Title: Multi-dimensional microstructural imaging offers novel in-vivo insights into brain pathology: an application to multiple sclerosis Authors Kasper Winther Andersen1, Samo Lasič1,2, Henrik Lundell1, Markus Nilsson3, Daniel Topgaard4, Filip Szczepankiewicz2,3, Lars G. Hanson1,8, Hartwig Roman Siebner1,5,6, Morten Blinkenberg7, Tim B. Dyrby1,9 1 Danish Research Centre for Magnetic Resonance, Centre for Functional and Diagnostic Imaging and Research, Copenhagen University Hospital Hvidovre 2 Random Walk Imaging, AB, Lund, Sweden 3 Clinical Sciences, Lund, Department of Radiology, Lund University, Lund, Sweden 4 Division of Physical Chemistry, Department of Chemistry, Lund University, Lund, Sweden 5 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen 6 Department of Neurology, Copenhagen University Hospital Bispebjerg 7 Danish Multiple Sclerosis Center, Copenhagen University Hospital Rigshospitalet, Copenhagen 8 Center for Magnetic Resonance, DTU Electrical Engineering, Technical University of Denmark 9 Department of Applied Mathematics and Computer Science, Technical University of Denmark HYPOTHESIS Multi-dimensional in-vivo microstructural imaging provides specific and broad microstructural insight into the underlying pathology of multiple sclerosis (MS), which is normally only obtained with histology. METHODS Thirty MS patients (age-range 22-66) and 17 healthy controls (HC, age-range 22-63) were scanned with a comprehensive MR-protocol including: • Microscopic fractional anisotropy (µFA - sensitive to cell shape or microscopic anisotropy); • Apparent exchange rate (AXR - sensitive to cell-membrane permeability); • Multi-shell diffusion weighted imaging - allowing to extract measures of intra-cellular volume fraction

(ICVF), orientation dispersion (OD), and isotropic signal fraction (ISOSF); • Fractional anisotropy (FA) using DTI; • Magnetization transfer (MT) and proton density (PD) (sensitive to myelination). Parameters were compared between HC white matter (HC-WM), MS normal-appearing white matter (MS-NAWM), and MS lesions (MS-L). RESULTS AND DISCUSSION MS-NAWM was associated with lower µFA and ICVF compared to HC-WM. This is consistent with histology showing that diffuse axonal damage is the dominant pathology occurring in MS-NAWM. In MS-NAWM primary demyelination is sparse, but myelin damage is a consequence of axonal damage. This is also reflected in our data, which shows no reduction in the myelin-sensitive parameter (MT) and no changes in permeability (AXR). In MS-lesions all parameters were significantly different from HC-WM and MS-NAWM. As expected, myelination (MT) was decreased. Increased AXR suggests increased cell permeability due to demyelinated axons or/and increasing number of glial cells. ICVF was clearly reduced compared with NAWM suggesting significant neurodegeneration. However, µFA=0.6 suggests that a fraction of demyelinated axons remain present. Less axonal dispersion could be due to less crossing fibers due to neurodegeneration or systematic degeneration of a specific axonal size population. In general, it is expected that free water is increased in MS-lesions, which is supported by higher ISOSF and PD as well as lower FA. CONCLUSION Despite its high sensitivity, the conventional diffusion tensor imaging suffers from low specificity to changes in microstructural features such as myelination, cell permeability, and fiber dispersion. Using novel MR sequences and models, each probing different microstructural features, allow us to extract more specific tissue information, which is normally only obtainable with histology.

Molecular signature of brain lesion evolution and fate in progressive MS Elkjaer M1, Frisch T2, Burton M3, Reynolds R4, Kruse T3, Thomassen M3, Baumbach J2, Illes Z1 1Department of Neurology, OUH/SDU; 2Department of Mathematics and Computer Science, SDU; 3Department of Clinical Genetics, OUH/SDU; 4Division of

Brain Science, Imperial College, London

We hypothesized that the different lesion types in the brain of progressive MS patients can be characterized by specific transcriptome signatures. These could unmask mechanisms that drive the evolution and fate of lesions, and lead to discovery of biomarkers and potential drug targets that can halt progression. With immunohistochemistry, we classified 98 brain areas covering lesion evolution (NAWM and active lesions), lesion fate (inactive, chronic active, repairing) and control WM from 10 MS and 5 non-neurological diseased brains. We created the transcriptome profile of each lesion type by next generation RNA sequencing. We produced clusters and networks of significant genes using KeyPathwayMiner and TiCoNE to find genes important for the evolution and fate of the lesions. We extracted molecules that could influence either the maintenance or halt of active inflammation. Out of 18000 detected genes, over 4000 were differentially expressed between MS and controls (FDR<0.05). During the evolution of active lesion from NAWM, over 3000 genes changed significantly (FDR<0.05). For fate of active lesions, we discovered a radical change in the transcriptome when active lesions were compared to chronic active lesion, and less prominent changes compared to inactive and repairing lesions. Next, we retrieved the major hubs in networks, when an active lesion developed into a chronic active, inactive or repairing lesion. Lastly, we extracted genes uniquely expressed in the different lesion types, and compared them with our CSF proteome database of MS patients in the early and progressive disease stages. Our data support lesion type specific transcriptome signatures. We used de novo network analysis to identify gene clusters and hubs that are important for lesion evolution, and for its fate. Our data implies that the NAWM in the MS brain is much more similar to the WM from control subjects than to lesions within the same brain, and that development of a chronic active lesion is very uniquely regulated on gene expression level. Future functional studies on recognized key molecules of evolution and fate are needed to confirm their role, and that may lead to potential targets to halt progression. Lastly, the protein products of lesion specific genes that were also found in the CSF proteome of MS should be validated in cohorts for potential biomarkers of progression.

Titel: Neurofilament light chain niveauer i CSF og blod hos patienter med multipel sklerose før og efter

behandlingmeddimethylfumaratForfattere:TobiasSejbaek1,2,3,HelleHvilstedNielsen1,3,NellieA.Martin3,MariaL.Elkjaer1,3,MadsRavnborg1,Zsolt

Illes1,31:Neurologiskafdeling,OdenseUniversitetsHospital,Odense2:Neurologiskafdeling,SydvestjyskSygehus,Esbjerg3:Kliniskinstitut,SyddanskUniversitet,OdenseHypotese:Niveauafneurofilamentlightchain(NfL)korrelerermedaksonalskadeogerderforforhøjetvedmultipel

sklerose (MS). MS-Behandling som er anti-inflammatorisk og neuroprotektiv kan medføre reduktion af NfLniveau. Studiet ønsker at vurdere NfL niveau i blod og CSF hos MS patienter i forhold til raske kontroller.Desudenønskesundersøgtændring iNfLniveauefterbehandlingmeddimethyl fumarat(DMF)ogprædikativværdiafNfL-niveauiforholdklinisksygdomsaktivitet.

Metode: IetprospektivtfaseIVstudiehospatientermedattakvisMS(RRMS)indsamledeviplasmaogCSFførog

efterbehandlingmedDMF.CSFblevindsamletvoluntærtvedbaselineogefter12måneder(n=24).Plasmablevindsamletvedbaseline,1,3,6og12månedersbehandling(n=50).Prøverneblevbehandletefterinternationaleguidelinesforbiobankindsamling.NfLblevanalyseretpåimmunoassaysfraECLMesoscaleogSimoaQuanterix.

Resultater: Baseline niveauet af NfL var højere hos patienter med RRMS sammenlignet med raske kontroller

(middelværdi 1500±216,0 pg/ml versus 255±23,3 pg/ml, p<0,001). Receiver operating characteristic (ROC)kurveanalysedefineredeencut-offværdipå462pg/mlmedensensitivitetpå80,6%ogspecificitetpå100%.DMFreduceredekoncentrationenafNfLmed1382±449,0pg/ml(p<0,01)efterétårsbehandling.Patientermedværdier over normalområdet efter et års behandling med DMF havde en forøget relativ risiko forsygdomsaktivitet(signifikantMRog/ellerklinisksygdomsaktivitet)på5,4.

Diskussion:NfLerforhøjethospatientermedMSogerspecifiktilatskelneraskefrasyge,dogmedensensitivitet

på80,6%.NfLerforhøjetvedCNSsygdommedergiveraksonalskaderogdeterderforikkeforventeligtatNfLbliverendiagnostiskmarkørforMS.NfLfalderbetydeligtiCSFefterbehandlingmedDMFognormaliseringafNfL værdier prædikterer godt klinisk respons på DMF. NfL er en potentiel biomarkør for ”personalizedmedicine” ved behandling af RRMS. Normalisering af NfL fremfor blot reduktion er formentlig afgørende iforholdtilatforudsigesygdomsaktivitethospatientermedMS.

Title: Association between aerobic capacity and cognitive performance in people with multiple sclerosis – A cross-sectional study. Authors: Martin Langeskov-Christensen, Søren Eskildsen, Egon Stenager, Henrik Boye Jensen, Helle Hvilsted Nielsen, Thor Petersen, Lars Grøndahl Hvid, Päivi Hämäläinen, Ulrik Dalgas. Hypothesis: In persons with multiple sclerosis (PwMS) it was hypothesized that aerobic capacity would be associated with (1) cognitive performance in the domain of information processing, and (2) a global Z-score of the Brief Repeatable Battery of Neuropsychological tests (BRB-N). Methods: The current study presents baseline data from an ongoing randomized controlled trial. Subjects were recruited through MS clinics in Aarhus, Kolding, Viborg and Odense. After completing the BRB-N all subjects performed a maximal oxygen consumption (VO2peak) test. Uni- and multivariate (adjusting for age, sex and education level) regression analyses were performed to evaluate the relationship between aerobic capacity and cognitive performance. Based on published norms for healthy controls Z-scores were computed for each individual BRB-N test. Subjects were categorized as cognitively impaired if the Z-score of one or more of the BRB-N tests were below 1.5 SD of healthy controls. Finally, composite Z-scores for the BRB-N and its subdomains were calculated. Results: Baseline data from 84 subjects were included (44.9±9 years, 28.4±7 ml O2/min/kg, 16.3±2 education years, EDSS: 2.6±1.4, MS type (RR, PP, SP): 73/6/5, disease duration: 9.9±7 years). Regression analyses revealed no significant association between aerobic capacity and cognitive performance in the BRB-N tests. An average global Z-score of -0.2±0.66 indicated a cognitively well-functioning sample. A significant, but weak, relationship was found between the composite processing speed Z-score and aerobic capacity (R2=0.06, P = 0.02). When comparing the cognitively impaired (34.5%) to the non-impaired group (65.5%) lower aerobic capacity (25.9±1 vs. 29.7±1 mlO2/min/kg, P = 0.02) and higher age (48.8±8 vs. 42.9±9, P < 0.01) were found. Discussion: Exercise has been suggested as a potential intervention to improve cognitive performance in PwMS. The current study only provide limited support to the association between cognitive performance and aerobic capacity. However, the present findings must be considered in relation to methodological limitations including the cross-sectional design, inclusion of cognitive performance as a secondary outcome and only mildly cognitively impaired subjects.

Title Early versus later treatment start in multiple sclerosis - a register based cohort study.

Authors: Chalmer T1,2; Baggesen LM3; Nørgaard M3; Koch-Henriksen N2,3; Magyari M1,2; Sorensen PS 1, 2, on behalf of the Danish Multiple Sclerosis Group

Affiliations: 1Danish Multiple Sclerosis Center, Department of Neurology, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK-

2100 Copenhagen, Denmark. 2The Danish Multiple Sclerosis Registry, Department of Neurology, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen,

Denmark 3Department of Clinical Epidemiology, Aarhus University Hospital, Aarhus, Denmark.

Objective: To assess long-term treatment effectiveness of disease-modifying therapy (DMT) initiated early in the

disease course compared with later treatment start.

Method: We retrieved data on all Danish MS patients (n=3,795) treated with DMT from two nationwide population-

based MS registries. The cohort consisted of early treated patients, defined as treatment start within two years after

the first MS symptom (n=2,316) and later treated patients, defined as treatment start between two and eight years

from clinical onset (n=1,479). We compared time from treatment start to progression to Expanded Disability Status

Scale (EDSS) 6 and mortality between cohorts as hazard ratio (HR) using a Cox proportional hazards model with

adjustment for inverse probability of treatment weights. Several sensitivity analyses were conducted.

Results: Median follow-up time was 10 years (range 1-20) for both the EDSS 6 outcome and mortality.

Patients with later treatment start showed 28% increased hazard of reaching EDSS 6 compared with the

early treated patients (HR 1.28; 95% CI 1.06-1.53). When stratified by sex the increased hazard among

later treated women persisted (HR 1.39; 95% CI 1.10-1.75), while the HR decreased in men (1.09; 95% CI

0.81-1.47). Mortality did not differ between early and later treatment start (HR 1.02; 95% CI 0.63-1.83).

Discussion: Patients who started treatment with DMT later had shorter time to reaching EDSS 6 compared

with patients who started early, but the delay did not influence mortality. Our results support the scheme of

early treatment.

IFN-β isessentialformitochondrialfissioninneurons

Emilie Tresse-Gommeaux, Elham Jaberi, Lluis Riera-Ponsati, Guinevere Sew and Shohreh Issazadeh-

Navikas

Neuroinflammation Unit, Biotech Research & Innovation Centre; University of Copenhagen; Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark

Multiple Sclerosis (MS) initiation and progression has been associated to adaptive changes in

mitochondrial function and morphology and ROS production. Interferon (IFN)-β is a cytokine used to treat

MS but the associated molecular mechanism still remains to be elucidated. We demonstrated previously

that mice lacking interferon (IFN)-β (Ifnb–/–) or its receptor (Ifnar–/–) display neurodegeneration during

ageing and experience behavioural deficits. In particular, defective mitochondria accumulate in Ifnb–/–

neurons. We hypothesized that IFN-β was necessary for mitochondrial homeostasis.

We found that total mitochondrial mass is increased in Ifnb-/- cortical neurons, due to an increase in the

size of individual mitochondria. We demonstrated that this was due to impairment of mitochondrial fission,

a process allowing the creation of new mitochondria, the removal of defective ones and controlling

oxidative stress in postmitotic neurons. In cultured cortical neurons and brain tissue from Ifnb-/- mice, we

showed a decrease of the active form of Dynamin-like protein-1 (Drp-1), a key protein involved in the

mitochondrial fission. Additionally, we observed that IFN-β treatment of the cortical neurons promotes the

activation of Drp-1 and its relocation to mitochondria. This was associated with an instability of the

mitochondria-ER platform through a Signal transducer and activator of transcription 5 (STAT5) dependent

mechanism.

Altogether, our results demonstrate that IFN-β is promoting mitochondrial fission in neurons, through a

STAT5-Drp-1 pathway. This suggests that IFN-β administration to MS patients might be beneficial by

compensating mitochondrial dysfunction and that targeting mitochondrial homeostasis more specifically in

these patients might be beneficial.

A.1. EndogenousIFNβregulatesCNSbarrierintegrity

Henrik Hasseldam1, Olivia Anna Lie-Andersen1, Veronika Drabova1, ShohrehIssazadeh-Navikas11NeuroinflammationUnit,BiotechResearch&InnovationCentre(BRIC),HealthScienceFaculty,UniversityofCopenhagen,OleMaaløesVej5,DK-2200CopenhagenN,DenmarkIFNβwasamongthefirsttreatmentswithbeneficialeffectsonrelapseratesinrelapsing-remittingmultiplesclerosis.Whileaplethoraofimmunomodulatoryeffectshavebeendemonstrated,IFNβsroleinrestoringtheintegrityofthebloodbrainbarrierisnotwellestablished.Severalinvitrostudieshaveindicatedadirectbarrier-tighteningeffectofIFNβ.Furthermore,MSpatientspresentdecreasedfrequenciesofnewgadolinium-enhancinglesionsonMRIaftertreatmentwithIFNβ,signifyingthatIFNβmightaffectbarrierintegrityinadirectfashion.However,nosolidproofofspecificeffectsormechanismshasbeenestablishedinvivo,andmoreimportantly,thereisnodatatosupporthowendogenousIFNβcouldregulateBBBintegrity.Inthisstudy,wedemonstratethatphysiologicallevelsofendogenousIFNβarecriticalformaintainingtheCNSbarrierintegrity.IFNβ-/-miceexhibitsevereCNSbarrierdefects,includingchangesinbarrier-associatedtightjunctionproteinsandincreasedbarrierpermeability.Thetightjunctionsproteins,claudin3and5arebothsignificantlydecreasedinBBBendotheliumandchoroidplexusependymain6monthsoldIFNβ-/-mice.Occludinexpression,ontheotherhand,isnotreducedbutinsteadexhibitahighlydisturbedandjaggedexpressionpattern,alreadyfrom3monthsofage.ThesechangesareassociatedwithincreasedCNSbarrierspermeabilityintheformofsignificantaccumulationofthefluorescenttracersodiumfluoresceinandIgGinbrainparenchyma.ThisisfurthermoresubstantiatedbyMRIwhereincreasedgadofluorineleakageisobserved,mostsignificantintheorbitalarea.OurdatashowthatIFNβisimportantforCNSbarrierhomeostasis,andsuggeststhatasignificantpartofthedisease-reducingeffectsofIFNβtreatmentisrelatedtoCNSbarrierstabilization.

Requirements for FoxA1 regulation of Pdl1 promoter in T cells to impose suppressive anti-inflammatory functions of FoxA1+Tregulatory cells Louise Munk Rasmussen1.*, Mahdieh Hadi1,*, Michael Kosicki1, Robert Carlsson1, Shohreh Issazadeh-Navikas1

1Neuroinflammation Unit, Biotech Research & Innovation Centre (BRIC), Health Science Faculty, University of Copenhagen, Copenhagen Biocentre, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark * Equal contribution ABSTRACT Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system, mediated by autoreactive T cells. Regulatory T (Treg) cells, particularly FoxA1+Treg cells have an anti-inflammatory and protective role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). The suppressive capacity of FoxA1+Treg cells is mediated by expression of the programmed death-ligand 1 (PD-L1) which binds to PD-1 on activated T cells. IFNβ is inducing the transcription factor FoxA1 in T cells, which in turn is essential for identity and suppressive capacity of the FoxA1+Treg cells via regulation of PD-L1. However, the mechanism by which FoxA1+Treg cells control PD-L1 expression is unknown, and it is therefore central to understand how FoxA1 is regulating the Pdl1 gene. Here, we report that FoxA1 is sufficient to activate the Pdl1 promoter both in primary human and murine T cells. More importantly, we identified that FoxA1 regulates the Pdl1 gene in T cells by binding to a 27 basepair (bp) site in the Pdl1 gene promoter. We also identified that FoxA1 in synergy with IFNβ utilizes a responsive minimal promoter (60 bp), which is sufficient to fully recover the promoter activity of the full Pdl1 promoter. IFNβ significantly synergizes FoxA1’s activation of the 60 bp Pdl1 minimal promoter by recruitment and nuclear translocation of FoxA1 together with phosphorylated STAT1/2/3 that act as co-factors for FoxA1. In conclusion, we have identified a minimal Pdl1 promoter region sufficient to recover the full Pdl1 promoter activity, and therefore could hold strong promises as a new potential target to regulate the T-cell PD-L1 expression and thereby to secure the anti-inflammatory capacity of FoxA1+Treg cells.

Title: Cytosolic DNA-sensing through stimulator of interferon genes (STING) has a protective role in focal brain lesions in experimental autoimmune encephalomyelitis (EAE). Authors: Marlene Thorsen Mørch, Line S. Reinert, Reza Khorooshi, Mark Burton, Mads Thomassen, Søren R. Paludan. Nasrin Asgari, Trevor Owens Hypothesis: RNA sequencing shows that the cytosolic DNA-sensing pathway is upregulated in focal brain lesions in EAE. We propose that this pathway has a protective effect by signaling through STING. Methods: Adult female C57BL/6J and STINGgt/gt mice were immunized for EAE. On day 10 post immunization mice received an intra-corpus callosum (CC) needle stick and on day16 they were sacrificed. Brains were isolated for histology or CC was isolated for RNA extraction. Results: Focal CC lesions in EAE in C57BL/6J mice showed demyelination (loss of MOG staining) as well as infiltrates. Demyelinating pathology correlated with the size of infiltrates. As expected CC pathology did not correlate with symptoms of EAE. Multi-dimensional principal component analysis showed that focal CC lesions in EAE have a distinctive transcriptional profile. Several Type I interferon (IFN) signaling related genes were significantly upregulated in focal CC pathology - 73 of the top 100 upregulated genes were regulated by Type I IFN (Interferome database v2.01). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed several significantly upregulated pathways, one of which was the cytosolic DNA-sensing pathway. Signaling through STING is part of this pathway, which results in increased Type I IFN signaling. Histological analysis of focal lesions in mice lacking functional STING revealed that these mice have significantly higher percentage of demyelination in CC, and a tendency towards more infiltration. Interestingly in mice lacking STING, the correlation between demyelination and infiltration was absent. Discussion: Multiple sclerosis (MS) is an inflammatory demyelinating disease, characterized by the formation of focal demyelinated plaques in the white matter of the central nervous system. The CC is a structurally distinct white matter tract and is often affected in MS. Regional demyelination and axonal loss in the CC correlate with cerebral white matter lesion volume and distribution in MS. An understanding of the appearance and regulation of lesions in the CC may provide insight into MS pathogenesis. Here we show that focal CC lesions in EAE with demyelination and infiltration in CC are reduced by activation of STING, which also leads to transcription of Type I interferons and related genes.

Title: Re-programming Th17 cells to FoxA1+Tregs, prospect for MS treatment Authors: Yawei Liu, Ugnė Kuliešiūtė, Sonia Simón Serrano, Shohreh Issazadeh-Navikas

Neuroinflammation Unit, Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen Biocentre, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.

abstract Hypothesis: T helper 17 (Th17) cells and their downstream pathways are implicated in the pathogenesis of

central nervous system (CNS) autoimmunity in Multiple sclerosis (MS). Th17 cells can efficiently cross

the blood-brain barrier (BBB), promote its disruption, and induce the activation of other inflammatory cells

in the CNS. Th17 cells have high plasticity, in vitro generated Th17 cells only poorly maintain their

differentiation program and can be reprogrammed into other T-cell lineages. The fate of Th17 cells is

mainly depending on the microenvironment conditions. Here we aim to understand conditions that leading

to reprogramming of Th17 cells, and their possible conversion to novel FoxA1+T regulatory cells

(FoxA1+Tregs) with anti-inflammatory capacity.

Methods: Th17 in vitro polarization, Flow cytometry, Amaxa Transfection, Western blotting, Co-

Immuniprecipitation, Luciferase report assay, Suppression assay, Immunofluorence staining, Adoptive

transfer EAE.

Results: Previously we have shown that recombinant IFNβ can convert naïve T cells to FoxA1+Tregs by

triggering the lineage transcription factor FoxA1. In the present study, we show that IFNβ is capable to

reprogram polarized Th17 cells to FoxA1+Tregs. FoxA1 is required for re-generation of FoxA1+Tregs from

Th17 cells by physically binding to Rorg, a lineage transcription factor for Th17 cells. Moreover,

regenerated FoxA1+Tregs from Th17 cells have shown a suppressive function by inhibiting activated T cells

proliferation in vitro. Most importantly, Th17-converted FoxA1+Tregs maintain their function in vivo and

prevent adoptive transfer of EAE induced by Th17 cells.

Discussion: These findings are strongly indicating that MS-pathogenic Th17 cells could be targeted to

convert to FoxA1+Tregs, which would be a promising potential for MS-therapy.

Titel: Baseline Lymfocytter og høj alder predikterer lymfopeni hos dimethyl fumarate-behandlede patienter med multipel sklerose Forfattere: Caroline Holst Bloch1,2, Matthias Kant3, Henrik Boye Jensen4, Preben Borring Andersen5, Annett Petersen6, Monika Katarzyna Góra7, Morten Blaabjerg1,2, Tobias Sejbæk1,2,6

1 Klinisk Institut, Syddansk Universitet, Odense 2 Neurologisk Afdeling, Odense Universitetshospital, Odense 3 Neurologisk Afdeling, Sygehus Sønderjylland, Sønderborg 4 Neurologisk Afdeling, Lillebaelt Hospital, Kolding 5 Neurologisk Afdeling, Sjællands Universitetshospital, Roskilde 6 Neurologisk Afdeling, Sydvestjysk Sygehus, Esbjerg 7 Neurologisk Afdeling, Slagelse Hospital, Slagelse Hypotese: Undersøge faktorer som predisponerer til udvikling af lymfopeni under dimethyl fumarate (DMF) behandling. Herunder associationen imellem lavt body mass index (BMI), baseline lymfocytter og/eller alder hos patienter med multipel sklerose (MS) i forhold til udvikling af lymfopeni undervejs i behandlingen med DMF. Metode: Retrospektivt kohorte-studie med patienter fra skleroseklinikkerne i Region Sjælland (Roskilde og Slagelse) og Region Syddanmark (Esbjerg, Kolding, Odense og Sønderborg). Patienter der startede behandling med dimethyl fumarate i perioden 05/2014 til 09/2017 (n=452) er inkluderet. Resultater: 30,8 % udviklede lymfopeni grad II-IV heraf 6,4 % grad III-lymfopeni. Én patient udviklede grad IV-lymfopeni. Der kunne ikke findes association imellem lav BMI og tendens til udvikling af DMF-induceret lymfopeni. Patienter ældre end 55 år havde en odds ratio (OR):3,58 [95% CI:1.79-7.62], p< 0.001, ift. udvikling af lymfopeni. Patienter med lymfocytter under normalt eller lavt i normalområdet havde generelt højere risiko for lymfopeni. For gruppen mellem 1,0-1,49·109/l var OR:5.48 [CI:3.17-9.19], p≤0.0001. Patienter med høje baseline-lymfocytter var i markant lavere risiko; således var der i gruppen >2,5·109/l en OR:0.11 [CI:0.01-0.66], p<0,01. 32.3% af patienterne i studiet ophørte DMF-behandling (25.9% og 48.8% i hhv. Region Syddanmark og Region Sjælland). OR for ophør i Region Syddanmark vs. Region Sjælland var 2.72 [CI:1.77-4.18], p≤0.0001. Hvis graviditet og attak som ophørsårsager udelukkes i beregningen stiger OR:4.25 [CI:2.66-6.81], p≤0.0001. Diskussion: Lymfopeni i varierende grad (II-III) er almindeligt ved behandling med DMF, grad IV lymfopeni er sjælden (n=1). Patienter med alder over 55 år og lymfocytværdier lavt i normalområdet har en forhøjet risiko for udvikling af lymfopeni. Generelt er BMI er ikke en afgørende faktor i forhold til udvikling af lymfopeni, og der er således ikke en dosis-respons association, idet alle patienter modtager samme dosis DMF. Patienter >55 år samt patienter med lave baseline lymfocytter har øget risiko for lymfopeni. Hos disse patientgrupper kan man overveje at initiere andre immunologiske behandlinger. Den betydelige forskel i OR for ophør af behandling på baggrund af lymfopeni indikerer, at der er forskellig klinisk praksis i regionerne, selvom der er ens nationale guidelines. Flere studier er påkrævet, inden der ændres generel praksis for anvendelse af DMF i MS.

Asymmetries in structural connectivity correlates with fatigue in multiple sclerosisChristian Bauer1, 2, Olivia Svolgaard1, Kathrine S. Madsen1, 2, Tim B. Dyrby1, 5, Finn Sellebjerg3, Hartwig R. Siebner1, 4, Kasper W. Andersen1 1 Danish Research Centre for Magnetic Resonance, Copenhagen University Hospital, Hvidovre, Denmark 2 Metropolitan University College, Copenhagen, Denmark 3 Danish Multiple Sclerosis Centre, Department of Neurology, Copenhagen University Hospital Rigshospitalet, Copenhagen,

Denmark 4 Department of Neurology, Copenhagen University Hospital Bispebjerg, Copenhagen, Denmark 5 Department of Applied Mathematics and Computer Science, Technical University of Denmark, Copenhagen, Denmark Hypothesis Structural connectedness in the bilateral CST can predict motor fatigue among patients with multiple sclerosis (MS). Methods Forty-six mildly disabled MS patients with relapsing remitting MS were recruited from the Danish Multiple Sclerosis Centre, Rigshospitalet. A group of 25 age- and sex-matched individuals were enrolled as controls. Effort-independent (trait) fatigue ratings were evaluated with the motor sub-score of the Fatigue Scale for Motor and Cognitive functions (FSMC). Additionally, all participants underwent a battery of tests, questionnaires and a magnetic resonance imaging (MRI) examination. The MRI included diffusion weighted imaging (DWI) and conventional structural scans. The DWI data were analysed using anatomical connectivity mapping (ACM) – a novel method to assess indices of structural connectivity. ACM reflects how well each voxel is connected with the rest of the brain and is a surrogate measure of white matter brain connectedness. Bilaterally, the cortico-spinal tracts (CST) were chosen as region of interests due to its functional importance of the motor system. The MS group was split into fatigue (FMS, n=29) and non-fatigue (NFMS, n=17) subgroups. The mean ACM in left and right CST was compared using between group (HC, FMS, NFMS) repeated-measures (left and right hemisphere) ANOVA. In addition, we calculated lateralization index (LI) between left and right CST, which was analysed using between groups ANOVA. Results We found a main-effect of group (F=5.83, p=.005), group by hemisphere interaction (F=3.98, p=.023), but no main-effect of hemisphere (F=.060, p=.441). Post-hoc tests showed increased ACM in the left CST in FMS compared to NFMS (F=5.68, p=.022), but no between-group differences in the right CST. For the LI, we found a main effect of group (F=3.384, p=.040). This was caused by a higher LI in the FMS group relative to the NFMS group. Discussion Increased ACM was found in left CST as well as increased lateralization index (left greater than right) in patients with motor fatigue. ACM is a complex measure, reflecting the integration of microstructural changes and fiber dispersion along the axonal fiber tract (e.g., CST), but can also reflect less crossing fibers. Thus our results could be driven by a degeneration of association fibres.

Alcohol consumption in adolescence is associated with lower risk of multiple sclerosis

in a Danish cohort

C. Andersena, H. B. Søndergaarda, D. B. Oturai, J. H. Laursena, S. Gustavsena, N. Larsena, M. Magyaria,b, E.

Just-Østergaardc, L. W. Thørnerd, F. Sellebjerga, H. Ullumd, A. B. Oturaia

Background and objective: Environmental factors are known to be important in the etiology of multiple

sclerosis (MS). However, studies of the association between alcohol consumption and MS have shown

conflicting results. Due to the possible existence of a vulnerable period of MS susceptibility in adolescence

and the fact that Danish teenagers have high alcohol consumption, we investigated the association between

alcohol consumption at ages 15 to 19 and the risk of developing MS.

Methods:Weconducteda case-control study including1,717patientswithMSand4,685healthy

blooddonors,who filled inacomprehensiveenvironmentaland lifestylequestionnaire.Datawere

analysed by logistic regressionmodels and adjusted for selected confounders. Due to interaction

betweensexandalcoholconsumptiontheanalyseswereperformedwithstratificationbysex.

Results: We found an inverse association between alcohol consumption in adolescence and risk of

developing MS in both women (p<0.001) and men (p=0.013). Women with low alcohol consumption had

an odds ratio (OR) of 0.58 (95% CI: 0.62-0.92) compared with non-drinking women. The ORs were similar

for women with moderate (OR=0.55, 95% CI: 0.43-0.70) and high consumption (OR=0.61, 95% CI: 0.41-

0.90).

Men with low alcohol consumption had an OR of 0.71 (95% CI: 0.55-0.92) compared with non-drinking

men but no decreased risk was found for men with moderate (OR=0.97, 95% CI: 0.68-1.37) and high

alcohol consumption. (OR=1.10, 95% CI: 0.68-1.74).

Conclusion: We found that alcohol consumption at ages 15 to 19 was associated with lower risk of

developing MS among Danes. Whether this reflects a causal relationship or an association with other

etiologically important factors remains to be established.

Title:SensitiveassessmentofacuteopticneuritisbyanewdigitalflickertestAuthors:GormPihl-Jensen,IliyaIvanov,DesireeHinze,SusanneTrauzettel-Klosinski,JetteFrederiksenHypothesis:TheAulhornflickertest(AFT)effectivelydiagnosesacuteopticneuritis(AON).Anew,digitalizedversionoftheAFT(DFT)hasnotpreviouslybeenexaminedinAON.TheobjectiveofthestudywastoexaminethediagnosticaccuracyandreproducibilityoftheDFTinAONMethods:IntheDFTsubjectivebrightnessofaflickeringfield(0-60Hz)isrecorded.Innormalcases,brightnessenhancementisshownatmedialfrequencieswhereasinAONdarknessenhancement(DE)ishypothesized.AFTandDFTmeasurementswereobtainedinAONpatients(≤1month)withDEasaquantitativecovariate.Results:95untreatedfirst-timeAONpatientsand55healthycontrolswereexamined.AFTandDFTwereperformed14.9days(SD:7.7)followingONonset.TheDFTshowedasensitivityof94.7%toaspecificityof93%.87of95patientswerere-examined3-6monthsfollowingON.Resultsshoweda52%decreaseindarknessenhancement.NocorrelationwasshownbetweenDFTandvisualacuity.Intra-classcorrelationoftheDFTendpointinhealthysubjectswas>0.80.Discussion:Wepresentanew,digitalflickertestinacuteONdisplayingahighsensitivityof94.7%toaspecificityof93%.Whilefurthertestingisneededourstudyindicatesanaccurateandeasy-to-usetoolinAON.

How much does balance and muscle strength impact walking in persons with multiple sclerosis? Callesen J1,2, Dalgas U2, Brincks J1, Cattaneo D3

1 VIA University College, Faculty of Health Science, Department of Research in Rehabilitation and Health Promotion, Aarhus, Denmark 2 Aarhus University, Department of Public Health, Section of Sport Science, Aarhus, Denmark 3 Larice Lab, Don Gnocchi Foundation, Gait and Balance rehabilitation Lab, Milan, Italy Background: Gait impairment is a frequent symptom in multiple sclerosis (MS), and is considered the most disabling problem by patients. Despite the obvious perspectives in the development of new effective interventions targeting gait impairments, only little is known about how muscle strength and balance impacts different gait outcomes. Objective: To determine the impact of balance and strength on different gait outcomes in persons with MS. Methods: Seventy persons with MS were enrolled in this cross-sectional study (53 women; mean age = 49 (SD: 11.2) years; median Expanded Disability Status Scale score = 3.5 (range: 1.5 - 6.5). Spearman correlation coefficients and multivariate linear regression analyses were used to determine associations between balance and muscle strength parameters and three gait outcomes. The dependent variables were the six-minute walk test (6MWT), Timed 25 Foot Walk (T25FW), and The Six Spot Step Test (SSST). Dynamic balance (Mini-BESTest), static balance (force plate measures of postural sway), and maximal muscle strength of the knee-extensors, knee-flexors, plantar-flexors and dorsi-flexors obtained from gold-standard dynamometry, were the independent explanatory variables. Results: Static balance were weakly related to the dependent outcome measures (r=0.35–0.40; p<0.05). Weak to moderate relationships with the dependent outcomes were found in muscle strength of the knee-extensors, knee-flexors, plantar-flexors and the dorsi-flexors (r=0.31-0.60; p<0,001) while a strong relationship with dynamic balance was observed (r>0.76; p<0.001). Multivariate regression models including dynamic balance and muscle strength of the plantar-flexors accounted for 72% and 73% of the variability in the 6MWT and T25FW, respectively. Standardized beta coefficients for balance in 6MW and T25FW were in both outcomes 0.70 while 0.33 and 0.15 in strength, respectively. A similar multivariate regression model with SSST as outcome found only an association with dynamic balance (beta=0.65; p<0.001). Conclusions: Dynamic balance and muscle strength are significant associated to gait function when performing fast regular forward walking. However, when habitual walking is interrupted (as during the SSST), the association with muscle strength declines, suggesting that motor control to a greater extend determines the functional outcome.

Title: Mental health status among children of mothers diagnosed with multiple sclerosis: a Danish cohort and register-based study Authors: Johanna Balslev Andersen, Julie Yoon Moberg, Janni Niclasen, Bjarne Laursen, Melinda Magyari

Abstract: Background: Multiple sclerosis (MS) is associated with an increased risk of developing physical, cognitive

and mental health problems. Current studies have demonstrated an adverse association between parental

MS mental health problems and their offspring’s mental health development.

Objective: The purpose of this study was to investigate whether the maternal multiple sclerosis is

associated with mental health status of their child.

Methods: Data from the Danish National Birth Cohort (DNBC) were merged with information from the

Danish Multiple Sclerosis Registry. Two proxies – total difficulties score and prediction of any psychiatric

diagnosis, based on the Strength and Difficulties Questionnaire, were used to measure mental health status

of the children.

Results: For the total difficulties score the control and exposed group consisted of respectively 42,016

children of mothers without MS and 40 children of mothers with MS. For the prediction of any psychiatric

diagnosis, 16,829 children of mothers without MS were in the control group and 17 children in the exposed

group. We found no statistical significant association between maternal MS and mental health status on

neither of the proxies.

Discussion: Unlike previous studies, it seems that in our cohort maternal MS is not associated with mental

health status of the children up to age 11. However, we did not have information about maternal mental

health status, which in other studies shows a mediating effect on mental health status of their children, and

it may be that participation of more resourceful women in the DNBC favours our results.

Across-sectionalstudyontherelationshipbetweencardiorespiratoryfitness,diseaseseverityandwalkingspeedinpersonswithMultipleSclerosis.LauritsT.Madsen1,UlrikDalgas1,LarsG.Hvid1,JensBansi2

1DepartmentofPublicHealth,SectionofSportScience,AarhusUniversity,Aarhus,Denmark.2DepartmentofNeurology,KlinikenValens,Valens,Switzerland.

Background:InpersonswithMultipleSclerosis(PwMS)lowercardiorespiratoryfitnesshasbeenassociatedwithdiseaseseverity,walkingcapacityandcomorbidities.However,currentevidenceisofmoderatequalityandalarge-scalesingle-centerstudyisneededtofurtherelucidatetheserelationships.Objective:Thepurposeofthestudywasto1)examinetherelationshipbetweencardiorespiratoryfitnessanddiseaseseverityinPwMS;2)investigatetherelationshipbetweencardiorespiratoryfitnessandwalkingspeed;and3)examinethepotentialimpactofMultipleSclerosis(MS)diseasetypeontheserelationships.Methods:Datawasextractedfromadatabaseconsistingofdatafrom700inpatientsatValensRehabilitationCenter,Switzerland.VO2peak(cardiorespiratoryfitness),informationondiseasecourseandMStype,walkingperformance,comorbiditiesandanthropometrywereeligiblefrom242PwMS.Results:CardiorespiratoryfitnessandExpandedDisabilityStatusScale(EDSS)wasinverselyrelated(r=-.465,p<.01).Multiplelinearregressionanalysisrevealedthatanincreaseof1pointontheEDSSscorewasassociatedwithadecreaseof1.88mL·kg-1·min-1andexplained36%ofthevariance(adjustedfortimesincediagnosis,gender,age,phenotypeofMS).Cardiorespiratoryfitnessandwalkingspeedwassignificantlycorrelated(r=.584,p<.01)withtherelapseremittingMS(RRMS)groupwalkingfasterthanthesecondaryprogressiveMS(SPMS)group(1.12±0.42m/svs.0.91±0.37m/s,p<.05)Conclusion:InPwMSasignificantrelationshipbetweencardiorespiratoryfitnessanddiseaseseveritywasconfirmed,andanincreaseof1EDDSpointdecreasescardiorespiratoryfitnessby1.88mL·kg-1·min-1.Furthermore,cardiorespiratoryfitnesswasrelatedtowalkingspeed,andforbothEDSSandwalkingspeedMSphenotypewasofinfluence.

Multiple sclerosis in first- and second-generation immigrants to Denmark A population-based cohort study. Nete Munk Nielsen1, Giulia Corn1, Nils Koch-Henriksen2,3, Egon Stenager2,4,5,6,7, Jan Wohlfahrt1, Morten Frisch1, Melinda Magyari*2,8, Mads Melbye MD*1. *The two last authors; Melinda Magyari and Mads Melbye contributed equally to the study and should be considered at co-last authors. Department of Epidemiology Research, Statens Serum Institut, Copenhagen Denmark1. The Danish Multiple Sclerosis Registry, Rigshospitalet, Copenhagen, Denmark2. Department of Clinical Epidemiology, Clinical Institute, University of Aarhus, Aarhus, Denmark3. Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark4. Multiple Sclerosis Clinic of Southern Jutland (Sønderborg, Kolding, Esbjerg), Dept. Neurology, Sønderborg, Denmark5. National Institute of Public Health, University of Southern Denmark, Copenhagen, Denmark6. Focused Research Unit in Neurology; Department of Neurology, Hospital of Southern Jutland, Denmark7 .Danish Multiple Sclerosis Centre, Department of Neurology, University of Copenhagen, Danish Multiple Sclerosis Research Centre, Department of Neurology, Neuroscience Centre, Rigshospitalet, Denmark8. Abstract Hypothesis The etiology of Multiple Sclerosis (MS) is still not known, but environmental factors, together with genetic susceptibility is believed to have an important role, and childhood has been suggested to be a period of special vulnerability to environmental factors of relevance for MS. The incidence of MS varies considerably worldwide, being lowest in Sub-Saharan Africa and East Asia and highest in North America and Europe. Denmark is a high incidence country, which could suggest that certain environmental factors in the Danish society might contribute to the etiology of MS. We therefore examined MS incidence rates among first-generation immigrants in Denmark, and their Danish born children (second-generation immigrants), to evaluate the importance and timing of environmental exposures for the risk of MS. Methods Using information from the Danish Civil Registration System, we established a cohort comprising 9 million individuals who lived in Denmark in the period 1968 to 2015 and defined first- and second-generations immigrants according to own and parental birthplaces. MS cases were identified in the Danish MS registry. Associations between history of immigration and MS risk were evaluated using incidence rate ratios (IRR) obtained in log-linear Poisson regression analysis. Results 10,242 cases of MS were identified in the study cohort, of which 578 and 106 were among first- and second-generation immigrants, respectively. Compared to Danish-born of Danish-born parents, first-generation immigrants were at a lower risk of MS (IRR=0.49, 95% CI: 0.45-0.53), whereas second-generation immigrants were at a higher risk of MS (IRR=1.28 (1.06-1.56)). IRRs among first-generation immigrants reflected their original MS risk and were modified by age at immigration. Thus, persons who immigrated to Denmark before the age of 15 years were at a 80% higher risk of MS compared to first-generation immigrants, who entered Denmark at a later age (IRR<15/IRR≥15years =1.84 (1.42-2.38). Conclusions Our study supports previous findings suggesting that childhood including fetal life might be a period of special vulnerability to environmental factors of relevance to MS. Word counts: 325

Rygning, GPR15 ekspression og multipel sklerose

Forfattere: Cecilie Ammitzbøll, Marina R. von Essen, Lars Börnsen, Eva R. Petersen, Oskar McWilliam, Rikke Ratzer, Jeppe Romme Christensen, Annette B. Oturai, Helle B. Søndergaard, Finn Sellebjerg

Baggrund: Rygning er påvist at være en disponerende faktor ved udvikling af Multipel Sklerose (MS), men hvordan rygning påvirker sygdomspatogenesen ved MS vides ikke. Cirkulerende immunceller menes at spille en væsentlig rolle i immunpatogenesen særligt ved attakvis MS (RRMS) og det er derfor interessant at undersøge hvordan rygning påvirker cirkulerende immunceller i blodet hos patienter med MS i forhold til raske individer. Med udgangspunkt i genekspressionsanalyser udvalgte vi specifikke celletyper og foretog yderligere undersøgelser og phenotype-karakterisering ved hjælp af flow cytometri.

Hypotese: Gen- og proteinekspression i cirkulerende immunceller er ændret ved rygning og disse ændringer er forbundet med MS.

Metoder: Vi analyserede microarray data fra patienter med MS, og lavede herefter PCR analyser på en konfirmatorisk kohorte af raske individer og patienter med både progressiv og attakvis MS. For at undersøge sammenhænge mellem rygerelaterede gener og MS-relevante cellepopulationer (karakteriseret ved flow cytometri), udførte vi korrelationsanalyser. Flow cytometri anvendtes til at undersøge overflade-proteiner svarende til de fundne gener samt til at karakterisere cellerne yderligere. Vi udførte T-celle proliferationsassays for at undersøge reaktiviteten af rygespecifikke celletyper overfor auto-antigener (myelin basisk protein og myelin oligodendrocyt glykoprotein).

Resultater: Ved genekspressionsanalyser fandt vi opregulering af genet G-protein coupled receptor 15 (GPR15) ved rygning og RRMS. GPR15 genekspressionen fandtes endvidere at korrelere positivt med cirkulerende CCR6+CXCR3-CD4+ T-celler hos rygere. Frekvensen af cirkulerende GPR15+CD4+ T-celler og GPR15+ CCR6+ CXCR3- CD4+ T-celler var sammenlignelig hos raske og MS patienter, men ved 7 dages kultivering med og uden antigen havde rygere og MS patienter højere frekvenser af ikke-prolifererende GPR15+CD4+ T-celler og GPR15+ CCR6+ CXCR3- CD4+ T-celler. Frekvensen af GPR15+ CD4+ T-celler var højere i cerebrospinal væske i forhold til blod hos patienter med attakvis MS. Konklusion: Effekten af rygning på systemisk immunaktivering er karakteriseret af GPR15 gen- og proteinekspression. GPR15 ekspressionen er desuden øget ved RRMS og forbundet med en CCR6+CXCR3-CD4+ T-celletype. Frekvensen af GPR15+CD4+ T-celler er øget hos MS patienter ved uspecifik stimulering og ”enriched” i CSF. GPR15 er en kemokinlignende receptor med en nyopdaget ligand (C10orf99) forbundet med lymfocyt ”homing” til inflammeret væv. Vi fortsætter undersøgelserne af denne CCR6+CXCR3-CD4 T-celle type med migratorisk potentiale forbundet med både rygning og MS.

Effect of Multiple Sclerosis Associated Interleukin 2 Receptor Alpha (IL2RA) Gene Variants on DNA Methylation Hannah-Marie Laigaard, Sophie Buhelt, Annette Oturai, Marina von Essen, Finn Sellebjerg, Helle Bach Søndergaard. Background: Genome wide association studies (GWAS) and fine mapping have demonstrated association between single nucleotide polymorphisms (SNP) within the IL2RA gene and risk of developing multiple sclerosis (MS). Yet, little is known about how and when these genetic variants affect MS susceptibility. DNA methylation is a reversible chemical modification of the DNA sequence that regulates gene expression without changing the coding sequence and the modification is highly influenced by environmental factors. Individual SNPs can affect local and more distant DNA methylation patterns, a phenomenon known as allele specific methylation (ASM). Thus DNA methylation may provide a logical interface for environmental factors and genetic traits to interplay and induce disease phenotype. Objective: We have investigated the relation between the IL2RA loci SNPs - rs2104286 and rs11256593 - and DNA methylation and the effect on IL2RA gene expression in CD8 T cells. Method: Total CD8 T cells collected from 12 MS cases and 52 controls homozygous or heterozygous for the risk (T) or the protective (C) allele of rs2104286 and rs11256593 were included in the analysis. DNA methylation status of six CpG sites in the IL2RA promoter and one at the rs2104286 SNP were determined using PyroMarkQ24 pyrosequencing machine and PyroMarkQ24 2.0.7 software. A Nanostring NCounter® PanCancer Immune Profiling panel was used to measure IL2RA gene expression levels. Results: We found a highly significant allele specific methylation at the rs2104286 CpG-site (chr.10:6,057,082) in CD8 T cells in both cases and controls. Analyses of six CpG sites within the promoter of IL2RA showed that one CpG site (chr10:6,062,684) was significantly higher methylated in healthy CC carriers compered to healthy TT carriers and this DNA methylation correlated with rs2104286 CpG methylation status. However, we observed no correlation as hypothesized between the IL2RA genotypes and IL2RA gene expression in CD8 T cells. Conclusion: These results show that differential methylation of CpG-sites in the IL2RA gene in CD8 T cells is associated with the gene variants rs2104286 and rs11256593, which could represent one mechanism to how they increase MS susceptibility, however the lack of effect on IL2RA gene expression is paradoxically and will be investigated further in more cell populations and in different states of activation.

Title: Extended antigen panel called the EZMMR panel in the diagnosis of Multiple Sclerosis Authors: Julie Heiden1, Jette L. Frederiksen2 and Gunnar Houen1

Hypothesis: B cells infected by Epstein-Barr virus (EBV) enter the central nervous system at the time of and triggered by acute EBV infection. The entering B cells reflect the different kinds of B cells that are circulating in the peripheral system – producing different virus antibodies. The aim of this project was to get closer to the etiology of Multiple Sclerosis (MS) and to identify potential biomarkers. Methods: The immune response towards the EZMMR panel, consisting of Epstein-Barr Virus (EBV), Varicella Zoster Virus (VZV), Measles virus (MeV), Mumps virus (MuV) and Rubella virus (RuV) RuV was analysed by ELISA technique in serum and cerebrospinal fluid (CSF) from patients diagnosed with Multiple Sclerosis (MS), Optic Neuritis (ON) and various controls. Results: The results showed a significantly elevated level of serum and CSF IgG antibodies directed against EBNA-1, VZV, MeV and MuV in relapsing-remitting MS (RRMS) patients. The specific antibody index (AI) was calculated, to determine if the specific antibodies were intrathecally synthesized. The sensitivity of the EZMMR panel regarding RRMS patients was approximately 85% if two or more positive AIs were required. Compared with the MRZ (measles, rubella, varicella zoster) panel where only 50% of the RRMS patients had two positive AIs, the EZMMR panel revealed improved sensitivity. Furthermore, three of the included RRMS patients were negative of oligoclonal bands (OCBs), but they were positive of the EZMMR reaction. Combining OCB and EZMMR determinations gave a sensitivity of 100%. Regarding the ON patients, the sensitivity was 85% with the combination of EZMMR panel and OCB presence. Unfortunately, it was not possible to determine the specificity. Discussion: To determine the specificity of the EZMMR panel, an appropriate control cohort has to be included. More RRMS patients with negative OCBs are necessary to determine if the EZMMR and OCB in combination have a sensitivity of 100%. In conclusion, the results obtained in this study supports the theory of the impact of viruses according to MS etiology, and especially EBV. 1 Department of Autoimmunology and Biomarkers, Statens Serum Institut 2 Rigshospitalet Glostrup and University of Copenhagen

Differential intrathecal inflammatory markers in acute optic neuritis and later conversion to

multiple sclerosis MN. Olesen1,2,3,4, K. Soelberg1,2,4,5,6,7, AC. Nilsson8, S. Jarius9 , JS. Madsen1,3, J. Grauslund6,10, TJ. Smith11, ST. Lillevang8, I. Brandslund1,3, F. Paul12, N. Asgari1,2,4,7

1 Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark 2 Department of Neurobiology, Institute of Molecular Medicine, University of Southern Denmark,

Denmark 3 Department of Clinical Immunology and Biochemistry, Lillebaelt Hospital, Vejle, Denmark 4 Department of Neurology, Slagelse Hospital, Slagelse, Denmark 5 Department of Neurology, Lillebaelt Hospital, Kolding, Denmark 6 Department of Ophthalmology, Odense University Hospital, Odense, Denmark 7 OPEN, Odense Patient data Explorative Network, Odense University Hospital, Odense, Denmark 8 Department of Clinical Immunology, Odense University Hospital, Odense, Denmark 9 Molecular Neuroimmunology Group, Department of Neurology, University Hospital Heidelberg,

Germany 10 Department of Clinical Research, University of Southern Denmark, Denmark 11 Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, USA 12 Clinical and Experimental Multiple Sclerosis Research Center and NeuroCure Clinical Research

Center, Department of Neurology, Charité - Universitätsmedizin Berlin, Germany; Experimental and Clinical Research Center, Max Delbrueck Center for Molecular Medicine and Charité – Universitätsmedizin Berlin, Berlin, Germany

Hypothesis: Optic neuritis (ON) is an early inflammatory, demyelinating event often associated with multiple sclerosis (MS). We propose that (a) patients with MS-related ON (MS-ON) and isolated ON (ION) have different cytokine/chemokine signatures at symptom onset, and that (b) this signature predicts conversion to MS in patients presenting with a first episode of ON. Methods: Forty (27 female/13 male) patients with acute ON participated in a population-based prospective cohort. All underwent clinical examination, and blood/cerebrospinal fluid (CSF) samples were taken in the acute phase prior to any immunomodulatory treatment (median interval from onset of symptoms: 14 days, range 2-38). Within three months from onset, 12 were diagnosed with MS (MS-ON). After one year, another four had converted to MS-ON. The remaining 24 were considered isolated ON (ION). Blood and CSF IL-1b, IL-6, IL-10, IL-17A, CXCL13 and NF-L were measured on the Simoa™ platform (Quanterix). CXCL13 was measured with a commercial ELISA kit (Euroimmun, sensitivity cutoff: 10 pg/ml). For statistical analyses, multiple t-tests or Fishers exact test were used followed by Holm-Šidák p-value correction. Spearman correlation p-values were not adjusted. Results: CSF levels of IL-10 and CXCL13 were significantly increased in MS-ON patients compared to ION patient (p-values: 0.027 and 0.029, respectively; median IL-10 was 0.27 pg/ml vs 0.083 pg/ml. Median CXCL13 was 18.8 pg/ml vs <10 pg/ml). Moreover, MS-ON patients had more CSF leukocytes than ION (p-value: 0.0018 and median 21.5/µl CSF vs 2.5/µl CSF), as well as higher frequency of oligoclonal bands (p-value: 0.015; detected in 13/16 vs 6/24) and higher IgG index (p-value: 0.018; median IgG indices 1.1 vs 0.52, respectively). Levels of IL-10, TNF-a, IL-17A and CXCL13 in CSF were all highly correlated with leukocyte counts (Spearman r>0.69 and p<0.002 for all), but not to albumin ratio (Spearman |r|<0.23 and p>0.40 for all). Discussion: Our data indicate subtle intrathecal immune processes in MS-ON that are not seen in ION, and this may be part of the underlying disease mechanisms. The data suggest further studies in order to dissect these immunological discrepancies in more detail.

Dimethyl fumarate suppresses B cell responses in patients with relapsing-remitting multiple sclerosis

Authors:RikkeHolmHansen,HeleneChow,FinnSellebjerg,MarinaRodevonEssen

Institution:TheDanishMultipleSclerosisCenter,DepartmentofNeurology,Rigshospitalet,UniversityofCopenhagen,Blegdamsvej9,2100Copenhagen,Denmark Dimethylfumarate(DMF),marketedasTecfidera,isadiseasemodifyingdrugusedtotreatpatientswithrelapsing-remittingmultiplesclerosis(RRMS).Despiteitspositivetreatmenteffect,theimpactofDMFontheBcellpopulationremainsuncertain.BcellsareimportantcontributorstothepathogenesisofRRMS,wheretheyregulatetheinflammatoryimmuneresponseandparticipateindevelopmentoflesionsinthecentralnervoussystem.InthisstudywethereforeevaluatedtheimmunomodulatoryeffectofTecfideraonBcellsubpopulationsandtheireffectorfunction.Analyzingthebloodfrom21Tecfideratreatedand18untreatedpatientswithRRMSshowedthatTecfiderareducesthefrequencyofperipheralantigen-experiencedBcells.Wealsofoundthatthereductioninantigen-experiencedBcellslikelywasduetoareducedfrequencyoffollicularhelperT(TFH)cellsandanincreasedfrequencyoffollicularregulatoryT(TFR)cells,whosefunctionistorestraintheactivityofTFHcells.Studyingtheimpactofmonomethylfumarate(MMF),theprimarymetaboliteofDMF,onBcelleffectorfunctioninvitroshowedthatMMFincreasedthefrequencyofTGF-βproducingcellsanddecreasedthefrequencyofLTα,TNFα,IL-6andtoalesserextentIL-10producingBcells.Altogether,thesedatademonstrateananti-inflammatoryroleofDMFanditsmetaboliteMMFontheBcellcompartment.

Pro-inflammatory monocyte subsets are augmented in incipient and progressed Multiple Sclerosis Mikkel Carstensen Gjelstrup1,2, Morten Stilund2, Thor Petersen2, Christian Muchardt3, Holger Jon Møller4, Tove Christensen1. 1Department of Biomedicine, Aarhus University; 2 Department of Neurology F, Aarhus University Hospital; 3Unit of Epigenetic regulation, Institut Pasteur, Paris; 4Department of Clinical Biochemistry, Aarhus University Hospital. BackgroundPeripheralbloodmononuclearcellsholdthepotentialtounveilnewaspectsofmultifactorialiseasessuchasmultiplesclerosis(MS).Themonocytesubsetsareparticularlyimportantduetotheircentralrolesininflammatorydiseases.Anin-depthcharacterisationofthedifferentiationofmonocytesubsetsduringMSbyanalysisofsurfaceproteinexpression,combinedwithanalysesofgeneticandepigeneticregulation,willcontributenovelinsightsinMSpathogenesisanddiagnostics.ThisisparticularlyrelevantconsideringtherecenttherapeuticstrategiesforMS,involvingdepletionofspecificcellpopulations.MethodsFlowcytometryisusedforanalysisofmonocytedifferentiation(CD14,CD16,CD40,CD163,andCD192)aswellasexpressionofHumanEndogenousRetroVirus(HERV)-H/Fand-WEnvepitopes.ThiswillbecombinedwithanalysisofgeneticandepigeneticregulationpatternsusingPrecisionNuclearRun-On(PRO-seq)andChromatinImmunoprecipitations(ChIP).ResultsInitialresultsfromflowcytometricanalysesofsamplesfrompatientswithMSandcontrolsndicatethatseveralpro-inflammatorymarkersofmonocytedifferentiation(CD40,CD163,andCD192)aresignificantlymodulated(P<0.004),particularlyonthenon-classicallyactivatedmonocytesubset,asistheexpressionofHERV-H/FandHERV-WEnvepitopes(P<0.0005),indicatingacommonregulatorymechanism.PerspectvesFlowcytometricanalysesholdthepotentialtoaidorevenreplacesomeofthelaboratediagnosticwork-upnecessaryfortheMSdiagnosisandmay,togetherwiththeotherproposedanalyses,contributetoabetterunderstandingofthedisease

Abnorm B celle fænotype og cytokinproduktion ved attakvis multipel sklerose Oskar McWilliama, Finn Sellebjerga*, Hanne V. Marquartb, Marina Rode von Essena Dansk Multipel Sclerose Centera, Neurologisk Klinik og Klinisk Immunologisk Afdelingb, Rigshospitalet, Københavns Universitet. Præsenterende forfatter* Hypotese: Der er ved multipel sklerose (MS) øget intrathekal IgG syntese og B celleaktivering, og B celledepleterende antistoffer har markant effekt ved attakvis MS. Vores hypotese er, at cirkulerende B celler ved MS har en abnorm fænotype, og at denne kan knyttes til øget cytokinproduktion og potentiale for rekruttering til centralnervesystemet. Metoder: Vi undersøgte patienter med ubehandlet attakvis MS og raske kontrolpersoner. Fænotypen for cirkulerende B celler og B celler i cerebrospinalvæske (CSF) blev analyseret ved flow cytometri. Cytokinproduktionen blev undersøgt i cirkulerende B celler og undertyper af B celler (fraktioneret ved fluorescensaktiveret cellesortering) ved intracellullær flow cytometri efter aktivering med toll-like receptor ligander og ionomycin/PMA. Resultater: Ved analyse af CD27/CD38 ekspression kunne vi identificere plasmablaster og 5 subtyper af B celler i blod (transitionelle, naïve, delvist aktiverede, memory og sen memory). Der var ikke forskel på frekvensen af disse subtyper mellem raske kontrolpersoner og patienter med MS, men der var flere delvist aktiverede B celler med en IgD-IgM+ fænotype hos patienter med MS (p=0,002). Delvist aktiverede og memory B celler udtrykte adhæsionsmolekyler og kemokinreceptorer forbundet med migration til CSF, og der var hos patienter med MS en højere forekomst af særligt sene memory B celler i CSF end i blod (p<0,0001). Efter stimulation producerede flere B celler fra patienter med MS lymfotoxin (LT)-α (p=0,002) og mere transforming growth factor (TGF)-β (p=0,0002), mens der ikke var forskel i produktionen af interleukin (IL)-6, IL-10 og tumor nekrose faktor-α. TGF-β produceredes hovedsagelig af transitionelle B celler mens LT-α i særlig grad produceredes af memory B celler. Diskussion: Ved attakvis MS er der kun relativt beskedne ændringer i forekomsten af B celle subtyper i blod, men der er øget forekomst af en IgD-IgM+ fænotype, der er tidligere sat i forbindelse med Epstein-Barr virus infektion og autoantistofproduktion ved andre sygdomme. Derudover er der ved MS øget potentiale for produktion af visse cytokiner.

AbstractDAREMUS2018Title: Frequency and immunophenotype of IL10 producing regulatory B cells in Optic Neuritis Authors: Sara Lundqvist, Signe Modvig, Emilie A. Fischer, Jette L. Frederiksen, Matilda Degn Scleroseklinikken, Neurologisk afdeling, Rigshospitalet Glostrup. Hypothesis: Optic Neuritis (ON) is a common first clinical manifestation of Multiple Sclerosis (MS). Regulatory B-cells (Bregs) secreting Interleukin 10 (IL10) can protect against autoimmune disease. Animal models of MS have shown the importance of Bregs in inhibiting disease initiation and progression. Decreased frequency and functionality of Bregs correlate with disease activity in MS. The percentage of IL10 producing Bregs decreases during relapse and normalizes in remission. IL10 producing Bregs may be crucial in the transition from ON to MS. Methods: B-cells purified from 27 ON patients were sampled close to symptom onset and from13 healthy controls (HC). The B-cells were stimulated and cultured for 48 hours with CD40ligand and CpG before measurement of intracellular IL10 and the surface markers CD19, CD1d, CD5, CD24, CD38 and CD27 by flow cytometry. The frequency of B-cell subsets were analysed in the peripheral blood and in the CSF of patients. Results: 81% of the IL10 producing Bregs co-expressed CD1d, CD24 and CD38, suggesting that the naïve B-cells are the primary source of IL10 in the B-cells culture in HC and patients. In ON the Bregs ability to produce IL10 are normal compared with HC and had a comparable frequency of Bregs competent cells of the naïve CD19+CD24+CD38+CD1d+ phenotype. Bregs function did not correlate with the presence of brain MRI lesions or CSF Oligoclonal bands indicative of a higher risk of conversion to MS. The frequencies of IL10 producing B-cells did not correlate with the conversion to MS at two years follow-up. Discussion: Cells isolated from patients with ON or HC have the same frequency of naive Bregs and an equivalent capacity to produce IL10. The frequency of Bregs does not associate with the risk of progression to MS as determined by the presence of MRI and CSF risk factors. HC and patients Bregs have similar immunophenotypes with more than 80% being CD24+CD38+CD1d+. Bregs function is not affected at onset of MS, but may become compromised later by a mechanism independent of the Bregs frequency at disease onset.