Danish Multiple Sclerosis Center Annual Report 2013

Transcription

1 Department of Neurology THE Neuroscience Centre Copenhagen University Hospital Rigshospitalet Copenhagen, Denmark Danish Multiple Sclerosis Center Annual Report 2013

2 DMSC missions and aims The mission of Rigs hospitalet is to be the leading hospital in Denmark for patients in need of highly specialized treatment The missions of the Danish Multiple Sclerosis Center (DMSC) are: The aims of the DMSC are: to be the leading multiple sclerosis (MS) center in Denmark to be at the forefront of highly specialized management of MS to carry out research and development in MS at an advanced international level to collaborate scientifically and exchange knowledge in MS research to educate staff to a highly specialized level in their relevant fields to contribute with professional advice on MS to the healthcare community to meet people with MS at their terms with openness and respect to provide the optimal interdisciplinary patient care to all MS patients in the region and to patients from other regions in need of highly specialized therapy to carry out high quality research in MS with focus on clinical research, new therapies, MS genetics, neuroimmunology and MS pathology to teach undergraduate students and PhDstudents and stimulate their interest in MS research to educate post docs, MS physicians, nurses, secretaries and other professionals to a high level of knowledge of MS in their relevant expert fields to lead the national research in Denmark in partnership with other Danish researchers and to establish a broad international collaboration with MS research groups in Europe and from overseas. DMSC staff seminar DANISH Multiple sclerosis CENTER annual report 2013

3 Table of contents Annual Report DMSC missions and aims A short review of 2013 in the Danish Multiple Sclerosis Center About the Danish Multiple Sclerosis Center Organization diagram Research activities 2013 Clinical research Neuro Imaging Neurogenetics Neuroimmunology Routine analyses in Neuroimmunology Laboratory Scientific publications Honorary offices Scientific collaboration Collaboration with pharmaceutical companies Acknowledgements DANISH Multiple sclerosis CENTER annual report

4 Professor Per Soelberg Sørensen, MD, DMSc Director of the Danish Multiple Sclerosis Center (DMSC) A short review of 2013 in the Danish Multiple Sclerosis Center In 2013 Copenhagen hosted the 29th congress of the European Committee on Treatment and Research in Multiple Sclerosis (ECTRIMS) and the 18th Annual Conference of Rehabilitation in Multiple Sclerosis. Per Soelberg Sørensen was chairman of the local organizing committee and several colleagues at the Danish Multiple Sclerosis Center (DMSC) were involved as members of the local organizing committee. The congress took place in the Bella Congress Center and became the largest MS meeting ever with more than 7600 active delegates from 93 countries. Her Majesty Queen Margrethe II attended the opening ceremony and enjoyed the performance of the Safri Duo. The scientific programme included 93 oral presentation, 967 posters, 14 teaching courses, and 11 satellite symposia. Overall, the congress was very successful and it was appointed Congress Host of the Year at the Copenhagen Congress and Event Award 2014 by the Municipality of Copenhagen and the Wonderful Copenhagen committee. DMSC got its second professor in May 2013 when Finn Sellebjerg was appointed professor in neuroimmunology and multiple sclerosis at University of Copenhagen, sponsored by the Danish Multiple Sclerosis Society. Moreover, Annette Oturai was appointed associate research professor from July From January 2014 the Danish Multiple Sclerosis Register that is supported by the Danish Multiple Sclerosis Society will be associated with DMSC and the leader of the register will be employed as consultant at DMSC. In 2013 we completed the refurbishment of the MS Clinic with significant improvement in space and comfort for our patients and better working conditions for the personnel. Two new treatments for relapsing-remitting multiple sclerosis were Opening of the 29th ECTRIMS congress 4 DANISH Multiple sclerosis CENTER annual report 2013

5 approved in Europe and taken in use at DMSC in 2013: Aubagio, the first 1.-line oral treatment, and Lemtrada, a very effective therapeutic antibody that only needs to be administered as a short series of intravenous infusions every year. The MS Research Unit has completed a placebocontrolled, double-blind clinical trial of EPO in patients with progressive multiple sclerosis with the aim to test if EPO could improve neurological deficits, and the results will be reported at the next ECTRIMS congress. Another trial in patients with progressive multiple sclerosis has used monthly oral methylprednisolone courses to decrease inflammation and disease progression. The study will be completed early Our study of mesenchymal stem cells for treatment of relapsing-remitting MS, which is as a part of a large international collaborative investigator driven study, has proceeded as planned in 2013 and approximate half of the patients have been enrolled. In collaboration with the Novo Nordisk Foundation Center for Basic Metabolic Research, Section of Metabolic Genetics at University of Copenhagen we have initiated an ambitious project: Studies of the role of altered gut bacteria flora in the pathogenesis of multiple sclerosis, in which 200 multiple sclerosis patients from DMSC will have their stools examined to study whether patients with multiple sclerosis harbour a diseasespecific gut microbiota that promotes production of immune-modulating metabolites and inflammatory markers and plays an important role in the pathogenesis of multiple sclerosis. Jeppe Romme Christensen defended his ph.d. thesis Systemic and intrathecal activation in multiple sclerosis Emphasis on progressive multiple sclerosis in February Other ph.d. studies comprise molecular biology studies on immune activation in progressive MS and studies of the interaction between genes and environmental factors of importance for MS with focus on vitamin D and vitamin D metabolism. Together with the Danish Multiple Sclerosis Register we have completed a ph.d. study of gender aspect of multiple sclerosis including the protective effect of childbirth. This thesis will be defended in A junior Consultant in DMSC, Mikkel Anthonisen, has succeeded in realising his project, Sailing Sclerosis, in The project implies sailing people with multiple sclerosis around the world in a sailing boat. The boat will leave Copenhagen in June 2014, cross the Atlantic and arrive at the multiple sclerosis Congress (ACTRIMS-ECTRIMS) in Boston in September Members of the staff in DMSC will serve as neurologist to the people on board at some of the legs of the journey. I hope you will enjoy reading the annual report of DMSC. Per Soelberg Sørensen Congress Host of the Year Award DANISH Multiple sclerosis CENTER annual report

6 About The Danish Multiple Sclerosis Center 6 DANISH Multiple sclerosis CENTER annual report 2013

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8 The Danish Multiple Sclerosis Center About the Danish Multiple Sclerosis Center ACtive patients in DMSC The Danish Multiple Sclerosis Center (DMSC) is the leading centre for treatment and research in multiple sclerosis in Denmark and provides multidisciplinary care for more than 2000 MS patients, offering both basic and highly specialized therapy. We serve patients from the Copenhagen capital region and many patients from neighbouring regions and from all over Zealand are referred for regional and highly specialized therapy. DMSC is composed of an MS Clinic and an MS Research Unit. The MS Clinic is located at the 8th floor of the main complex of Rigshospitalet where the offices of professors and consultants are located. The MS Clinic contains the reception desk, the secretary offices, the nurse offices, as well as the outpatient consultation rooms, facilities for intravenous infusion-therapy with disease modifying drugs, and rooms for invasive procedures. We have been designated highly specialized function in providing therapy with strong immunosuppressants and experimental medications. From 2011 we have been offering immunoablative therapy followed by autologous stem cell transplantation to patients with very active MS and accumulation of disability despite immunomodulatory or immunosuppressive therapy DMSC is the Eastern Denmark centre for treatment of children and adolescence with MS and has a highly specialized function in treating neuromyelitis optica. DMSC also offers treatment of severe spasticity with an intrathecal baclofen pump, not only to patients with MS but also to other patients with diseases or traumatic injuries causing severe spasticity. It is the aim of the MS Clinic to provide highquality multi-disciplinary care for all our patients with openness and respect. The staff comprises two professors, one associate professor, 5 consultants, 1 staff neurologist and several external consultants working part time in the MS Clinic. There is one leading nurse and 9 MS specialist nurses, 3 secretaries, a neuropsychologist, a physiotherapist and a medical social counsellor. The MS Research Unit is located partly in the proximity of the MS Clinic, where most patientrelated clinical research takes place and where the offices of 3 research nurses and 1 research secretary are embedded. The remaining part of the MS Research Unit is located on the first floor in the Michaelsen Building 63 and in the basement of building 93. These facilities contain the Neuroimmunology Laboratory and offices for the research staff. The laboratory is equipped with an 8-colour flow cytometer and facilities for doing real-time polymerase chain reaction (PCR). Further, the facilities contain the MS Biobank and the neurogenetics laboratory for DNA preparation, and facilities for making routine laboratory tests. The focus of the research in DMSC is clinical research, neuroimmunology, neurogenetics and, in particular, translational research aiming at implementing the findings in neurogenetics and immunology into new therapies of MS. 8 DANISH Multiple sclerosis CENTER annual report 2013

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10 Organization Danish Multiple Sclerosis Research Center Director: Professor Per Soelberg Sørensen MS clinic MS Research Unit Neurologists Professor Per Soelberg Sørensen Professor Finn Sellebjerg Consultant Morten Blinkenberg Ass. Professor Annette Oturai Consultant Karen Schreiber Consultant Ana Voldsgaard Consultant Melinda Magyari Staff neurologist Henrik Mathiesen Staff neurologist Peter Roos PhD student Eva Rosa Petersen PhD student Rikke Ratzer PhD student Julie Maria Hejgaard PhD student Cecilie Ammitzbøll Ext. Consultant Sarah Taudorf Ext. consultant Mikael Lund Ext. consultant Mikkel Anthonisen Ext. consultant Janus Kaufmann Ext. Consutant Lars-Henrik Krarup Ext. Consultant Tina Dysgaard Ext. consultant Stephan Bramow Ext. consultant Cecilia Rajda MS nurses Leading nurse Anne Hansen Dorthe Stauning Rasmussen Anette Husted Pedersen Lene Almind Julie Yoon S. Moberg Louise Nathalie Christiansen Rie Forsberg Pedersen Sidsel Nielsen Mette Harborg Karina Jørgensen Secretaries Annette Larsen Malene Møllesøe Pia Maria Sandstød Ibsen Maiken Leth Svane Neuropsychologist Agnete Jønsson Lisbet Marstrand Physiotherapist Lis Albrechtsen Medical social counselor Keld Stage Clinical research Professor Per Soelberg Sørensen Consultant Karen Schreiber Consultant Ana Voldsgaard Neuropsycologist Agnete Jønsson PhD student Melinda Magyari Research nurses Vibeke Jespersen Joan Pietraszek Sidsel Nielsen Research secretary Annette Larsen Neuroimaging research Consultant Morten Blinkenberg Staff neurologist Henrik Mathiesen Genetic research Ass. Professor Annette Oturai Senior research fellow Helle Bach Søndergaard PhD students Julie Maria Hejgaard Neuroimmunology research Professor Finn Sellebjerg Senior research fellow Helle Bach Søndergaard Senior research fellow Lars Börnsen Senior research fellow Marina Rode Von Essen Part-time post doc Jeppe Romme Christensen PhD students Rikke Ratzer Cecilie Ammitzbøll Julie Maria Hejgaard Eva Rosa Petersen Neuroimmunology Laboratory Head of Laboratory Poul Erik Hyldgaard Jensen Laboratory technicians Leading Laboratory technician Joy Mendel-Hartvig Michael K. Jensen Vibeke Lindgaard Fuglholt Rikke Larsen Freja Melissa Bekner Betina Gall 10 DANISH Multiple sclerosis CENTER annual report 2013

11 Professor Per Soelberg Sørensen Professor Finn Sellebjerg Consultant Morten Blinkenberg Ass. professor Annette Oturai Consultant Karen Schreiber Consultant Ana Voldsgaard Consultant Melinda Magyari Staff neurologist Henrik Mathiesen Laboratory leader Poul Erik Hyldgaard Jensen Senior research fellow Helle Bach Søndergaard Senior research fellow Lars Börnsen Senior research fellow Marina Rode Von Essen Leading laboratory technician Joy Mendel- Hartvig Leading nurse Anne Hansen DANISH Multiple sclerosis CENTER annual report

12 Research activities 2013 Clinical research Clinical research Neuroimaging Neurogenetics Neuroimmunology Routine analyses in Neuroimmunology Laboratory 12 DANISH Multiple sclerosis CENTER annual report 2013

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14 Research activities 2013 Clinical and epidemiological research Clinical research group: Per Soelberg Sørensen, Morten Blinkenberg, Finn Sellebjerg, Annette Oturai, Ana Voldsgaard, Karen Schreiber, Henrik Mathiesen, Melinda Magyari, Lars Bornsen, Jeppe Romme Christensen, Rikke Ratzer, Julie Maria Hejgaard, Vibeke Jespersen, Joan Pietraszek, Sidsel Walther Nielsen, Anne Hansen, Annette Larsen. Therapeutic trials of new medicine Most of our efforts in therapeutic trials of new medicine have been directed towards treatment of patients with progressive forms of MS. We are currently performing several single-centre studies in patients with progressive MS in order to try to find treatment options for this phase of the disease that currently lacks effective therapy. These studies are performed in collaboration with the Danish Research Centre for Magnetic Resonance, Hvidovre Hospital. We published the results of a proof-of-concept phase II open-label study (Neurology 2014) of the effects of treatment with natalizumab 300 mg every 4 weeks for 60 weeks in patients with progressive MS. Compared with baseline findings natalizumab significantly reduced inflammatory molecules in the cerebrospinal fluid, and also markers of axonal damage (neurofilament light) decreased significantly. In addition, we showed a reduction in disability (mean EDSS), reduction in grey matter atrophy on MRI and increase in magnetisation transfer ratio. The findings suggest a role of systemic inflammation in progressive MS, and natalizumab treatment may have favourable clinical effect in progressive MS patients. In this regard we also studied the effect of erythropoietin (EPO) on disability in patients with progressive MS in a randomized double-blind trial. 56 patients with primary or secondary progressive MS have been included and treated with either EPO or placebo for 24 weeks. The primary outcome measure is the change from baseline to 24 weeks in a composite measure of maximum gait distance, a test of hand dexterity, and a neuropsychological test. Secondary endpoints include clinical relapses, disability, and several MRI measures. The results will be presented at the ECTRIMS/ACTRIMS congress in September DANISH Multiple sclerosis CENTER annual report 2013

15 We also completed a study of 30 progressive MS patients in a pilot study of methylprednisolone administered orally in monthly courses of 500 mg for 3 days. The primary endpoint is the changes in inflammatory molecules in the cerebrospinal fluid (osteopontin, CXCL13 and matrix metalloproteinase-9), but also markers of axonal damage (neurofilament light) and demyelination (myelin basic protein) will be studied. In addition, we use conventional and non-conventional MRI markers (magnetization transfer ratio and diffusion tensor imaging) as well as reduction in disability as secondary endpoints. The results of this study will also be reported at the ECTRIMS/ACTRIMS congress i September In collaboration with the Stem Cell Unit at the Department of Clinical Immunology, the Blood Bank, Rigshospitalet and the MRI Department, Hvidovre Hospital we have initiated a trial of intravenous therapy with autologous mesenchymal stem cells in MS patients. The primary objective of the study is to assess the safety and the activity in terms of reduction as compared to placebo in the total number of contrast-enhancing lesions on MRI over 24 weeks. The secondary objectives of the study are to gather preliminary information on the efficacy of the experimental treatment in terms of combined MRI activity, clinical efficacy measures and immunological markers. The study is a randomized double-blind study comparing treatment with autologous mesenchymal stem cells vs suspension media at 24 weeks. After 24 weeks patients initially assigned to suspension media treatment will be shifted to the treatment with mesenchymal stem cells, and a secondary analysis will compare the number of adverse events and gadoliniumenhancing lesions in weeks 0-23 and in these patients. The study is a part of an international collaboration between centres in Spain, England, Sweden, Denmark, France, Germany and Canada comprising in all 160 patients, of whom 25 patients will be Danish. We have recruited half of the patients and enrolment will be completed in Currently, we take part in an EU supported study ABIRISK (Immunogenicity: assessing the clinical relevance and risk minimization of antibodies to pharmaceuticals). The project aims to provide an integrated approach to anti-drug immunization, bringing together, in an extensive and coordinated manner, a large network of clinicians from various DANISH Multiple sclerosis CENTER annual report

16 Research activities 2013 specialties with broad experience in the care of patients, biologists and scientists specialized in the mechanisms of immunogenicity, and, in addition, collaboration with a large network of private pharmaceutical companies. In addition to these investigator-driven therapeutic trials, DMSC are taking part in clinical trials of new drugs sponsored and driven by the pharmaceutical industry. We are currently involved in trials of new indications for treatment with natalizumab, and in the development of three new monoclonal antibodies, alemtuzumab, daclizumab and ofatumumab, with strong effects on disease activity. Epidemiological research In collaboration with the Danish MS Register we have performed extensive analysis of possible factors that might have contributed to the increase of MS in women. At the American Academy of Neurology Meeting in April 2013, using data from the Danish Multiple Sclerosis Registry, we presented the results of a nationwide study comprising 1403 MS patients aged at clinical onset between 2000 and We showed that childbirths within five years before clinical onset reduced the risk of MS in women, but not in men. Considering the possibility of reversed causation, which was not supported by the data, pregnancy could exert a certain protection against MS on a biological basis, lasting up to five years. We did not identify any physical or social environmental factors which could explain the gender discrepancy in the incidence increase. Further studies are needed to reveal possible etiological occupational exposures in MS. Recently we performed a pilot study of the impact of co-morbidity in MS patients and the importance of co-morbidity has evolved into a ph.d. study. The studies in women with MS continue with exploration of environmental factors, such as smoking habits and vitamin D intake. Vitamin D that seems to yield some protection against encountering MS may also influence the disease activity in established MS, and in 2010 we initiated a study exploring the role of vitamin D on clinical disease activity in MS. Paediatric MS Paediatric MS carries a relatively higher mortality and morbidity than adult MS but little is known about symptoms and paraclinical findings at the first demyelinating event in a Danish setting and the nationwide incidence of paediatric MS have never been described in Denmark. In 2013 we initiated two studies aiming at answering these questions and results are expected in DANISH Multiple sclerosis CENTER annual report 2013

17 Neuro Imaging 2013 Neuroimaging research group: Morten Blinkenberg, Henrik Mathiesen, Per Soelberg Sørensen Magnetic resonance spectroscopic imaging (MRSI) provides in vivo information about neuronal damage, loss, or dysfunction by measuring decreases of N-acetyl aspartate (NAA). In the human brain NAA is almost exclusively found within neurons and neuronal processes and therefore serves as a marker of neurodegeneration and disease progression in MS. In 2013 we completed data evaluation of a cohort of newly diagnosed relapsing remitting MS patients studied by serial MRI and MRSI for two years and clinical examinations performed up to 7 years after the primary MRI. The study shows a relationship between baseline MSRI and clinical disability after 2 and 7 years and in this way MRSI may be used as a predictor of long-term disease outcome in MS. Studies on resting-state fmri, assessing functional brain connectivity, were continued in 2013 in collaboration with Danish Research Centre for Magnetic Resonance, PhD Anne-Marie Dogonowski. Our studies show, that worsening of disability in MS results in increased coupling between regions of importance to motor function, which might reflect an adaptive mechanism to maintain motor function, as a consequence of disease progression. Furthermore, people with MS have better integrity of cortical motor connectivity compared with deep brain regions, again speaking for a relative higher preservation of motor function compared with other neural networks. Finally, studies of local neural connectivity in people with MS show disintegration in the cerebellum, underlining the integrative role of the cerebellum in motor function, processing input from spinal as well as cerebral motor regions. In conclusion, our data provide a better understanding of the functional reorganization of distinct brain networks in MS patients as a consequence of the neurodegenerative disease, which may find use in current and future rehabilitation programs. All results have been published in 2013 and data processing from resting-state motor connectivity studies during recovery from an acute motor relapse is still ongoing. In 2013 we also initiated an imaging study with a quite different focus than the brain. Corticosteroids are widely used in the treatment of MS, both for acute relapses and as add on treatment to interferon-beta. A rare side effect of corticosteroids is avascular osteonecrosis characterized by focal necrosis of bone tissue and followed by collapse of architectural bony structure. A former study has reported a high prevalence of osteonecrosis in MS patients (15.2%) receiving a high cumulated dose of methylprednisolone, although this has never been confirmed. In the current study our aim is to determine the prevalence of avascular osteonecrosis in a well characterized cohort of MS patients, treated with monthly courses of high dose methylprednisolone or placebo, as add on treatment to interferon beta-1a i.m (the MECOMBIN study). Bilateral femoral MRI is performed in order to determine the presence of avascular osteonecrosis. Results of the study are expected in DANISH Multiple sclerosis CENTER annual report

18 Research activities 2013 Neurogenetics Neurogenetics research group: Annette Bang Oturai, Helle Bach Søndergaard, Julie Hejgaard, Eva Petersen, Finn Sellebjerg, Per Soelberg Sørensen MS-genetics One major step towards revealing the genetic component in MS was taken in 2013 identifying 48 new MS genetic variations. We participated in the study as part of the International Multiple Sclerosis Genetics Consortium (IMSGC) investigating thousands of MS patients and controls in the Immuno-chip project (study of common autoimmune disease genes). We are now looking forward to the next step investigating the first so called MS-chip (replication and fine-mapping of all known MS genes). The study includes more than 1000 Danish MS patients/1000 controls, all together a total of more than MS patients and controls and more than single nucleotide polymorphisms (SNPs). Gene-environment interaction Several of our studies focus on investigating geneenvironmental interactions, based on information from a newly collected questionnaire from 2000 Danish MS patients and 5000 Danish blood donors including more than 100 questions on lifestyle and environmental factors (e.g. smoking, alcohol, mononucleosis, BMI, sun habits, geographical upbringing). Vitamin D Vitamin D is known to have a strong immunomodulatory potential and accumulating evidence supports a beneficial effect of vitamin D in the pathogenesis and disease course of MS. Genomewide association studies (GWAS) have shown significant associations between serum levels of 25(OH)D and SNPs in several key genes in the vitamin D metabolism. In 1500 MS patients we have examined the association between 25(OH)D and six GWAS SNPs, season, age, sex, eye colour, body mass index, vitamin D supplements, smoking, fish intake, sun habits and severity of MS. We found effects of environmental factors on 25(OH)D levels and to our knowledge for the first time, effects of SNPs in genes important for 25-hydroxylases in the liver (CYP2R1, gene) and vitamin D metabolism (GC, gene). Smoking It is known that both HLA genes and smoking increases the risk of MS. However, the effect and interaction of smoking and HLA type on treatment response have not yet been investigated. We have investigated if smoking status and HLA type are associated with disease activity and disease severity in interferon-beta (IFN-beta) treated relapsingremitting MS patients and demonstrate for the first time a higher disease activity and severity in MS patients who smoked before and during treatment with IFN-beta. Furthermore, the effect seems to be modulated by HLA-DRB1*15:01. The previously reported interaction between HLA genes, smoking and MS susceptibility may therefore also affect disease course BMI, mononucleosis and alcohol In order to determine the most important genetic and environmental-lifestyle factors for age at MS disease onset in Danish MS patients we investigated about 1500 patients and tested the predictive value for age of MS onset for: body mass index, smoking habits smoking, alcohol consumption sex, previous mononucleosis, education, HLA-DRB1*15:01 and HLA-A*02:01. We found that high BMI at age 20, previous mononucleosis and high alcohol consumption between age are the most important known factors for lowering age at onset in Danish MS patients Genetic influence on oligoclonal band status The presence of oligoclonal bands (OCB) in cerebrospinal fluid (CSF) is a typical finding in MS. In a Scandinavian collaboration, genetic differences in MS relating to OCB status were studied. Data from earlier GWAS were compared in 1367 OCB positive and 161 OCB negative MS patients, and nine of the most associated SNPs were genotyped for replication in 3403 Scandinavian MS patients. Results showed that SNPs from the HLA complex and six other loci were associated to OCB status. The study confirmed both shared and distinct genetic risk for MS subtypes in the Scandinavian population de- 18 DANISH Multiple sclerosis CENTER annual report 2013

19 fined by OCB status and indicates different clinical characteristics between the groups. This suggests differences in disease mechanisms between OCB negative and OCB positive MS patients with possible implications for patient management. Genetic influence on treatment and clinical outcome Investigations of the genetic influence on how patients respond to various disease modifying treatments are important to guide the most effective treatment for each patient. Previously, six single nucleotide polymorphisms were suggested to be associated with the response to treatment with IFN-β. We investigated these variants in 575 patients with relapsing-remitting MS followed prospectively after the initiation of their first treatment with IFN-β. These gene variants in IRF5, IRF8 and GPC5 did not show association with risk of relapse or disease progression in this patient cohort. However, the analyses showed that the pre-treatment relapses and clinical disease activity during the first two years of treatment may be associated with disease progression in MS patients treated with IFN-β. The chemokine receptor CCR5 may be important for the recruitment of pathogenic T cells to the CNS in multiple sclerosis (MS). A deletion in the CCR5 gene, which results in lower gene expression of this receptor, could possibly lower disease activity in MS patients. We investigated the impact of CCR5Δ32 in patients treated with Natalizumab (tysabri) and found a less severe disease in patients carrying the deletion, but no effect on disease activity in the investigated cohort. DANISH Multiple sclerosis CENTER annual report

20 Research activities 2013 Neuroimmunology Neuroimmunology research group: Finn Sellebjerg, Cecilie Ammitzbøll, Sophie Buhelt, Lars Börnsen, Marina Rode von Essen, Poul Erik Hyldgaard Jensen, Ditte Jonesco, Annette Bang Oturai, Eva Rosa Petersen, Rikke Ratzer, Birgitte Romme Nielsen, Jeppe Romme Christensen, Helle Bach Søndergaard, Per Soelberg Sørensen Neuroimmunology of MS The extent to which inflammation is involved in the pathogenesis of MS remains controversial. There is compelling evidence that treatment strategies targeting systemic immune activation or migration of immune cells to the brain and spinal cord prevent relapses in MS. Some researchers do, however, argue that primary neurodegenerative processes are more important, and that inflammation and relapses have no major impact on the long-term evolution of the disease. In our research we focus on the role of immune activation and inflammation in the pathogenesis of MS. This is achieved by studying the activation of blood cells, cerebrospinal fluid (CSF) cells and soluble inflammatory mediators in blood and CSF. We compare these in patients with relapsing-remitting, primary and secondary progressive MS, study how treatment alters these factors, and whether they are influenced by genetic, environmental and lifestyle factors associated with the risk of developing MS. Furthermore, we study whether immune activation correlates with clinical and magnetic resonance imaging measures of disease activity. In order to investigate these processes in detail, we combine molecular biology studies with flow cytometry studies of leukocyte phenotypes and functional studies of antigen reactivity, migration and cytokine secretion. The overall aim of the neuroimmunology research at DMSC is to identify immune activation processes that drive the disease evolution in MS and to understand differences between the different subtypes of MS, in this way describing the effects of MS treatment on the immune system, and how genetic and environmental factors increase the risk of MS and influence immune activation and inflammation. Molecular biology studies In the molecular biology studies we investigate mrna and protein expression and measure the expression and function of micrornas, which are small, non-coding RNA molecules that regulate the degradation and translation of mrna. In 2013 we published a study on the expression of microrna 20 DANISH Multiple sclerosis CENTER annual report 2013

21 in blood cells, serum and plasma in relapsing-remitting MS. This study showed that several micro- RNAs are differentially expressed in blood cells and plasma, and that one specific molecule (mir-145) may be a useful biomarker for diagnosing MS. In another study we compared gene expression by Affymetrix DNA arrays, and found that gene expression is quite comparable in patients with relapsingremitting, primary and secondary progressive MS. Differentially expressed genes were associated with immunoinflammatory processes, arguing for a role of these processes in all subtypes of MS. Ongoing studies further investigate the role of micrornas in MS and investigate how MS-associated genetic variants influence gene expression and immune cell functions in MS. These studies include: Studies of microrna and immune activation in pregnant patients with MS Studies of microrna biomarkers in serum and CSF Studies of microrna effect of treatment with interferon-beta and glatiramer acetate Studies of the effects of an MS-associated genetic variant in the gene encoding the interleukin-2 receptor alfa-chain in patients with MS and healthy control subjects Studies of the effects of MS-associated genetic variants on the expression of pro-inflammatory and immunoregulatory cytokines Immune activation in MS We have conducted a series of studies addressing the immune activation in blood cells and CSF in patients with relapsing-remitting, primary and secondary progressive MS. These studies complement our microarray studies, and implicate several subtypes of T lymphocytes in the pathogenesis of MS. Interestingly, a recently discovered T lymphocyte subset (follicular helper cells), which are important drivers of antibody responses, seems to be particularly important in patients with relapsing-remitting onset of the disease. We are now exploring these findings further in a series of ongoing studies: Activation of naïve, central memory and effector T cells in MS the effect of smoking and other exogenous risk factors on immune activation in MS the effect of genetic risk factors on immune activation in MS immune activation and autoreactive T cells in relapsing-remitting MS immune activation and autoreactive T cells in progressive MS Cytokine effects on T cells in MS Effects of MS therapy and biomarkers The development of biomarkers that can be used as surrogate outcomes in clinical trials and identify patients with different responses to specific treatments is an important research area in MS. We have sought to identify biomarkers for the response to treatment with glatiramer acetate and interferonbeta for several years, and published studies in 2012 and 2013 suggesting that flow cytometry may be useful to identify effects associated with an insufficient response to treatment. We have also tried to identify genetic variants associated with the response to treatment with natalizumab and interferon-beta, but until now these attempts have not been successful. In parallel, we have studied CSF biomarkers for use in early trials in progressive MS, and have identified factors that are associated with tissue damage and which are inhibited by treatment with natalizumab in progressive MS. We recently completed a study investigating the effect of monthly pulse methylprednisolone treatment in progressive MS, and are currently analyzing these results in detail. Other ongoing studies investigate the effects of treatment with mesenchymal stem cells as part of an ongoing, multicentre phase 2 study, and we continue our attempts to identify biomarkers for treatment response for other MS therapies. These studies investigate: effects of mesenchymal stem cell treatment on immune activation and autoreactive T cells in MS immune activation and autoreactive T cells in MS patients treated with natalizumab immune activation and autoreactive T cells in MS patients treated with fingolimod immune activation and autoreactive T cells in MS patients treated with dimethyl fumarate Chemokines and gene expression in patients treated with interferon-beta gene expression and disease activity in patients treated with glatiramer acetate DANISH Multiple sclerosis CENTER annual report

22 Research activities 2013 Routine analyses in the Neuroimmunology Laboratory Neuroimmunology Laboratory research group: Poul Erik H. Jensen, laboratory technicians: Joy Mendel-Hartvig, Michael Kolbjørn Jensen, Vibeke Fuglholt, Rikke Larsen, Freja Melissa Bekner, Betina Gall. Diagnostic evaluation The presence of oligoclonal IgG-bands in CSF is of interest in the diagnosis of MS. In 2013 we have analyzed 1304 patient samples, using isoelectric focusing of CSF and corresponding plasma samples for the characterization of immunoglobulin G bands. In the autoimmune disease myasthenia gravis, autoantibodies against the acetylcholine receptor (AChR) may cause a diminished binding of ACh on muscular surfaces and thereby a reduced impulse transmission to the postsynaptic membrane of the neuromuscular endplate occurs. For diagnostic and therapeutic purposes, we measure the concentrations of these autoantibodies from patient serum samples, using a radio-immunoassay kit, and in year 2013 we analyzed 1011 patient samples. The measurements of antibody concentration against aquaporin-4 are used for the diagnosis of neuromyelitis optica (NMO) and patient samples analyzed in 2013 increased in number to 81. Measurement of neutralizing antibodies Subgroups of MS patients, treated with interferonbeta (IFN-β) or Tysabri, generate neutralizing antibodies, which diminish the therapeutic effects. IFN-β molecules bind to leucocytes and a specific up-regulation of MxA mrna in the cells occur. Neutralizing antibodies may abolish this effect, and therefore we measure the neutralization of IFN-β by antibodies in acell culture-based based on luciferase-induced expression, and further by the MxA mrna-expression as a biological response to treatment with IFN-ß. In 2013 we have analyzed 1263 patient samples for neutralizing antibodies, and 85 patient samples for MxA mrna expression. The action of Tysabri differs from IFN-β, since it blocks mononuclear cell binding to endothelial cells. In this way Tysabri inhibits mononuclear cells from entering the central nervous system. The generation of neutralizing antibodies to Tysabri in MS patients blocks the biological effects of Tysabri. In 2013 we analyzed 496 blood samples for the presence of antibodies to Tysabri by ELISA. 22 DANISH Multiple sclerosis CENTER annual report 2013

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24 Scientific publications, prizes, collaboration, acknowledgements Scientific publications Prizes Honorary offices Scientific collaboration Acknowledgements

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26 Scientific publications Publications Peer reviewed original papers Ali S, Paracha N, Cook S, Giovannoni G, Comi G, Rammohan K et al. Reduction in Healthcare and Societal Resource Utilization Associated with Cladribine Tablets in Patients with Relapsing-Remitting Multiple Sclerosis: Analysis of Economic Data from the CLARITY Study. Clinical Drug Investigation. 2012;32(1): Blinkenberg M, Mathiesen HK, Tscherning T, Jønsson A, Svarer C, Holm S et al. Cerebral metabolism, magnetic resonance spectroscopy and cognitive dysfunction in early multiple sclerosis: an exploratory study. Neurological Research jan;34(1):52-8. Blinkenberg M, Akeson P, Sillesen H, Lövgaard S, Sellebjerg F, Paulson OB et al. Chronic cerebrospinal venous insufficiency and venous stenoses in multiple sclerosis. Acta Neurologica Scandinavica. Supplementum dec;126(6): Braendstrup P, Langkilde A, Schreiber K, Ravnborg M, Sellebjerg F, Vindeløv L. Progression and CSF Inflammation after Eradication of Oligoclonal Bands in an MS Patient Treated with Allogeneic Hematopoietic Cell Transplantation for Follicular Lymphoma. Case Reports in Neurology. 2012;4(2): Brambilla P, Esposito F, Lindstrom E, Sorosina M, Giacalone G, Clarelli F et al. Association between DPP6 polymorphism and the risk of progressive multiple sclerosis in Northern and Southern Europeans. Neuroscience Letters. Supplement. 2012;530(2): Börnsen L, Christensen JR, Ratzer R, Oturai AB, Sørensen PS, Søndergaard HB et al. Effect of Natalizumab on Circulating CD4(+) T-Cells in Multiple Sclerosis. P L o S One. 2012;7(11):e Cohen JA, Reingold SC, Polman CH, Wolinsky JS, International Advisory Committee on Clinical Trials in Multiple Sclerosis, Sørensen PS. Disability outcome measures in multiple sclerosis clinical trials: current status and future prospects. Lancet Neurology Network. 2012;11(5): Coles AJ, Twyman CL, Arnold DL, Cohen JA, Con favreux C, Fox EJ et al. Alemtuzumab for patients with relapsing multiple sclerosis after diseasemodifying therapy: a randomised controlled phase 3 trial. Lancet. 2012;380(9856): Comi G, Cook SD, Giovannoni G, Rammohan K, Rieckmann P, Sørensen PS et al. MRI outcomes with cladribine tablets for multiple sclerosis in the CLARITY study. Journal of Neurology. Supplement Jensen PEH, Koch-Henriksen N, Sellebjerg F, Sørensen PS. Prediction of antibody persistency from antibody titres to natalizumab. Multiple Sclerosis. 2012;18(10): Jensen PEH, Sellebjerg F, Søndergaard HB, Sørensen PS. Correlation between anti-interferon-β binding and neutralizing antibodies in interferon-β-treated multiple sclerosis patients. European Journal of Neurology. 2012;19(10): Kondziella D, Hansen K, Gonzalez T, Gideon P, Christiansen I, Sellebjerg F. Antithyroideaantistof hos to patienter med subakut dementiel udvikling, ataksi og myoklonus. Ugeskrift for Laeger. 2012;174(9): Lund H, Jønsson A, Andresen JG, Rostrup E, Paulson OB, Sørensen PS. Cognitive deficits in multiple sclerosis: correlations with T2 changes in normal appearing brain tissue. Acta Neurologica Scandinavica. Supplementum. 2012;125(5): Lyksborg M, Larsen R, Sørensen PS, Blinkenberg MB, Garde E, Siebner HR et al. Segmenting Multiple Sclerosis Lesions Using a Spatially Constrained K- Nearest Neighbour Approach. Campilho A, Kamel M, red. I: ICIAR 12 Proceedings of the 9th international conference on Image Analysis and Recognition Volume Part II. Springer s Martinelli-Boneschi F, Esposito F, Brambilla P, Lindström E, Lavorgna G, Stankovich J et al. A genome-wide association study in progressive multiple sclerosis. Multiple Sclerosis. 2012;18(10): Romme Christensen J, Börnsen L, Khademi M, Olsson T, Jensen PE, Sørensen PS et al. CSF inflammation and axonal damage are increased and correlate in progressive multiple sclerosis. Multiple Sclerosis Romme Christensen J, Börnsen L, Hesse D, Krakauer M, Sørensen PS, Søndergaard HB et al. Cellular sources of dysregulated cytokines in relapsing-remitting multiple sclerosis. Journal of Neuroinflammation. 2012;9:215. Sellebjerg F, Krakauer M, Limborg S, Hesse D, Lund H, Langkilde A et al. Endogenous and recombinant type I interferons and disease activity in multiple sclerosis. P L o S One. 2012;7(6):e Sellebjerg F, Hesse D, Limborg S, Lund H, Søndergaard HB, Krakauer M et al. Dendritic cell, monocyte and T cell activation and response to glatiramer acetate in multiple sclerosis. Multiple Sclerosis feb;19(2): DANISH Multiple sclerosis CENTER annual report 2013

27 Sellebjerg F, Krakauer M, Khademi M, Olsson T, Sørensen PS. FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4(+) CD25(high) T cells in multiple sclerosis. Clinical and Experimental Immunology. Supplement. 2012;170(2): Sørensen PS, Bertolotto A, Edan G, Giovannoni G, Gold R, Havrdova E et al. Risk stratification for progressive multifocal leukoencephalopathy in patients treated with natalizumab. Multiple Sclerosis. 2012;18(2): Sørensen PS. Effects of neutralizing antibodies to interferon beta in multiple sclerosis: a logical paradox. Multiple Sclerosis. 2012;18(2): Sørensen PS. Deaths and disability from natalizumab are no longer tolerable: No - (they can be avoided). Multiple Sclerosis. 2012;18(8): Sørensen PS. Dødsfald i forbindelse med tvangsfastholdelse af svært agiterede personer bør kunne undgås. Ugeskrift for laeger. 2012;174(40):2367. Thirup P, Koch-Henriksen N, Sørensen PS. Sclerosebehandlingsregistret. Ugeskrift for laeger. 2012;174(42):2537. Bo Baslund, Ulla Feldt-Rasmussen, Jens Kastrup, Per Soelberg Sørensen (editors). Textbook of Medicine (Lærebog i Medicin). FADLS Forlag, Copenhagen Author of several chapters. International Multiple Sclerosis Genetics Consortium, Sørensen PS. Network-based multiple sclerosis pathway analysis with GWAS data from 15,000 cases and 30,000 controls. American Journal of Human Genetics jun 6;92(6): International Multiple Sclerosis Genetics Consortium (IMSGC). Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis. Nat Genet 2013; 45: Beecham AH, Patsopoulos NA, Xifara DK, Davis MF, Kemppinen A, Cotsapas C et al. Analysis of immunerelated loci identifies 48 new susceptibility variants for multiple sclerosis. Nature Genetics ov;45(11): Blinkenberg M, Sellebjerg F, Leffers AM, Madsen CG, Sørensen PS. Clinically silent PML and prolonged immune reconstitution inflammatory syndrome in a patient with multiple sclerosis treated with natalizumab. Multiple sclerosis (Houndmills, Basingstoke, England) aug;19(9): Comi G, Cook SD, Giovannoni G, Rammohan K, Rieckmann P, Sørensen PS et al. MRI outcomes with cladribine tablets for multiple sclerosis in the CLARITY study. Journal of Neurology. 2013;260(4): Dogonowski A-M, Siebner HR, Sørensen PS, Wu X, Biswal B, Paulson OB et al. Expanded functional coupling of subcortical nuclei with the motor restingstate network in multiple sclerosis. Multiple Sclerosis apr;19(5): Dogonowski A-M, Andersen KW, Madsen KH, Sørensen PS, Paulson OB, Blinkenberg M et al. Multiple sclerosis impairs regional functional connectivity in the cerebellum. NeuroImage. Clinical nov 27;4: Dogonowski A-M, Siebner HR, Sørensen PS, Paulson OB, Dyrby TB, Blinkenberg M et al. Resting-state connectivity of pre-motor cortex reflects disability in multiple sclerosis. Acta Neurologica Scandinavica mar 6;128(5): Hegen H, Millonig A, Albrecht N, Bertolotto A, Comabella M, Giovannoni G, Guger M, Hoelzl M, Khalil M, Lindberg R, Polman CH, Rudzki D, Schautzer F, Sellebjerg F, Skrobal A, Sørensen PS, Deisenhammer F. Early detection of neutralizing antibodies to interferon-beta in multiple sclerosis patients. Mult Scler [E-pub ahead of print]. Leone MA, Barizzone N, Esposito F, Lucenti A, Harbo HF, Goris A, Kockum I, Oturai AB, Celius EG, Mero IL, Dubois B, Olsson T, Søndergaard HB, Cusi D, Lupoli S, Andreassen BK; International Multiple Sclerosis Genetics Consortium; Wellcome Trust Case Control Consortium 2, Myhr KM, Guerini FR; PROGEMUS Group; PROGRESSO Group, Comi G, Martinelli-Boneschi F, D Alfonso S. Association of Genetic Markers with CSF Oligoclonal Bands in Multiple Sclerosis Patients. PLoS One Jun 13;8(6):e doi: /journal. pone Print PMID: [PubMed in process] Lund H, Krakauer M, Skimminge A, Sellebjerg F, Garde E, Siebner HR et al. Blood-brain barrier permeability of normal appearing white matter in relapsing-remitting multiple sclerosis. P L o S One. 2013;8(2):e Magyari M, Koch-Henriksen NI, Pfleger CC, Sørensen PS. Reproduction and the risk of multiple sclerosis. Multiple sclerosis (Houndmills, Basingstoke, England) okt;19(12): DANISH Multiple sclerosis CENTER annual report

28 Scientific publications Magyari M, Søndergaard HB, Sellebjerg F, Sørensen PS. Preserved in vivo response to interferon-α in multiple sclerosis patients with neutralising antibodies against interferon-β (REPAIR study). MSARD 2013; 2: Mathiesen HK, Sorensen PS. Prolonged-release fampridine improves walking in a proportion of patients with multiple sclerosis. Expert review of neurotherapeutics dec;13(12): Mechelli R, Umeton R, Policano C, Annibali V, Coarelli G, Ricigliano VAG et al. A candidate-interactome aggregate analysis of genome-wide association data in multiple sclerosis. P L o S One. 2013;8(5):e Mero I-L, Gustavsen MW, Sæther HS, Flåm ST, Berg- Hansen P, Søndergaard HB et al. Oligoclonal band status in Scandinavian multiple sclerosis patients is associated with specific genetic risk alleles. P L o S One. 2013;8(3):e Modvig S, Degn M, Horwitz H, Cramer SP, Larsson HBW, Wanscher B et al. Relationship between cerebrospinal fluid biomarkers for inflammation, demyelination and neurodegeneration in acute optic neuritis. P L o S One. 2013;8(10):e Møller M, Søndergaard HB, Koch-Henriksen N, Sorensen PS, Sellebjerg F, Oturai AB. The chemokine receptor CCR5 Δ32 allele in natalizumab-treated multiple sclerosis. Acta Neurol Scand [E-pub ahead of print]. Olsson T, Achiron A, Alfredsson L, Berger T, Brassat D, Chan A et al. Anti-JC virus antibody prevalence in a multinational multiple sclerosis cohort. Multiple sclerosis (Houndmills, Basingstoke, England) okt;19(11): Paulson OB, Sørensen PS. Neuroonkologi. I Paulson OB, Thage O, Waldemar G, red., Neurologi i 100 år: beretninger fra Rigshospitalets neurologiske afdeling. [Nationalt Videnscenter for Demens] s Pedersen EG, Hallas J, Hansen K, Jensen PEH, Gaist D. Late-onset myasthenia not on the increase: a nationwide register study in Denmark, European Journal of Neurology. 2013;20(2): Pedersen EG, Pottegård A, Hallas J, Friis S, Hansen K, Jensen PEH et al. Use of azathioprine for non-thymoma myasthenia and risk of cancer: a nationwide casecontrol study in Denmark. European journal of neurology: the official journal of the European Federation of Neurological Societies jun;20(6): Ratzer R, Søndergaard HB, Christensen JR, Börnsen L, Borup R, Sørensen PS et al. Gene expression analysis of relapsing-remitting, primary progressive and secondary progressive multiple sclerosis. Multiple sclerosis (Houndmills, Basingstoke, England) dec;19(14): Romme Christensen J, Börnsen L, Khademi M, Olsson T, Jensen PE, Sørensen PS et al. CSF inflammation and axonal damage are increased and correlate in progressive multiple sclerosis. Multiple Sclerosis. 2013;19(7): Romme Christensen J, Börnsen L, Ratzer R, Piehl F, Khademi M, Olsson T et al. Systemic inflammation in progressive multiple sclerosis involves follicular T-helper, Th17- and activated B-cells and correlates with progression. P L o S One. 2013;8(3):e Schreiber K, Voldsgaard A, Sørensen PS. Der er begrænset effekt af interferonbeta til behandling af sekundær progressive multipel sklerose--en gennemgang af et Cochranereview. Ugeskrift for laeger maj 6;175(19): Sellebjerg F, Hesse D, Limborg S, Lund H, Søndergaard HB, Krakauer M et al. Dendritic cell, monocyte and T cell activation and response to glatiramer acetate in multiple sclerosis. Multiple sclerosis (Houndmills, Basingstoke, England) feb;19(2): Svenningsson A, Falk E, Celius EG, Fuchs S, Schreiber K, Berkö S et al. Natalizumab treatment reduces fatigue in multiple sclerosis. Results from the TYNERGY trial; a study in the real life setting. P L o S One. 2013;8(3):e Søndergaard HB, Hesse D, Krakauer M, Sørensen PS, Sellebjerg F. Differential microrna expression in blood in multiple sclerosis. Multiple sclerosis (Houndmills, Basingstoke, England) dec;19(14): Søndergaard HB, Hesse D, Krakauer M, Sørensen PS, Sellebjerg F. Differential expression of microrna in multiple sclerosis. Mult Scler 2013; 19: Sørensen PS. Multipel sklerose. I Paulson OB, Thage O, Waldemar G, red., Neurologi i 100 år: beretninger fra Rigshospitalets neurologiske afdeling. [Nationalt Videnscenter for Demens] s Theibich A, Dreyer L, Magyari M, Locht H. Demyelinizing neurological disease after treatment with tumor necrosis factor alpha-inhibiting agents in a rheumatological outpatient clinic: description of six cases. Clinical rheumatology nov DANISH Multiple sclerosis CENTER annual report 2013