Environmental risk factors for multiple sclerosis. Results from animal and human studies on diet, vitamin D and Epstein-Barr virus
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Multiple Sclerosis (MS) is an immune mediated disease of the central nervous system (CNS). The aetiology is unknown, but environmental factors as well as multiple separate genetic loci contribute to the disease susceptibility. Much research in the last decades has focused on the genetic basis of MS, but increasing evidence is emerging for major impact of environmental risk factors. Research on environmental risk factors is important since they offer a potential for disease prevention. The present study was designed to evaluate and add new knowledge to the role of environmental risk factors associated with the disease. The specific objectives were: A) Explore whether diet intervention with n-3 polyunsaturated fatty acids (PUFAs) could influence MRI disease activity, microglia activation, de- and remyelination in the cuprizone model for demyelination (Paper I). B) Assess if diet intervention with n-3 PUFAs from two different sources could prevent behavioural changes in the cuprizone model for demyelination (Paper II). C) Describe and characterise a case series of patients with co-occurrence of vitamin D dependent rickets (VDDR1) and MS (Paper III), D) Study if relapses in MS were associated with reactivation of latent Epstein-Barr virus (EBV) (Paper IV). E) Examine changes in gene expression in cortical lesions, normal appearing grey matter and meninges of MS patients. Perform an immunohistochemical characterisation of these brain sections and determine if active replication or latent infection with EBV was present in the sections examined (Paper V). The prevalence of MS is lower in the northern than in the southern parts of Norway. Since the prevalence of MS is known to normally increase with the distance from equator, this is an anomalous distribution of the disease compared to most other parts of the world. One widely accepted hypothesis for this distribution is that the inhabitants in the northern parts of Norway have a higher consumption of fatty fish than in the south, and that some substances in the fish (vitamin D or n-3 fatty acids) offer protection against MS. Two of the papers in this thesis investigate the role of nutrition and diet modification for the susceptibility to demyelinating diseases. We used an animal model for MS (the cuprizone model) in order to study how diet modification with fatty fish or fish oils could influence the disease course, MRI activity, de- and remyelination in this animal model. Sixty-three mice were fed either a diet enriched with 1) salmon filets, rich in n-3 fatty acids, 2) cod liver oil rich in n-3 fatty acids or 3) soybean oil rich in n-6 fatty acids. Cuprizone was added to the different diets. In the first study (Paper I), we found that the mice fed a salmonenriched diet had less MRI disease activity and demyelination than the two other groups. In the second study behavioural changes to cuprizone treatment were assessed with the elevated-plus-maze test (EPM). There were striking differences in weight loss, anxiety behaviour and activity levels, with a more favourable clinical outcome for the mice given a diet enriched with salmon filets (Paper II). The findings from these two studies indicated that diets rich in fatty fish could have protective effects against demyelination. There were no effects of giving n-3 supplements in the form of cod liver oil, indicating that other substances than the fatty acids could have accounted for the effect of a salmon based diet. Future studies should aim at isolating the active component in the fish diet, as this could offer a valuable treatment supplement for demyelinating diseases. Three of the papers deal with the most studied and recognised environmental triggers of MS, Vitamin D and Epstein-Barr virus (EBV). The third study (Paper III) identified three patients with a rare hereditary form of rickets (VDDR1). All of these patients later developed MS. These findings indicated rickets as a possible risk factor for MS and supported the theory that early childhood or the intrauterine periods are the main susceptibility periods for low levels of vitamin D. The next study (Paper IV) was designed to analyse whether there was an association between exacerbations in MS and reactivation of latent EBV. Sixty-one patients were followed for one year and blood samples were taken in the case of exacerbations or if magnetic resonance imaging (MRI) indicated evidence of new enhancing lesions. All the 61 patients (100%) were anti-viral capsid antigen (VCA) IgG positive, one (2%) was anti-VCA IgM positive and 60 (98%) were anti- Epstein Barr virus nuclear antigen (EBNA) positive. Mean anti-early antigen (EA)-D IgG at baseline was 0.57 (range 0.12-2.70) and at the time of exacerbations 0.61 (range 0.11-2.70). As demonstrated in earlier studies, all the patients in this sample had evidence of previous EBV infections, indicating that EBV could have a role in the pathogenesis of MS. There were, however, no signs of EBV-reactivation at the time of relapse. Thus, this study concluded that EBV reactivations do not play a significant role in MS exacerbations. The last study (Paper V) was designed to examine changes in gene expression in cortical lesions and in normal appearing grey matter from patients with MS. We observed a strong activation of immunoglobulin (Ig)-related genes in cortical sections of MS patients. The Ig-genes with the strongest activation were mostly variable and constant regions of the kappa and lambda light chains, but also included genes encoding heavy chains for IgM and IgG. No upregulation of Fc-receptor (FcR) genes was found. The cortical sections were immunohistochemically stained for plasma cells, Igs, T- and B-lymphocytes. The stainings revealed Ig-deposition in the meninges of the MS-patients compared to the controls. There were few B-lymphocytes and no B-lymphocyte follicles. Plasma cells were present in the meninges of all the MS patients but not in any of the controls. The activation of Ig-genes observed in the present study was highly interesting, as the synthesis of oligoclonal IgGs have been hypothesised to be caused by activation of EBV infected B-lymphocytes. The samples were screened for the presence of EBV by quantitative real time polymerase chain reaction (qPCR) and immunohistochemistry, but no evidence of active or latent EBV infection was detected. This study demonstrated that genes involved in the synthesis of Igs are upregulated in MS patients, and that this upregulation seems to be caused by a small number of plasma cells located in the meninges. Further, it concluded that EBV infected cells were not necessary for this Ig-upregulation. The findings indicated that the oligoclonal band (OCB) producing B-lymphocytes found in the cerebrospinal fluid (CSF) of MS-patients could have meningeal origin.
Paper I: Experimental Neurology 215(1), Torkildsen, Ø.; Brunborg, L. A.; Thorsen, F.; Mørk, S.; Stangel, M.; Myhr, K. M.; Bø, L., Effects of diet intervention on MRI activity, de- and remyelination in the cuprizone model for demyelination, pp. 160-166. Copyright 2008 Elsevier. Full text not available in BORA due to publisher restrictions. The published version is available at: http://dx.doi.org/10.1016/j.expneurol.2008.09.026Paper II: Clinical Nutrition 28(1), Torkildsen, Ø.; Brunborg, L. A.; Milde, A. M.; Mørk, S. J.; Myhr, K. M.; Bø, L., A salmon based diet protects mice from behavioural changes in the cuprizone model for demyelination, pp. 83-87. Copyright 2008 Elsevier. Full text not available in BORA due to publisher restrictions. The published version is available at: http://dx.doi.org/10.1016/j.clnu.2008.10.015Paper III: Archives of Neurology 65(6), Torkildsen, Ø.; Knappskog, P. M.; Nyland, H. I.; Myhr, K. M., Vitamin D dependent rickets as a possible risk factor for multiple sclerosis, pp. 809-811. Copyright 2008 American Medical Association. Full text not available in BORA due to publisher restrictions. The published version is available at: http://archneur.ama-assn.org/cgi/content/abstract/65/6/809Paper IV: European Journal of Neurology 15(1), Torkildsen, Ø.; Nyland, H.; Myrmel, H.; Myhr, K. M., Epstein-Barr virus reactivation and multiple sclerosis, pp. 106-108. Copyright 2007 The Author(s), copyright journal compilation 2007 EFNS. Published by Blackwell. Full text not available in BORA due to publisher restrictions. The published version is available at: http://dx.doi.org/10.1111/j.1468-1331.2007.02009.xPaper V: Brain Pathology 20(4), Torkildsen, Ø.; Stansberg, C.; Angelskår, S.; Kooi, E. J.; Geurts, J. J. G.; van der Valk, P.; Myhr, K. M.; Steen, V.; Bø, L., Upregulation of immunoglobulin related genes in cortical sections from multiple sclerosis patients, pp. 720-729. Copyright 2009 The Authors; Journal Compilation 2009 International Society of Neuropathology. Published by Wiley-Blackwell. Full text not available in BORA due to publisher restrictions. The published version is available at: http://dx.doi.org/10.1111/j.1750-3639.2009.00343.x