, Genzyme and Sanofi. She also received support for research activities from the National Institutes of Health, National Multiple Sclerosis Society, National Science Foundation, Department of Defense, EMD Serono, Biogen Idec, Teva Neuroscience, Cyberonics, Novartis, Accorda and the Jog for the Jake Foundation. EH received honoraria and consulting fees from Genzyme, Biogen Idec, Sanofi-Aventis, Merck Serono, Roche, Teva and Novartis for consulting services, speaking and serving on a scientific advisory boards. MT, JK, MV and ZS have received financial support for research activities from Biogen Idec. MR received research funding or consulting fees from EMD Serono, Biogen Idec, Allergan, Netezza, Pfizer, Novartis, Monsanto, the National Multiple Sclerosis Society, the Department of Defense, Jog for the Jake Foundation, the National Institutes of Health and National Science Foundation. He received compensation for serving as an Editor from the American Association of Pharmaceutical Scientists. These are unrelated to the research presented in this report.

  • Ethics approval Charles University in Prague Ethical Committee and by Ethical Committees at each of the eight participating sites.

  • Provenance and peer review Not commissioned; externally peer reviewed.

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.