The role of biomarkers in the diagnosis of Multiple Sclerosis

Gustavo Miguel da Rocha Rodrigues The role of biomarkers in the diagnosis of Multiple sclerosis 2011/2012 março, 2012 Gustavo Miguel da Rocha Rodrigues The role of biomarkers in the diagnosis of Multiple Sclerosis Mestrado Integrado em Medicina Área: Neurologia Trabalho efetuado sob a Orientação de: Dra. Joana da Cruz Guimarães Ferreira de Almeida Trabalho organizado de acordo com as normas da revista: Multiple Sclerosis and related Disorders março, 2012 IIIPORTO Projeto de Opção do 6° ano - DECLARAÇÃO DE INTEGRIDADE !FMUP! FACULDADE DE MEDICINA UNIVERSIDADE DO PORTO UNIDADE CURRICUlAR PROJETO DE o e c z o DISSERTAÇÃO f MONOGRAFIA Eu, Gustavo Miguel da Rocha Rodrigues, abaixo assinado, nOmecanográfico 060801225, estudante do 6 0 ano do Mestrado Integrado em Medicina, na Faculdade de Medicina da Universidade do Porto, declaro ter atuado com absoluta integridade na elaboração deste projeto de opção. Neste sentido, confirmo que NÃO incorri em plágio (ato pelo qual um indivíduo, mesmo por omissão, assume a autoria de um determinado trabalho intelectual, ou partes dele). Mais declaro que todas as frases que retirei de trabalhos anteriores pertencentes a outros autores, foram referenciadas, ou redigidas com novas palavras, tendo colocado, neste caso, a citação da fonte bibliográfica. Faculdade de Medicina da Universidade do Porto, 1 b /ó'? / ~o 1), Assinatura: DlpORTO FMUP FACULDADE DE MEDICINA UNIVERSIDADE DO PORTO Projecto de Opção do 6° ano - DECLARAÇÃODEREPRODUÇÃO Nome: Gustavo Miguel da Rocha Rodrigues Endereço electrónico: gustavo [email protected] Telefone ou Telemóvel: 912248477 Número do Bilhete de Identidade: 13213739 Título da Monografia: The role of biomarkers in the diagnosis of Multiple Sclerosis Orientador: Dra. Joana da Cruz Guimarães Ferreira de Almeida Ano de conclusão: 2012 Designação da área do projecto: Neurologia É autorizada a reprodução integral desta Monografia para efeitos de investigação e de divulgação pedagógica, em programas e projectos coordenados pela FMUP. Faculdade de Medicina da Universidade do Porto, t0~ lo 11 Assinatura: L~josJ{) Dedicatória Dedico este trabalho á minha família pela disponibilidade e apoio incondicional ao longo de todo o meu percurso académico. MESTRADO INTEGRADO EM MEDICINA Ano letivo: 2011/2012 Nome do(a) Estudante: Gustavo Miguel da Rocha Rodrigues Orientador(a): Dra. Joana da Cruz Guimarães Ferreira de Almeida Área do Projeto: Neurologia Título do Projeto: The role of biomarkers in the diagnosis of Multiple Sclerosis. Resumo: Esclerose Múltipla (EM) é uma doença neurológica inflamatória desmielinizante importante devido à sua incidência, cronicidade e maior prevalência em adultos jovens. No estudo inicial de uma doença desmielinizante do Sistema Nervoso Central existe a necessidade de encontrar biomarcadores que possam melhor discriminar a EM de outras patologias. Atualmente, apenas as bandas oligoclonais (BOC) são utilizadas. Esta revisão pretende, através de uma pesquisa na base de dados PubMed, examinar a literatura publicada nos últimos 5 anos, sobre o papel dos biomarcadores no diagnóstico da esclerose múltipla. O uso de marcadores no líquido encéfalo-raquidiano, aumenta a especificidade e valor preditivo negativo, diminuindo os possíveis falsos positivos da, muito sensível, ressonância magnética. As BOC confirmaram o seu valor, apoiando o diagnóstico quando presentes e sugerindo a revisão do mesmo quando ausentes, mostrando contudo algumas limitações: variabilidade interpopulacional, laboratorial e metodológica, controvérsia sobre o uso do índice IgG, a interpretação de uma única BOC e a relação das BOC com a EM. Enquanto o vírus Epstein-Barr e os anticorpos antimielina falharam quando testados, as cadeias leves livres K demonstraram resultados promissores, especialmente, como todos os biomarcadores, quando combinadas com ressonância magnética. Na previsão de conversão do síndrome clínico isolado para EM, as BOC confirmaram o seu papel, enquanto CXC quimiocina motif 13 e a reação MRZ mostraram bons valores preditivos, e os marcadores de lesão axonal e anticorpos contra a mielina falharam. Além da deteção de anticorpos aquaporina-4 e a reação MRZ no líquido céfaloraquidiano a deteção de N-acetil aspartato no sangue mostraram bons resultados a distinguir EM de neuromielite ótica. Há uma procura para desenvolver métodos automatizados para estudar biomarcadores, e o desenvolvimento de técnicas proteómicas é promissor para a identificação de novos alvos moleculares para ensaios de diagnóstico e terapêutica. Concluindo, o principal contributo dos biomarcadores no diagóstico de EM, consiste no aumento de especificidade e valor preditivo. É necessário corrigir qualquer possível erro metodológico, tentando estandardizar ao máximo de modo a obter resultados mais consistentes. Apesar dos esforços na pesquisa de novos biomarcadores é necessário compreender melhor a EM de modo a encontrar um teste simplificado. Palavras-chave: Esclerose Múltipla, Síndrome clínico isolado, bandas oligoclonais, líquido encéfaloraquidiano, biomarcadores, diagnóstico. The role of biomarkers in the diagnosis of Multiple Sclerosis Rodrigues, Gustavo Faculty of Medicine of Porto University (FMUP)* Neurology Department, Faculty of Medicine, University of Porto, Hospital de São João, Alameda Professor Hernâni Monteiro, 4200, Porto, Portugal Faculty of Medicine of Porto University Telephone and Fax: + 00351 225511200 *Corresponding author, Praça, 135 Vila Nova, 4465-767 Praia da Vitória, Açores - Portugal E-mail: [email protected]; Telephone: +00351 912248477 Abstract Multiple Sclerosis (MS) is an important inflammatory demyelinative neurologic disease, due to its, incidence, chronicity and higher prevalence among young adults. In the initial approach to a demyelinative disease of the Central Nervous System there is a urge for biomarkers to best discriminate MS from other diseases. Despite the broad range of biomarker studies, only oligoclonal bands (OCB) are used by routine in differential diagnosis. This review aims to access the published literature in the last 5 years (trough a search in PubMed database), regarding the role of biomarkers in the diagnosis of MS. Regarding MS differential diagnosis, the use of cerebrospinal fluid markers (CSF) increases specificity and negative predictive, diminishing the possible false positives from the high sensible magnetic resonance imaging (MRI). OCB confirmed they value, supporting the MS diagnosis when present and suggesting revision of this, but some limitations appeared regarding inter-population, interlaboratories and inter-methods detection variability and there’s controversy concerning the current use of IgGindex, interpretation of a single weak OCB and the relation of OCB with MS. While Epstein Barr Virus (EBV) and anti-myelin antibodies failed when tested, Kappa free light chains (KFLC) performed in a promising way, specially, as all biomarkers, when combined with MRI. In this review, OCB confirmed they importance while C-X-C motif chemokine 13 (CXCL13) and MRZ reaction showed good predictive values results while axonal damage markers and antibodies against myelin didn’t perform well, predicting clinically isolated syndrome (CIS) conversion to MS. Besides aquaporin-4 antibody detection and MRZ reaction in CSF, N-acetyl aspartate detection in serum showed good results distinguishing MS from neuromyelitis optica (NMO). There has been a struggle to develop standard easy automated methods to biomarker studying and the development of more sensitive proteomic techniques is very promising for the identification of new molecular targets for diagnostic assays and therapies preventing axonal damage. Concluding, biomarkers major contribute is in terms of Specificity and predictive value. Methodological standardization, correcting any possible error, is vital for a more consistent research. Despite the efforts in this field, an accurate simple test is only possible when the pathophysiology of MS is better understood. Keywords: Multiple Sclerosis, Clinically isolated syndrome, oligoclonal bands, cerebrospinal fluid, biomarker, diagnosis. 1. Introducion 1.1 Historic note and state of art Multiple Sclerosis (MS) is an important inflammatory demyelinative neurologic disease, due to its incidence, chronicity and tendency to affect young adults.(1) MS features a broad heterogeneity of clinical, histopathological and immunological phenotypes, which urges a more accurate defining of patients by biological markers that reflect the underlying disease process and allow the prediction of disease course and treatment response.(2, 3) Thus, serious efforts are made in the field of biomarkers to improve the diagnostic discrimination, to determine prognostic factors, and to identify markers to monitor the clinical course and response to disease-modifying therapies.(3) Starting with Charcot (1868) and his triad (nystagmus, intention tremor and scanning speech), the diagnostic criteria for MS have evolved to reflect the breakthroughs in our understanding of the disease and the development of new diagnostic techniques, changing from purely clinical criteria to increasing dependency upon imaging of the central nervous system (CNS). Only in 1954 the first true diagnostic scheme appeared, published by Allison and Millar describing three categories: early, probable and possible disseminated sclerosis.(1) There was a need for reliable and reproducible diagnostic criteria in order to provide accurate epidemiological studies and therapeutic trials. A major step in that direction occurred in 1965, when Broman et al. outlined the principles that were the basis for following schemes.(4) They consisted of age of onset, the empiric significance of symptoms for an MS diagnosis, the multiplicity of lesions, the number of bouts, and the familial “recurrence” of MS. The results of the newly described electrophoretic examination of cerebrospinal fluid (CSF) protein, the precursor of oligoclonal bands (OCB), were also introduced. Additionally, all other possible diagnoses were to be considered and, if possible, excluded. For the first time was stated the twin principles of dissemination in time and in space, that to this day remain the fundamental criteria for the diagnosis of MS.(4) The further need for accurate diagnosis criteria, lead to the formation of an expert panel and elaboration of the Schumacher et al. criteria, used worldwide.(5) A adaptation of the Schumacher criteria emerged from a worldwide investigation by Bauer that was the first to include CSF OCB.(1) synthesis is directed against diverse antigens. An association of these antibodies with the cause of MS is still missing.(2) Epstein-Bar virus (EBV) infection has been suspected as an initial trigger of autoimmunity in MS and, thus, evidence of EBV infection could have relevance for MS diagnosis.(10) A number of studies have shown that almost 100% of MS patients have evidence of prior EBV infection, whereas approximately 90% of healthy adults also have evidence of EBV infection.(40) Therefore, the presence of EBV antibodies is not considered to be diagnostic relevant. Furthermore, CSF reactivity to EBV antigens in MS compared with healthy controls has not yielded any specific associations.(41) Current investigations are focused on determining whether MS patients have antibodies to unique determinants of EBV, or if viral-clearance responses to EBV infection in MS patients are abnormal.(42) If these investigations are fruitful, they could have application as diagnostic markers.(10) Additionally, Bartos et al. (2007) reported no difference in antibodies against light neurofilaments in all sub-types of MS and other diseases.(43) Nogo-A, an inhibitor of axonal regeneration, is a perfect example of the inconsistent and contradictory data gathered in this review. Jurewicz et al. (2007) reported that CSF Nogo-A may be specific (100%) for MS and its presence may predict failure of axonal regeneration within the CNS. Nogo-A was found in CSF of 96% of MS patients and wasn’t present in meningo-encephalomyelitis, control subjects wither neurologic, and autoimmune diseases.(44) These findings lacked confirmation by William et al. (2008) with the same methodology.(45) Correspondence between authors, showed high discordance in this subject. Numerous studies have demonstrated elevated kappa free light chains (KFCLs) in CSF of MS patients. However, so far only small cohorts have been examined, and generally only through qualitative KFLCs analysis. Determining a single quantitative diagnostic FLC parameter (KFLC index) by nephelometry (highly automated) and IEF, Presslauer et al. (2008) showed higher sensitivity (96% vs 91%) but lower specificity (86% vs 92%) compared with OCB, but was far more accurate than IgG index. Additionally, there was no correlation between MS type, EDSS score, disease duration, or number/localization of MRI lesions and the value of KFLC or KFLC index.(14) The combined use of three FLC indices accounting for monomeric FLC –k level and k/λ ratio values in the CSF and serum was found to be of promising diagnostic importance for differentiation of MS from other non-MS neurological diseases (specificity 93%, sensitivity 100% vs OCB 76% and 87% in the same cohort).(46) In contrast to the OCB test, where the result is interpreted as “positive”, “negative” or “trace”, this method provides quantitative evaluation of relative amounts of FLCs, as a measure of the severity of intrathecal immunoglobulin synthesis. This procedure for FLC analysis can serve as a basis of a new diagnostic test for MS. The described method is inexpensive, it requires no special requirement, and may be applied in clinical laboratories.(46) This procedure also allows preparation of samples in a dry state, that is helpful for prolonged storage and transportation of CSF and serum samples prior to their analysis.(46) As it is measured by automated, routinely available laboratory methods, KFLC quantification can provide a rapid and reproducible indication of intrathecal immunological processes supporting current MS diagnostic criteria.(14) These results strongly suggest that the KFLC index should be part of the routine MS diagnostic algorithm.(14, 46) Additional LFLC index determination can support the exclusion of alternative diagnoses. Alternative diagnoses should be considered if these are associated with concurrent significant elevation of LFLC levels.(14) Elevated CSF anti-MBP antibodies correlate well with acute myelin damage in the CNS, but are not specific to MS. Reproducible quantification of MBP has been difficult since immunoassays are not standardized and not commercially available.(3) CSF MBP has lacked specificity in most studies. The test was recently reevaluated by Tian et al. (2009) who reported a diagnostic sensitivity of 83.7% and specificity of 78.3%. These results need to be confirmed in further studies.(16, 47) Preliminary studies, approaching Gelsolin, leukemia inhibitory factor and 7ketocholesterol, showed good result, needing further evaluation.(48-50) In table 1, are displayed other biomarkers revised in this search. 3.1.3 Conversion of Clinically Isolated Syndrome to Clinically Definitive Multiple Sclerosis In a significant percentage of patients who later develop MS, the disease initially presents with an acute or sub-acute episode of neurological symptoms due to a single demyelinating lesion, known as clinically isolated syndrome (CIS). Predict the risk of future events, confirming the MS diagnosis is crucial to begin an important early treatment. In the last few years, the main focus has been on diagnostic sensitivity in order to early detect the patients in need for early therapeutics, resulting therefore in possible over diagnoses.(51) In this context, the most relevant statistical parameters to predict conversion from CIS to MS are positive predictive value (PPV) and negative predictive value (NPV).(51) MRI is the main exam used to this prediction but there is an ongoing search for accurate biomarkers for this conversion.(52, 53) Tintoré et al. (2008) showed that detection of OCB in CSF approximately doubles the risk of having a second attack, autonomously of MRI, but does not seem to influence the development of disability.(54) This higher risk is more significant when MRI is normal or when there is not MRI criteria for dissemination in space (DIS) (4% of patients with normal MRI and negative OCB converted and 23% of those with normal MRI and positive OCB converted).(54) The association of OCB to MRI maximizes the risk; therefore they can be used together to better predict conversion to CDMS. OCB associated with number of Barkhof criteria and number of lesions but the absence of OCB should suggest extending the diagnosis workup.(15, 17) Similar results were found by other displayed on table 2.(15, 55-58) Additionally, two of these studies suggested that patients with OCB converts faster to CDMS (55, 57), especially when IgM is associated.(55) Brettsschneider et al. (2010) in a prospective pilot study with 91 patients and 2 years of follow-up, showed that the presence of C-X-C motif chemokine 13 (CXCL13) in CSF has a better PPV (70%, augmented to 80% with Barkhof criteria) than OCB, Measles, Rubella and Varicella zoster reaction (MRZR, intra-thecal synthesis of specific antiviral IgG antibodies that supports the diagnosis) or Barkhof criteria.(59) A large clinical case-control cohort, including the whole spectrum of MS forms, as well as large numbers of inflammatory and non-inflammatory subjects with other neurologic diseases confirmed the potential role of CXCL13 as a MS disease activity marker. CXCL13 correlated with relapse rate, Expanded Disability Status Scale (EDSS), number of lesion on MRI and response to treatment.(60) However, this biomarker is not a disease-specific biomarker, but showed a close relationship with OCB and CSF cell counts.(59-61) Regarding antibodies against myelin, myelin oligodendrocyte glycoprotein (MOG), and myelin basic protein (MBP), discordant unexplained results have appeared.(62) Correlation ranged from highly significant (63), significant (64, 65), to not significant at all (62, 66). It is important to note that these controversial results were obtained with the same type of analysis of antimyelin antibodies, immunoblotting. Thus, these different results may primarily reflect differences in study cohorts rather than methodological problems.(2) Currently there is no proven value in the diagnostic and prognostic utility of CSF anti-myelin antibodies in patients with CIS.(16) CSF of patients with MS is characterized by a polyspecific, intrathecal B-cell response with a prominent antibody production against neurotropic viruses like measles, rubella and varicella zoster.(51) A prospective study with 89 CIS patients comparing MRZR with OCB and MRI lesion load showed that MRZR was significantly more frequent in CIS that developed MS during follow-up. Their data showed MRZR to have a high PPV. MRZ score, produced according to the different prognostic relevance of the three antibody included in MRZR, had the best single PPV (79%), augmented when added to MRI (≥2 lesions, 91%).(51) An important drawback of MRZR is that there’s no workable combined assay to determine these antibodies. Therefore, another study from the same authors, following 121 individuals for 2 years values more CXCL13 determination (PPV of 70%).(59) Axonal damage markers (tau and NfHSMI35) were measured by Brettschneider et al. (2006) in order to access their power to predict conversion from CIS to MS. CSF tau and NfHSMI35 were significantly elevated in CIS compared to controls, and correlated with disease activity.(67) The sensitivity predicting the conversion from CIS to MS was higher for the combination of CSF markers (either tau or NfHSMI35 elevated) than for MRI (40 versus 34%), but could be further increased to 60% if CSF and MRI criteria were combined. Similarly, the combination of tau and NfHSMI35 showed higher specificity (94%) than MRI (82%). Tau and NfHSMI35, according to this study, are valuable biomarkers for axonal damage in the CIS patients.(67) Other molecules showed results that need further evaluation: increased CSF cystatin C (in contrast with former studies), dissociation 14-3-3/tau protein in CSF, blood and CSF white blood cell count and neurofilament light.(68-71) In other hand, IgM and human leukocyte antigen G failed to be relevant in the evaluation of CIS.(72, 73) 3.1.4 Multiple Sclerosis vs Neuromyelitis optica (NMO) An example of success in researching biomarkers for differential diagnosis of MS is the aquaporin-4 antibody. Recently, highly specific serum IgG antibodies were detected by immunofluorescence staining of mouse brain tissue in a majority (73%) of patients with definite NMO and in 46% of patients at high risk for NMO.(74) NMO-IgG had 91% specificity for NMO and were suggested to be a useful diagnostic biomarker in patients with their first episode of myelitis.(75, 76) Most recently a number of confirmatory studies from different laboratories using diverse assays and different cohorts of patients were published.(77-80) A positive MRZR, defined by a combination of at least two antibody indices was found in 37/42 MS patients, while only in 1/20 NMO patients by Jarius et al. (2008) allowing the differentiation between MS and NMO.(81) Tortorella et al. (2011) demonstrated that MS patients show higher serum and CSF N-acetyl aspartate (NAA) levels than NMO patients, and higher serum NAA levels than healthy controls. High serum NAA values, exceeding the 95th percentile of serum NAA value in healthy controls, were found in all MS patients and in none with NMO. No differences in serum NAA levels were found between NMO and healthy controls.(82) Therefore, serum and CSF NAA may be valuable in the diagnostic workup to differentiate MS from NMO.(82) 3.1.5 Proteomics Multiple proteomic preliminary studies carried out in the past few years have revealed potential biomarkers candidates that may not otherwise have been discovered through function-based approaches.(10, 53, 83-88) For most of these candidate proteins, the pathophysiological relevance in MS remains to be clarified. Generally the disease specificity of these proteins is rather low, possibly due to common pathological mechanisms underlying different diseases.(53, 89) A problem of CSF proteome analysis is that high-abundance proteins, such as albumin and immunoglobulin, may overlay brain-specific low-abundance CSF proteins in the electrophoresis. Therefore, CSF has to be pre-processed to extract those extraproteins, possibly resulting in wasting proteins that are bounded do albumin.(53, 85) The advantages of mass spectrometry-based protein quantification are precision, sensitivity, throughput and convenience.(83) An important aspect in future CSF proteome studies is the standardization of sampling protocols, experimental design and data analysis, in order to reach consensus.(53) Further studies are needed to better characterize the correlation of these new candidate markers with MS.(88-90) 4. Conclusion Diagnosis of MS is currently based on clinical presentation, MRI findings, and CSF examination (OCB and IgG index) and requires the exclusion of alternative diagnosis.(45) CSF is especially important in not making a diagnosis of MS; that is, in excluding other conditions that mimic MS and as a predictor of conversion to CDMS. As long as we do not know what causes MS we will not have a simple diagnostic test .(91) To understand the relation of marker concentrations in body fluids and neuropathological processes, the underlying process need further investigation.(92) With increasing sensitivity and resolution of analytical methods, the demands of rigorous control of sample handling and storage also increase.(93) Often, early studies show great potential, but subsequent reports on the same marker yield inconsistent or even contradictory results. The reasons for this are mainly methodological although the heterogeneity and the lack of solid knowledge about MS also contribute.(10, 12, 65, 72, 94, 95) The use of OCB has some limitations and in result of MS heterogeneity, one marker possibly isn’t enough. In this respect, development of novel laboratory techniques and search for new reliable MS-specific markers remains of considerable diagnostic importance for MS.(46, 96) Other than OCB, MRZR and CXCL13 appeared like valuable markers for conversion of CIS to MS, and anti-myelin antibodies the opposite. Predicting conversion from CIS to MS can be improved combining MRI with CSF markers, but MRI remains as the major prognostic factor.(16, 54, 59, 67) CSF OCB utility was demonstrated in diagnosing and predicting MS, with the awareness of the frequency variability in the different communities. When present, is a strong support for MS but the diagnosis should be reviewed in it absence.(21, 33) The equivalent use of IgG index comparing with OCB is inadequate according with some authors.(17) Quantitative KFCL’s presented as a helpful marker for MS diagnosis, and should be further investigated.(46) Besides aquaporin-4 antibody, the presence of NAA in the serum can be important to distinguish MS from neuromyelitis.(82) Even though OCB provide important information regarding MS diagnosis, there are currently no CSF biomarkers that can reliably and definitively diagnose MS at the time of initial symptoms.(10) The development of more sensitive proteomic techniques is very promising for the identification of new molecular targets for diagnostic assays and therapies preventing axonal damage.(92) 5. Acknowledgements The help and availability of Dr Joana Guimarães were extremely helpful and welcome during this paper elaboration, reason why I am sincerely grateful. 6. 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MRI criteria in MS patients with negative and positive oligoclonal bands: equal fulfillment of Barkhof's criteria but different lesion patterns. Journal of neurology. 2009;256(7):1121-5. Epub 2009/03/03. 32. Villar LM, Masterman T, Casanova B, Gomez-Rial J, Espino M, Sadaba MC, et al. CSF oligoclonal band patterns reveal disease heterogeneity in multiple sclerosis. Journal of neuroimmunology. 2009;211(1-2):101-4. Epub 2009/05/16. 33. Siritho S, Freedman MS. The prognostic significance of cerebrospinal fluid in multiple sclerosis. Journal of the neurological sciences. 2009;279(1-2):21-5. Epub 2009/02/03. 34. Mares J, Herzig R, Urbanek K, Sladkova V, Sklenarova J, Bekarek V, et al. Correlation of the IgG index and oligoclonal bands in the cerebrospinal fluid of patients with multiple sclerosis. Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czechoslovakia. 2008;152(2):247-9. Epub 2009/02/17. 35. Wolinsky JS. The diagnosis of primary progressive multiple sclerosis. Journal of the neurological sciences. 2003;206(2):145-52. Epub 2003/02/01. 36. Dujmovic I, Mesaros S, Pekmezovic T, Levic Z, Drulovic J. Primary progressive multiple sclerosis: clinical and paraclinical characteristics with application of the new diagnostic criteria. European journal of neurology : the official journal of the European Federation of Neurological Societies. 2004;11(7):439-44. Epub 2004/07/20. 37. Martino G, Servalli C, Filippi M, Buscemi M, Martinelli V, Furlan R, et al. Absence of oligoclonally restricted immunoglobulins in tears from multiple sclerosis patients. Journal of neuroimmunology. 1993;44(2):149-55. Epub 1993/05/01. 38. Mehta PD, Patrick BA, Miller JA. Absence of oligoclonal IgA in CSF and serum of multiple sclerosis patients. Journal of neuroimmunology. 1984;6(1):67-9. Epub 1984/02/01. 39. Pohl D, Rostasy K, Reiber H, Hanefeld F. CSF characteristics in early-onset multiple sclerosis. Neurology. 2004;63(10):1966-7. Epub 2004/11/24. 40. Ascherio A, Munger KL. Environmental risk factors for multiple sclerosis. Part I: the role of infection. Annals of neurology. 2007;61(4):288-99. Epub 2007/04/21. 41. Torkildsen O, Stansberg C, Angelskar SM, Kooi EJ, Geurts JJ, van der Valk P, et al. Upregulation of immunoglobulin-related genes in cortical sections from multiple sclerosis patients. Brain pathology (Zurich, Switzerland). 2010;20(4):720-9. Epub 2009/11/19. 42. Jilek S, Schluep M, Meylan P, Vingerhoets F, Guignard L, Monney A, et al. Strong EBVspecific CD8+ T-cell response in patients with early multiple sclerosis. Brain : a journal of neurology. 2008;131(Pt 7):1712-21. Epub 2008/06/14. 43. Bartos A, Fialova L, Soukupova J, Kukal J, Malbohan I, Pitha J. Antibodies against light neurofilaments in multiple sclerosis patients. Acta neurologica Scandinavica. 2007;116(2):1007. Epub 2007/07/31. 44. Jurewicz A, Matysiak M, Raine CS, Selmaj K. Soluble Nogo-A, an inhibitor of axonal regeneration, as a biomarker for multiple sclerosis. Neurology. 2007;68(4):283-7. Epub 2007/01/24. 45. Lindsey JW, Crawford MP, Hatfield LM. Soluble Nogo-A in CSF is not a useful biomarker for multiple sclerosis. Neurology. 2008;71(1):35-7. Epub 2008/05/23. 46. Kaplan B, Aizenbud BM, Golderman S, Yaskariev R, Sela BA. Free light chain monomers in the diagnosis of multiple sclerosis. Journal of neuroimmunology. 2010;229(1-2):263-71. Epub 2010/09/28. 47. Tian ZJ, Zhao XX, Li ZH, Zhang F, Cao FT, Li SM, et al. [Evaluation of myelin basic protein levels with receiver operating characteristic curves for diagnosis of multiple sclerosis]. Nan fang yi ke da xue xue bao = Journal of Southern Medical University. 2009;29(2):250-2. Epub 2009/02/28. 48. Kulakowska A, Drozdowski W, Sadzynski A, Bucki R, Janmey PA. Gelsolin concentration in cerebrospinal fluid from patients with multiple sclerosis and other neurological disorders. European journal of neurology : the official journal of the European Federation of Neurological Societies. 2008;15(6):584-8. Epub 2008/05/14. 49. Mashayekhi F, Salehi Z. Expression of leukemia inhibitory factor in the cerebrospinal fluid of patients with multiple sclerosis. Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia. 2011;18(7):951-4. Epub 2011/05/17. 50. Diestel A, Aktas O, Hackel D, Hake I, Meier S, Raine CS, et al. Activation of microglial poly(ADP-ribose)-polymerase-1 by cholesterol breakdown products during neuroinflammation: a link between demyelination and neuronal damage. The Journal of experimental medicine. 2003;198(11):1729-40. Epub 2003/12/06. 51. Brettschneider J, Tumani H, Kiechle U, Muche R, Richards G, Lehmensiek V, et al. IgG antibodies against measles, rubella, and varicella zoster virus predict conversion to multiple sclerosis in clinically isolated syndrome. PloS one. 2009;4(11):e7638. Epub 2009/11/06. 52. Miller D, Barkhof F, Montalban X, Thompson A, Filippi M. Clinically isolated syndromes suggestive of multiple sclerosis, part I: natural history, pathogenesis, diagnosis, and prognosis. Lancet neurology. 2005;4(5):281-8. Epub 2005/04/26. 53. Tumani H, Lehmensiek V, Rau D, Guttmann I, Tauscher G, Mogel H, et al. CSF proteome analysis in clinically isolated syndrome (CIS): candidate markers for conversion to definite multiple sclerosis. Neuroscience letters. 2009;452(2):214-7. Epub 2009/04/23. 54. Tintore M, Rovira A, Rio J, Tur C, Pelayo R, Nos C, et al. Do oligoclonal bands add information to MRI in first attacks of multiple sclerosis? Neurology. 2008;70(13 Pt 2):1079-83. Epub 2007/09/21. 55. Villar LM, Garcia-Barragan N, Sadaba MC, Espino M, Gomez-Rial J, Martinez-San Millan J, et al. Accuracy of CSF and MRI criteria for dissemination in space in the diagnosis of multiple sclerosis. Journal of the neurological sciences. 2008;266(1-2):34-7. Epub 2007/09/22. 56. Perumal J, Zabad R, Caon C, MacKenzie M, Tselis A, Bao F, et al. Acute transverse myelitis with normal brain MRI : long-term risk of MS. Journal of neurology. 2008;255(1):89-93. Epub 2007/12/18. 57. Oligoclonal bands and MRI in clinically isolated syndromes: predicting conversion time to multiple sclerosis. Journal of neurology. 2010;257(7):1188-91. Epub 2010/02/17. 58. Bosca I, Magraner MJ, Coret F, Alvarez-Cermeno JC, Simo-Castello M, Villar LM, et al. The risk of relapse after a clinically isolated syndrome is related to the pattern of oligoclonal bands. Journal of neuroimmunology. 2010;226(1-2):143-6. Epub 2010/06/12. 59. Brettschneider J, Czerwoniak A, Senel M, Fang L, Kassubek J, Pinkhardt E, et al. The chemokine CXCL13 is a prognostic marker in clinically isolated syndrome (CIS). PloS one. 2010;5(8):e11986. Epub 2010/08/12. 60. Khademi M, Kockum I, Andersson ML, Iacobaeus E, Brundin L, Sellebjerg F, et al. Cerebrospinal fluid CXCL13 in multiple sclerosis: a suggestive prognostic marker for the disease course. Multiple sclerosis (Houndmills, Basingstoke, England). 2011;17(3):335-43. Epub 2010/12/08. 61. Ragheb S, Li Y, Simon K, VanHaerents S, Galimberti D, De Riz M, et al. Multiple sclerosis: BAFF and CXCL13 in cerebrospinal fluid. Multiple sclerosis (Houndmills, Basingstoke, England). 2011;17(7):819-29. Epub 2011/03/05. 62. Pelayo R, Tintore M, Montalban X, Rovira A, Espejo C, Reindl M, et al. Antimyelin antibodies with no progression to multiple sclerosis. The New England journal of medicine. 2007;356(4):426-8. Epub 2007/01/26. 63. Tomassini V, De Giglio L, Reindl M, Russo P, Pestalozza I, Pantano P, et al. Anti-myelin antibodies predict the clinical outcome after a first episode suggestive of MS. Multiple sclerosis (Houndmills, Basingstoke, England). 2007;13(9):1086-94. Epub 2007/05/01. AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 2 EDITORIAL BOARD . Chief Editors: B. Banwell, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8 G. Giovannoni, Neuroimmunology Unit, Neuroscience Centre; Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry; 4 Newark Street; London E1 2AT, United Kingdom C. H. Hawkes, Neuroimmunology Unit, Neuroscience Centre; Institute of Cell and Molecular Science Barts and The London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, United Kingdom F. D. Lublin, The Corinne Goldsmith Dickinson Center for Multiple Sclerosis, Mount Sinai School of Medicine, 5 East 98th Street, New York, NY 10029-6574, USA Section Editors: Experimental Models S. Amor, Amsterdam, The Netherlands Epidemiology A. Ascherio, Boston, MA, USA Neuro-opthalmology L. Balcer, Philadelphia, PA, USA Infection J. Berger, Lexington, KY, USA Neuroethics J. Bernat, Lebanon, NH, USA Pediatric MS R. Dale, Sydney, Australia Biomarkers F. Deisenhammer, Innsbruck, Austria Genetics / transcriptomics R. Hintzen, Rotterdam, The Netherlands Measurement / quality of life J. Hobart, Plymouth, United Kingdom Teaching M. Keegan, Rochester, MN, USA Rehabilitation J. Kesselring, Valens, Switzerland Clinical Neurology J.I. Kira, Fukuaka, Japan Psychology and Fatigue D. Langdon, Egham Surrey, United Kingdom Stem Cell Research G. Martino, Milan, Italy Therapy A. Miller, New York, NY, USA Immunology F. Sellebjerg, Copenhagen, Denmark Imaging J. Wolinsky, Houston, TX, USA Editorial Board: H. Butzkueven, Melbourne, Australia V. Brinar, Zagreb, Croatia AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 3 J. Correale, Buenos Aires, Argentina O. Fernandez, Malaga, Spain E. Havrdova, Prague, Czech Republic H.P. Hartung, Dusseldorf, Germany L. Kappos, Basel, Switzerland N. Koch-Henriksen, Aarhus, Denmark P. Li, Hong Kong, China J. Losy, Poznan, Poland C. Lubetzki, Paris, France A.H. Maghzi, Isfahan, Iran A. Miller, Haifa, Israel N. Prayoonwiwat, Bangkok, Thailand T. Saida, Kyoto, Japan B. Singhal, Mumbai, India S. Sveinbjornsdottir, Reykjavik, Iceland J. Torro, Bogota, Columbia C.P. Tsai, Taipei, Taiwan L. Vécsei, Szeged, Hungary E. Willoughby, Auckland, New Zealand B. Yamout, Beirout, Lebanon AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 4 GUIDE FOR AUTHORS . INTRODUCTION Types of article Original Research Articles Full length research papers will not normally be more than 3500 words in length from the Inotrduction through the Discussion section and will preferably be shorter. Submission of a paper to Multiple Sclerosis and Related Disorders will be held to imply that it represents original research not previously published (except in the form of an abstract or preliminary report), that it is not being considered for publication elsewhere, and that if accepted by Multiple Sclerosis and Related Disorders it will not be published elsewhere in the same form in any language without the consent of the Publisher. Major papers of topical content will be given priority in publication. Book Reviews These are normally submitted by the Book Review Editors, but they welcome suggestions of books for review. Case Reports Case reports should detail the clinical, laboratory and neuroimaging features of informative patients. Informative patients should provide insights that inform on genetic contributions to disease, rare clinical manifestations, novel laboratory or imaging features, or highlight important concepts in the differential of MS and related disorders. Case reports should be approximately 1200 words, and should have no more than five key references Comment Comments should focus on specific issues relevant to MS and related disorders, or should discuss recent publications. Comments should be less than 800 words and should reference the article(s) upon which the commentary is based. Clinical Trial papers Manuscripts detailing the results of clinical trials in MS and related disorders are encourage. The trial methodology should account for all screened participants, and analyses should observe an intentionto -treat model where appropriate. All sources of funding for the study must be disclosed, and the involvement of the study sponsor must be detailed. Clinical trial manuscripts should be a maximum of 3500 words. Editorials The Editors welcome suggestions for editorials which give personal and topical views on subjects within the Journal's area of interest. They should not normally exceed 1500 words in total, including references. Letters to the Editors These normally refer to articles previously published in the Journal. The Editors are also willing to consider letters on subjects of direct relevance to the Journal's interest. Letters should not exceed 1000 words in total and, where appropriate, must begin with the reference to the published article about which the author is commenting. Research letters should be submitted as 'letter to the Editors' Review Articles Review papers are normally 4000-5000 words in total. Authors are advised to consult one of the Editors with an outline before submitting a review. Contact details for submission Authors may send queries concerning the submission process, manuscript status, or journal procedures to the Editorial Office at: MSARD, Editorial Office, ELSEVIER. E-mail: [email protected] BEFORE YOU BEGIN Ethics in publishing For information on Ethics in publishing and Ethical guidelines for journal publication see http://www.elsevier.com/publishingethics and http://www.elsevier.com/ethicalguidelines. AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 5 Policy and ethics The work described in your article must have been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans http://www.wma.net/en/30publications/10policies/b3/index.html; EU Directive 2010/63/EU for animal experiments http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm; Uniform Requirements for manuscripts submitted to Biomedical journals http://www.icmje.org. This must be stated at an appropriate point in the article. 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Note that: (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the accepted manuscript in an online issue is suspended until authorship has been agreed. After the accepted manuscript is published in an online issue: Any requests to add, delete, or rearrange author names in an article published in an online issue will follow the same policies as noted above and result in a corrigendum. Clinical trial results In line with the position of the International Committee of Medical Journal Editors, the journal will not consider results posted in the same clinical trials registry in which primary registration resides to be prior publication if the results posted are presented in the form of a brief structured (less than 500 words) abstract or table. However, divulging results in other circumstances (e.g., investors' meetings) is discouraged and may jeopardise consideration of the manuscript. 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For this purpose, a clinical trial is defined as any research study that prospectively assigns human participants or groups of humans to one or more health-related interventions to evaluate the effects of health outcomes. Health-related interventions include any intervention used to modify a biomedical or health-related outcome (for example drugs, surgical procedures, devices, behavioural treatments, dietary interventions, and process-of-care changes). Health outcomes include any biomedical or health-related measures obtained in patients or AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 6 participants, including pharmacokinetic measures and adverse events. Purely observational studies (those in which the assignment of the medical intervention is not at the discretion of the investigator) will not require registration. Further information can be found at http://www.icmje.org. 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Formats Regardless of the application used, when your electronic artwork is finalised, please 'save as' or convert the images to one of the following formats (note the resolution requirements for line drawings, halftones, and line/halftone combinations given below): EPS: Vector drawings. Embed the font or save the text as 'graphics'. TIFF: Color or grayscale photographs (halftones): always use a minimum of 300 dpi. TIFF: Bitmapped line drawings: use a minimum of 1000 dpi. TIFF: Combinations bitmapped line/half-tone (color or grayscale): a minimum of 500 dpi is required. If your electronic artwork is created in a Microsoft Office application (Word, PowerPoint, Excel) then please supply 'as is'. Please do not: • Supply files that are optimised for screen use (e.g., GIF, BMP, PICT, WPG); the resolution is too low; • Supply files that are too low in resolution; • Submit graphics that are disproportionately large for the content. Color artwork Please make sure that artwork files are in an acceptable format (TIFF, EPS or MS Office files) and with the correct resolution. If, together with your accepted article, you submit usable color figures then Elsevier will ensure, at no additional charge, that these figures will appear in color on the Web (e.g., ScienceDirect and other sites) regardless of whether or not these illustrations are reproduced in color in the printed version. For color reproduction in print, you will receive information regarding the costs from Elsevier after receipt of your accepted article. Please indicate your preference for color: in print or on the Web only. For further information on the preparation of electronic artwork, please see http://www.elsevier.com/artworkinstructions. Please note: Because of technical complications which can arise by converting color figures to 'gray scale' (for the printed version should you not opt for color in print) please submit in addition usable black and white versions of all the color illustrations. Figure captions Ensure that each illustration has a caption. Supply captions separately, not attached to the figure. A caption should comprise a brief title (not on the figure itself) and a description of the illustration. Keep text in the illustrations themselves to a minimum but explain all symbols and abbreviations used. Tables Number tables consecutively in accordance with their appearance in the text. Place footnotes to tables below the table body and indicate them with superscript lowercase letters. Avoid vertical rules. Be sparing in the use of tables and ensure that the data presented in tables do not duplicate results described elsewhere in the article. References Citation in text Please ensure that every reference cited in the text is also present in the reference list (and vice versa). Any references cited in the abstract must be given in full. Unpublished results and personal communications are not recommended in the reference list, but may be mentioned in the text. If these references are included in the reference list they should follow the standard reference style of the journal and should include a substitution of the publication date with either 'Unpublished results' or 'Personal communication'. Citation of a reference as 'in press' implies that the item has been accepted for publication. Reference management software This journal has standard templates available in key reference management packages EndNote http://www.endnote.com/support/enstyles.asp and Reference Manager http://refman.com/support/rmstyles.asp. Using plug-ins to wordprocessing packages, authors only need to select the appropriate journal template when preparing their article and the list of references and citations to these will be formatted according to the journal style which is described below. Web references As a minimum, the full URL should be given and the date when the reference was last accessed. Any further information, if known (DOI, author names, dates, reference to a source publication, etc.), should also be given. Web references can be listed separately (e.g., after the reference list) under a different heading if desired, or can be included in the reference list. AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 10 References in a special issue Please ensure that the words 'this issue' are added to any references in the list (and any citations in the text) to other articles in the same Special Issue. Reference style Text: All citations in the text should refer to: 1. Single author: the author's name (without initials, unless there is ambiguity) and the year of publication; 2. Two authors: both authors' names and the year of publication; 3. Three or more authors: first author's name followed by 'et al.' and the year of publication. Citations may be made directly (or parenthetically). Groups of references should be listed first alphabetically, then chronologically. Examples: 'as demonstrated in wheat (Allan, 2000a, 2000b, 1999; Allan and Jones, 1999). Kramer et al. (2010) have recently shown ....' List: References should be arranged first alphabetically and then further sorted chronologically if necessary. More than one reference from the same author(s) in the same year must be identified by the letters 'a', 'b', 'c', etc., placed after the year of publication. Examples: Reference to a journal publication: Van der Geer J, Hanraads JAJ, Lupton RA. The art of writing a scientific article. J Sci Commun 2010;163:51–9. Reference to a book: Strunk Jr W, White EB. The elements of style. 4th ed. New York: Longman; 1979. Reference to a chapter in an edited book: Mettam GR, Adams LB. How to prepare an electronic version of your article. In: Jones BS, Smith RZ, editors. Introduction to the electronic age. New York: E-Publishing Inc; 2009. p. 281–304. Note shortened form for last page number. e.g., 51–9, and that for more than 6 authors the first 6 should be listed followed by "et al." For further details you are referred to "Uniform Requirements for Manuscripts submitted to Biomedical Journals" (J Am Med Assoc 1997;277:927–34) (see also http://www.nlm.nih.gov/bsd/uniform_requirements.html). Journal abbreviations source Journal names should be abbreviated according to Index Medicus journal abbreviations: http://www.nlm.nih.gov/tsd/serials/lji.html; List of title word abbreviations: http://www.issn.org/2-22661-LTWA-online.php; CAS (Chemical Abstracts Service): http://www.cas.org/sent.html. Video data Elsevier accepts video material and animation sequences to support and enhance your scientific research. Authors who have video or animation files that they wish to submit with their article are strongly encouraged to include these within the body of the article. This can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be placed. All submitted files should be properly labeled so that they directly relate to the video file's content. In order to ensure that your video or animation material is directly usable, please provide the files in one of our recommended file formats with a preferred maximum size of 50 MB. Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products, including ScienceDirect: http://www.sciencedirect.com. Please supply 'stills' with your files: you can choose any frame from the video or animation or make a separate image. These will be used instead of standard icons and will personalize the link to your video data. For more detailed instructions please visit our video instruction pages at http://www.elsevier.com/artworkinstructions. Note: since video and animation cannot be embedded in the print version of the journal, please provide text for both the electronic and the print version for the portions of the article that refer to this content. Supplementary data Elsevier accepts electronic supplementary material to support and enhance your scientific research. Supplementary files offer the author additional possibilities to publish supporting applications, highresolution images, background datasets, sound clips and more. Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products, including ScienceDirect: http://www.sciencedirect.com. In order to ensure that your submitted material is directly usable, please provide the data in one of our recommended file formats. Authors should AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 11 submit the material in electronic format together with the article and supply a concise and descriptive caption for each file. For more detailed instructions please visit our artwork instruction pages at http://www.elsevier.com/artworkinstructions. Submission checklist The following list will be useful during the final checking of an article prior to sending it to the journal for review. Please consult this Guide for Authors for further details of any item. Ensure that the following items are present: One author has been designated as the corresponding author with contact details: • E-mail address • Full postal address • Telephone and fax numbers All necessary files have been uploaded, and contain: • Keywords • All figure captions • All tables (including title, description, footnotes) Further considerations • Manuscript has been 'spell-checked' and 'grammar-checked' • References are in the correct format for this journal • All references mentioned in the Reference list are cited in the text, and vice versa • Permission has been obtained for use of copyrighted material from other sources (including the Web) • Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print, or to be reproduced in color on the Web (free of charge) and in black-and-white in print • If only color on the Web is required, black-and-white versions of the figures are also supplied for printing purposes For any further information please visit our customer support site at http://support.elsevier.com. AFTER ACCEPTANCE Use of the Digital Object Identifier The Digital Object Identifier (DOI) may be used to cite and link to electronic documents. The DOI consists of a unique alpha-numeric character string which is assigned to a document by the publisher upon the initial electronic publication. The assigned DOI never changes. Therefore, it is an ideal medium for citing a document, particularly 'Articles in press' because they have not yet received their full bibliographic information. The correct format for citing a DOI is shown as follows (example taken from a document in the journal Physics Letters B): doi:10.1016/j.physletb.2010.09.059 When you use the DOI to create URL hyperlinks to documents on the web, the DOIs are guaranteed never to change. Proofs One set of page proofs (as PDF files) will be sent by e-mail to the corresponding author (if we do not have an e-mail address then paper proofs will be sent by post) or, a link will be provided in the e-mail so that authors can download the files themselves. Elsevier now provides authors with PDF proofs which can be annotated; for this you will need to download Adobe Reader version 7 (or higher) available free from http://get.adobe.com/reader. Instructions on how to annotate PDF files will accompany the proofs (also given online). The exact system requirements are given at the Adobe site: http://www.adobe.com/products/reader/tech-specs.html. If you do not wish to use the PDF annotations function, you may list the corrections (including replies to the Query Form) and return them to Elsevier in an e-mail. Please list your corrections quoting line number. If, for any reason, this is not possible, then mark the corrections and any other comments (including replies to the Query Form) on a printout of your proof and return by fax, or scan the pages and e-mail, or by post. Please use this proof only for checking the typesetting, editing, completeness and correctness of the text, tables and figures. Significant changes to the article as accepted for publication will only be considered at this stage with permission from the Editor. We will do everything possible to get your article published quickly and accurately – please let us have all your corrections within 48 hours. It is important to ensure that all corrections are sent back to us in one communication: please check carefully before replying, as inclusion of any subsequent corrections cannot be guaranteed. Proofreading is solely your responsibility. Note that Elsevier may proceed with the publication of your article if no response is received.