MS Research Days 2016November 17 - 18, 2016 • VU Medical Center, Amsterdam

Program

The MS Research Days are made possible by the kind support of:

Golden Sponsors:

Silver Sponsor:

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Short program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Complete program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Poster walks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Invited speakers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Abstracts oral presentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Abstracts poster presentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Instructions for MS speed date session . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

CoNteNt

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Dear colleagues,

A warm welcome to the MS Research Days 2016!

We are happy to host this important meeting in the home of the VUmc MS Center Amsterdam this year .

And indeed, it is ‘meeting’ in all its facets .

It is meeting other researchers and listen to their already obtained results, but also to their future plans . Meeting experienced researchers in the field who will give keynote lectures with promising titles, which we all are looking forward to . Meeting patients’ perspective that will motivate us all to pursue a way out . Meeting potential collaborators, meeting different areas of MS research, meeting new ideas . And it is meeting friends even speed-dating with them!

What joins us is our fascination with MS, the disease, the underlying process, the pathological and imaging expression, the clinical presentation, the impact on many lives . And that motivates us to invest our time and good ideas to bring resolving the MS puzzle closer and conquering the disease within reach .

Progress has been made and will be made on many aspects of the disease making MS research exciting in this era . In this meeting you will hear more about these recent advancements .

I’m sure that the Scientific Advisory Board of the Dutch MS Research Foundation is equally looking forward to the Research Days 2016 . I’m sure we will be meeting their expectations .

On behalf of the VUmc MS Center Amsterdam,

Bernard UitdehaagProfessor of Neurology and Director VUmc MS Center Amsterdam

Introduction

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CoNGReSS VeNUeVU Medical CenterHospital building (main entrance)De Boelelaan 11171081 HV AmsterdamTel . +31-20-44 44444

DIReCtIoNSFor more information consult www .ns .nl (train) or www .9292ov .nl (all public transport)

By tram/metro Metro line 50 starts/ends at Isolatorweg and Gein and vice versa, calling at Amsterdam Sloterdijk train station . Get off at Amstelveenseweg . From there, it’s a ten-minute walk to the hospital . Metro line 51 starts/ends at Amsterdam Centraal train station and Amstelveen Westwijk and vice versa, calling at Amsterdam Amstel train station . Get off at De Boelelaan/ VU at the Buitenveldertselaan . From there, it’s a little over a five-minute walk to the hospital . tram 5 starts/ends at Amsterdam Centraal train station and Amstelveen Binnenhof and vice versa . Get off at the De Boelelaan/VU express tram stop at the Buitenveldertselaan . From there, it’s a little over a five minute walk to the hospital .tram 16 starts/ends at Amsterdam Centraal train station and De Boelelaan/VU and vice versa . Get off at the stop VUmc .tram 24 starts/ends at Amsterdam Centraal train station and De Boelelaan/VU and vice versa . Get off at the stop VUmc .

Location

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By bus Bus 62 runs between the Amsterdam Amstel and Lelylaan metro/railway stations and vice versa . Exit at VUmc . Bus 310 runs between Nieuw-Vennep and Amsterdam Zuid/WTC and vice versa . Exit at VUmc .Bus 346 runs between Amsterdam Zuid en Haarlem NS and vice versa . Exit at the De Boelelaan . Bus 142 runs between Amsterdam Central Station and Wilnis and vice versa . Exit at the De Boelelaan .Bus 170 bus runs between Amsterdam Central Station and Uithoorn and vice versa . Exit at the De Boelelaan . Bus 171 bus runs between Amsterdam Zuid and Hortensiaplein, in Aalsmeer and vice versa . Exit at VUmc .Bus 172 runs between Kudelstaat and Amsterdam Central Station and vice versa . Exit at the De Boelelaan

By car The VU University Medical Center website contains a route planner (www .vumc .com/site-wide-header/directions-contact/) . The A10 bypass to the south of Amsterdam is accessible from all directions . Leave the ring road at the exit S108 Zuid/VUmc . At the end of this exit, turn left to get to the VU University Medical Center . Parking spots might be occu-pied, The ANWB road signs indicate if there are available parking spots .

ParkingP1 VUmc The entrance to this car park is at Amstelveenseweg (see map) . Look for the road signs stating P1 VUmc . P2 VUmc The entrance to this car park is at the Gustav Mahlerlaan (see map) . Look for the road signs stating P2 VUmc . This car park’s headroom is 190 cm .P1 & P2 VUmc parking costs: e 1 per 17 minutes, daily rate e 30 . (pay in arrears, coins, banknotes, electronic cash system and PIN)

other parking spots Paid parking spots are available around VUmc (see map) . Monday to Friday between 9 .00 a .m . and 7 .00 p .m .: e 1 .40 maximum 3 hours . Paying in advance . Free parking after 7 .00 p .m . and at weekends . For more information on parking facilities and fees in Amsterdam, please go to www .parkereninamsterdam .nl .The parking meters on the street ask for your license plate number . So make sure you have it on hand, this prevents stagnation and delay . For more information: www .cition .nl/

Parking public road (free) Free parking during the day is only possible at Kalfjeslaan . After 7 .00 p .m . parking is

Location

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Location

free . Parking is also free at Amsterdamse Bos after 10 .00 p .m . From there on out, the VU University Medical Center is a ten-minute walk . For more information on parking facilities and fees in Amsterdam, please go to www .parkereninamsterdam .nl .Parking at the Stadionplein Additional parking spots close to the VU University Medical Center are available at the Stadionplein P+R facility (near to the olympic stadium) . Trams 16 and 24 will take you from this car park to the VU University Medical Center in 5 minutes . The parking fee including a tram ticket is e 8 . For more information, please go to www .gvb .nl .

Lecture hallsThe main lecture hall, the ‘Amstelzaal’, is located on ground level (room ZH 0A2) . The registration desk, posters and sponsor stands are located in the foyer adjacent to the main lecture hall . Coffee and lunch are served in in the foyer . The lecture hall the ‘Waver’ is located on level -1 (room ZH -1C118) .

Drinks & DinerThe Basket – Amsterdam(5 minute walk from VU Medical Center)De Boelelaan 1109-b1081 HV AmsterdamTel . +31 – 20 - 205 0966

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Short program

thURSDAy, 17 NoVeMBeR 2016

8:45–9:30 Registration and Coffee Foyer

9:30–9:40 Opening

9:40–10:30 Keynote Prof .dr . C . Stadelmann-NesslerCortical demyelinated lesions in MS - do they matter?

Amstelzaal

10:30–11:00 Thesis award & presentation

11:00–11:30 Break Foyer

11:30–12:00 PresentationsNeurodegeneration & Progressive MS

Amstelzaal

12:00–12:30 Presentation Dr . B . PlatelBram Platel, the ‘MS expert’

12:30–13:30 Lunch & poster viewing Foyer

13:15–14:00 Speed dates Waverzaal

14:00–15:00 PresentationsUnderlying genetic and biological processes (I)

Amstelzaal

15:00–15:30 Break Foyer

15:30–16:15 PresentationsTreatments and Therapies

Amstelzaal

16:15–17:00 ‘The Voice of MS Research’

17:00–17:10 Gemmy & Mibeth Tichelaar Award

17:15–18:00 DrinksPrepare proposal speed-dates

The Basket

18:00–20:00 Diner

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Short program

fRIDAy, 18 NoVeMBeR 2016

8:30–9:30 Welcome and CoffeePrepare proposal speed-dates

Foyer

9:30–10:15 Keynote Prof .dr . R .Q . HintzenGenetic and environmental factors in MS

Amstelzaal

10:15–11:00 PresentationsUnderlying genetic and biological processes (II)

11:00 Announcement nominees speed-dates

11:00–11:30 Break Foyer

11:30–12:15 PresentationsImaging and biomarkers

Amstelzaal

12:15–13:45 Poster walk & lunch Foyer

13:45–14:30 Keynote Prof .dr . J . van HorssenMultiple sclerosis pathogenesis 2016: Where are we now?

Amstelzaal

14:30–15:00 Presentation nominees speed date sessions Amstelzaal

15:00–15:15 Awards & Closure Amstelzaal

Golden sponsors

Silver sponsor

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Complete program

thURSDAy, 17 NoVeMBeR 2016

time topic Chairs Location

8:45–9:30 Registration and Coffee Foyer

9:30–9:40 Opening

Jeroen Geurts &Carlie de Vries

Amstelzaal9:40–10:30 Keynote Prof .dr . C . Stadelmann-Nessler

Cortical demyelinated lesions in MS - do they matter? (page 12)

10:30–11:00 Thesis award & presentation

11:00–11:30 Break Foyer

11:30–12:00 PresentationsNeurodegeneration & Progressive MS• Iron aberration, oxidative damage and mitochondrial

defects in Marmoset EAE; a model that reflects key patho-logical features of MS (page 14) Jordon Dunham

• The Phosphodiesterase 4 (PDE4) inhibitor roflumilast im-proves remyelination in a mouse model for MS (page 15) Tim Vanmierlo

• Resolution of neuro-inflammation: a solution for MS? (page 15) Gijs Kooij

Bert ‘t Hart & Jon Lamann

Amstelzaal

12:00–12:30 Presentation Dr . B . PlatelBram Platel, the ‘MS expert’ (page 13)

12:30–13:30 Lunch & poster viewing Foyer

13:15–14:00 Speed dates Wia Baron & Anne-Marie van Dam

Waverzaal

14:00–15:00 PresentationsUnderlying genetic and biological processes (I)• Oncostatin M protects against demyelination and is

essential for remyelination (page 17) Evelien Houben

• Persistent presence of galectin-4 in MS lesions disrupts the fine-tuning of remyelination (page 18) Charlotte de Jong

• Dysregulation of the macrophage migration inhibitory factor pathway in B cells during early development of multiple sclerosis (page 19) Liza Rijvers

• Age-associated B cells with proinflammatory characteristics are expanded in a proportion of MS patients (page 20) Judith Fraussen

• Identification and design of dendritic cells with blood-brain barrier crossing capacity: moving targets to treat multiple sclerosis (page 21) Maxime De Laere

Elga de Vries &Marvin van Luijn

Amstelzaal

15:00–15:30 Break Foyer

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Complete program

15:30–16:15 Presentationstreatments and therapies• High dose cholecalciferol (vitamin D3) oil as add-on

therapy in subjects with RRMS receiving subcutaneous interferon beta-1a (page 22) Joost Smolders

• Pediatric acute disseminated encephalomyelitis followed by recurrent optic neuritis (ADEM-ON): disease course and treatment (page 23) Yu Yi Wong

• Increased risk of false negative JC virus PCR in CSF of natalizumab-associated PML patients with low PML lesion volume (page 24) Martijn Wijburg

• Disease course after CIS in children versus adults: a prospective cohort study (page 25) Roos van der Vuurst de Vries

Brigitte de Jong & Jan Meilof

Amstelzaal

16:15–17:00 ‘The Voice of MS Research’Elga de Vries Amstelzaal

17:00–17:10 Gemmy & Mibeth Tichelaar Award

17:15–18:00 DrinksPrepare proposal speed-dates

The Basket

18:00–20:00 Diner The Basket

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Complete program

fRIDAy, 18 NoVeMBeR 2016

time topic Chair/ moderators

Location

8:30–9:30 Welcome and CoffeePrepare proposal speed-dates

Foyer

9:30–10:15 Keynote Prof .dr . R .Q . HintzenGenetic and environmental factors in MS (page 12)

Markus Kleine wietfeld & Inge Huitinga

Amstelzaal

10:15–11:00 PresentationsUnderlying genetic and biological processes (II)• Preferential recruitment of highly inflammatory Th17 .1

cells to human brain as underlying mechanism of early multiple sclerosis (page 27) Marvin van Luijn

• Impaired inflammatory signaling in macrophages from MS patients (page 28) Elien Wouters

• Neuronal Excitation / Inhibition balance in MS (page 29) Svenja Kiljan

• Selective changes in unconventional CD8+ T-cell subsets cultured from multiple sclerosis brain tissue (page 29) Malou Janssen

11:00 Announcement nominees speed-dates

11:00–11:30 Break Foyer

11:30–12:15 PresentationsImaging and biomarkers• Network changes underlying cognitive impairment in

patients with and without atrophy (page 31) Anand Eijlers

• Soluble CD27 level in CSF is a prognostic biomarker in CIS (page 32) Roos van der Vuurst de Vries

• Advanced glycation endproducts in the brain of multiple sclerosis patients (page 33) Suzan Wetzel

• Predicting pediatric MS in MRI-criteria negative patients: bands do aid (page 34) Yu Yi Wong

Hugo Vrenken &Joost Smolders

Amstelzaal

12:15–13:45 Poster walks & lunch Foyer

13:45–14:30 Keynote Prof .dr . J . van HorssenMultiple sclerosis pathogenesis 2016: Where are we now? (page 13)

Jeroen Geurts & Bernard Uitdehaag

Amstelzaal14:30–15:00 Presentation nominees speed date sessions

15:00–15:15 Awards & Closure

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PoSteR WALkS, 18 NoVeMBeR 2016, 12:45 – 13:45, foyeR

Walk A. Moderators: Hanneke Hulst & Mark Mizee

A-01 Liesbet Peeters Expansion of cytotoxic CD4+CD28null T cells is associated with worse disease prognosis in MS

page 35

A-02 Marlijn van der Poel

Transcriptional profiling of primary human microglia in normal appearing multiple sclerosis tissue

page 35

A-03 Jo Mailleux Liver X receptor activation in MS lesions page 36

A-04 Jamie van Langelaar

Class-switched memory B cells selectively accumulate and correlate with EBV load in natalizumab-treated MS blood

page 37

A-05 Dennis Lentferink Grey matter OPCs are more immature than white matter OPCs; consequences for remyelination

page 38

A-06 Claudio Derada Troletti

Molecular regulation of the blood-brain barrier phenotype . The role of endothelial to mesenchymal transition

page 39

A-07 Sabela Rodríguez Lorenzo

The role of the choroid plexus in multiple sclerosis page 40

A-08 Malou Janssen High and selective abundance of mature plasma cell subpopulations in multiple sclerosis leptomeninges

page 41

A-09 Inge Werkman Distinct cholesterol efflux in grey and white matter astrocytes: Implications for remyelination

page 42

Poster walks

Walk B. Moderators: Tim Vanmierlo & Gijs Kooij

B-01 Jeroen Bogie Collectin sub-family member 12 is a novel receptor involved in the uptake of myelin by phagocytes

page 43

B-02 Peng Wang Dysregulated MMP7 levels may contribute to the inability to clear remyelination-impairing fibronectin in MS lesions

page 44

B-03 Judith Derdelinckx mRNA electroporation is an effective tool to induce long-term myelin expression and presentation by human tolerogenic dendritic cells

page 44

B-04 Linda Rolf Vitamin D3 supplementation reduces antibody titers against the Epstein-Barr Virus nuclear antigen 1 (EBNA-1) in RRMS

page 46

B-05 Roos van der Vuurst de Vries

Fatigue persists after a first attack of suspected multiple sclerosis page 47

B-06 Gwendoline Montes Diaz

Immune regulation by dimethyl fumarate (DMF) in relapsing-remitting MS patients

page 48

B-07 Kim Meijer How do MS phenotypes differ in terms of cortical and deep grey matter functional connectivity?

page 49

B-08 Yu Yi Wong High levels of the T-cell activation marker sCD27 in CSF are associated with MS in childhood acquired demyelinating syndromes

page 50

B-09 Quinten van Geest Resting-state flexible brain connectivity and its relationship with cognitive functioning

page 51

B-10 Arun Thiruvalluvan

Survival and functionality of human induced pluripotent stem cell-derived oligodendrocytes in a non-human primate model for MS

page 52

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keyNote 1: ChRIStINe StADeLMANN-NeSSLeRProfessor in Neuropathology at the University Medical Center Göttingen, Göttingen, Germany

Cortical demyelinated lesions in MS - do they matter?Christine Stadelmann is an expert on the pathology of inflammatory and neurodegenerative diseases like multiple sclerosis (MS) . One of the central topics in her research is the pathological correlates of disease progression

in MS . She has performed studies on neuronal, axonal and synaptic damage in MS over the course of the disease . Furthermore, her interest lies in the cerebral cortex as a predilection site for lesion formation in MS .

keyNote 2: RoGIeR hINtzeNProfessor in MS and Neuroimmunology of the CNS and Head MS Center Rotterdam ErasMS, Erasmus Medical Center, Rotterdam, The Netherlands

Genetic and environmental factors in MSRogier Hintzen is a clinical neurologist and immunologist specialized in the biological determinants of the cause and the course of MS . The five central themes of his research are; 1) prediction of MS after a first attack 2) genes involved in cause and course of MS, 3) environmental factors that influence

cause and course of MS, 4) cellular immunology and biomarker research and 5) viral and systemic causes of CNS inflammation .

Invited speakers

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keyNote 3: JACk VAN hoRSSeNAssociate Professor at the VU Medical Center, Amsterdam, The Netherlands and visiting Professor at the University of Hasselt, Diepenbeek, Belgium.

Multiple sclerosis pathogenesis 2016: Where are we now? Jack van Horssen is a neurobiologist specialized in the pathology of neuro-degenerative disorders . His current research is focused on the identification of molecular pathways underlying impaired mitochondrial metabolism and reactive oxygen species production in MS and to examine the potential of antioxidant and mitochondrial protection to counteract oxidative stress and improve mitochondrial function .

BRAM PLAteLSenior Researcher Diagnostic Image Analysis Group at the Radboud University Medical Center, Nijmegen, The Netherlands

Bram Platel, the ‘MS expert’Bram Platel is an ‘expert’ on MS, a disease with which his family had to cope since Bram was two years old . Bram, working at the Radboudumc, is a biomedical engineer specialized in automated image analysis . Funded by a Dutch MS Research Foundation pilot grant, he is currently developing a system to delineate regions of diffusely abnormal white matter automatically . Two years ago Bram was diagnosed with MS . In his talk, Bram will elaborate on his private life with MS and his research .

Invited speakers

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NeURoDeGeNeRAtIoN & PRoGReSSIVe MSthursday 11:30h – 12:30h

IRoN ABeRRAtIoN, oxIDAtIVe DAMAGe AND MItoChoNDRIAL DefeCtS IN MARMoSet eAe; A MoDeL thAt RefLeCtS key PAthoLoGICAL feAtUReS of MSAuthors Jordon Dunham, MSc,1 Nikki van Driel, BSc,1 Jamie L . Lim PhD,3 Susanne M .A . van der

Pol, BSc,3 Jon D . Laman,2 PhD, Jan Bauer, PhD,4 Bert A . ‘t Hart, PhD,1 Don J . Mahad,

MD, PhD,5 Graham R . Campbell, PhD,5 Jack van Horssen, PhD,3 Yolanda S . Kap, PhD1

Affiliation 1Department of Immunobiology, Biomedical Primate Research Centre, Rijswijk,

The Netherlands; 2University Groningen, University Medical Center, Department of

Neuroscience, Groningen, The Netherlands; 3Department of Molecular Cell Biology

and Immunology, VU University Medical Center, Amsterdam, The Netherlands; 4Medical University of Vienna, Center for Brain Research, Vienna, Austria; 5Centre for

neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom .

Growing evidence supports a pathogenic role of oxidative damage and mitochondrial dys-function in multiple sclerosis (MS) disease progression . The oxidative damage and mito-chondrial alterations of the MS brain are not entirely replicated in current rodent models for MS, i .e . experimental autoimmune encephalomyelitis (EAE) and this has represented challenge with respect to developing therapeutics towards progressive stages of disease . It has previously been reported that the EAE model in the common marmosets (Callithrix jacchus) displays pathological hallmarks of (progressive) MS, namely multi-focal de-myelination of both white and cortical grey matter . Here, we examined the presence of oxidative damage, iron aberration, and mitochondrial dysfunction in an atypical marmo-set EAE model, i .e . EAE induced by myelin proteins emulsified in incomplete Freunds adjuvant . Lesion-associated expression of NADPH oxidase (p22phox, p47phox, gp91phox subunits), inducible nitric oxide synthase (iNOS), mitochondrial heat shock protein 70 (MtHSP70) and superoxide dimutases 1 and 2 (SOD1, SOD2) indicated the activation of the oxidative stress pathway in marmoset brain lesions . Turnbull’s blue staining and immunohistochemistry revealed iron accumulation, lesion-as-sociated liberation of iron, and changes in iron metabolic markers (FtH1, lactoferrin, hephaestin, ceruloplasmin) to be a prominent feature of marmoset EAE; a key factor in the amplification of oxidative tissue injury . Critically, we also observed profound accumu-lation of oxidized phospholipids and DNA oxidative damage; a key feature of MS desired in animal models . Oxidative injury in this model resulted in mitochondrial defects; ie Complex IV subunit (COX-1) loss, and partial Complex IV activity loss . In conclusion, this study demonstrates extensive oxidative injury, aberrant iron distribu-tion, and mitochondrial defects, as observed in MS, to be prominent pathological features of marmoset EAE . This data highlights the value of NHP in MS research and indicates marmoset EAE is a valuable model to test iron chelating and anti-oxidant therapies for progressive forms of disease .

Abstracts oral presentations

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the PhoSPhoDIeSteRASe 4 (PDe4) INhIBItoR RofLUMILASt IMPRoVeS ReMyeLINAtIoN IN A MoUSe MoDeL foR MULtIPLe SCLeRoSISAuthors Melissa Schepers1*, Jo Mailleux1*, Jeroen Bogie1, Nick van Goethem2, Niels Hellings1,

Jerome Hendriks1, Jos Prickaerts2, tim Vanmierlo1

Affiliation 1Dept . of Biochemistry and Neuroimmunology, BIOMED, Hasselt University, 3590

Diepenbeek, Belgium, 2Dept . of Psychiatry and Neuropsychology, School for Mental

Health and Neuroscience, Maastricht University, Maastricht, the Netherlands

*These authors contributed equally to this work

Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease of the central nervous system (CNS) that frequently coincides with cognitive impairment . Pathway anal-ysis shows that cyclic adenosine monophosphate (cAMP) directs oligodendrocyte precur-sor cell (OPC) differentiation into mature myelinating oligodendrocytes, a prerequisite for remyelination . Phosphodiesterase 4 (PDE4) inhibitors suppress the breakdown of cAMP, thereby augmenting cAMP levels and facilitating OPC differentiation . We hypothesize that the PDE4 inhibitor roflumilast promotes in vivo remyelination in an animal model for remyelination . The impact of roflumilast (5µM) on ex vivo remyelination was studied in lysolecithin-de-myelinated mouse brain slices . In vivo, cuprizone-induced demyelinated 10-week-old male C57bl6 mice (6 weeks, 0 .3% cuprizone w/w) were treated for seven days with subcutaneous roflumilast (1mg/kg or 3mg/kg) or vehicle injections upon withdrawal of the cuprizone . Immunohistochemistry for myelin basic protein (MBP) was applied to study (re)myelination . Moreover, cognitive performance (e .g . spatial memory in the object loca-tion task (OLT)) was monitored during de- and remyelination processes . Demyelinated brain slices showed an increase in remyelination after roflumilast (5µM) treatment compared to vehicle . In line herewith, we found in vivo a significant increase in corpus callosum remyelination in roflumilast (3mg/kg) treated mice, but not in vehicle or 1mg/kg roflumilast treated mice . Moreover, only the 3mg/kg roflumilast treated mice re-stored their cognitive impairment upon cuprizone withdrawal . The improved remyelination and spatial memory performance following roflumilast treatment at a dosage 100-fold the regular cognition enhancing dose, suggests that roflumilast boosts the endogenous repair mechanisms in cuprizone fed mice and thereby improves cognitive performances .

ReSoLUtIoN of NeURo-INfLAMMAtIoN: A SoLUtIoN foR MULtIPLe SCLeRoSIS?Authors Gijs kooij1, Sabela Rodriguez Lorenzo1, Paul C . Norris2, Gaby Enzmann3, Claudio Derada

Troletti1, Charlotte Teunissen1, Laura Peferoen1, Mohsen Khameni4, Ellen Iacobeus4,

Sandra Amor1, Christine D . Dijkstra1, Britta Engelhardt3, Helga E . de Vries1, and Charles

N . Serhan2

Affiliation 1Department of Molecular Cell Biology and Immunology, Neuroscience Campus

Amsterdam, VUmc MS Center Amsterdam, VU University Medical Center, Amsterdam,

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the Netherlands; 2Center for Experimental Therapeutics and Reperfusion Injury,

Harvard Institutes of Medicine, Department of Anesthesiology, Perioperative and Pain

Medicine, Brigham and Women’s Hospital (BWH) and Harvard Medical School, Boston,

Massachusetts, USA; 3Theodor Kocher Institute, University of Bern, Switzerland . 4Department of Clinical Neuroscience, Karolinska Institutet Stockholm, Sweden .

BackgroundInflammation is a fundamental part of the body’s intrinsic repair mechanism that not only ensures protection from life-threatening pathogens (by the action of pro-inflammatory cells) but also restores homeostasis of affected tissues by inflammation-resolving cells and specialized pro-resolving lipid mediators (SPMs), a process defined as resolution . In the chronic neuro-inflammatory disease multiple sclerosis (MS), the abundant presence of pro-inflammatory cells and wide-spread microglial activation within the central nervous system (CNS) suggests that this resolution process is impaired in MS . Consequently, the uncontrolled inflammatory response will acquire a chronic nature, leading to severe tissue damage (neurodegeneration) and disease progression . To date, fundamental insights into the regulation of CNS resolution processes and whether impairments in this system corre-late with MS progression remain elusive .

MethodsWe used liquid chromatography-tandem mass spectrometry (LC-MS-MS)-based lipid mediator metabololipidomics to reveal lipid mediator profiles in the cerebrospinal fluid (CSF) of RRMS (either in relapse or remission), SPMS and PPMS patients as well as age/sex matched controls .

ResultsHere we provide first evidence that the process that controls inflammation – resolution of inflammation – is deregulated in MS as reflected by a complete absence of brain specific SPMs in the CSF in all MS cases . We also defined the source of these lipid mediators as well as their target cells in the CNS during neuro-inflammation .

ConclusionBy using metabololipidomic profiling of human CSF, we here provide first evidence that impaired resolution pathways exists in MS and thereby provide a novel therapeutic opportunity to restore affected pathways and hamper disease progression . Moreover, our findings demonstrate an approach for identification of SPM pathways (e .g ., resolvins, protectins, and maresins) in human CSF, which may pave the way for the use and identifi-cation of CNS-lipid mediators as biomarkers .

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UNDeRLyING GeNetIC AND BIoLoGICAL PRoCeSSeS (I)thursday 14:00h – 15:00h

oNCoStAtIN M PRoteCtS AGAINSt DeMyeLINAtIoN AND IS eSSeNtIAL foR ReMyeLINAtIoNAuthors dr . Kris Janssens,1 evelien houben,1 dr . Anurag Maheswari,1 dr . Chris Van den Haute,2,3

prof . dr . Tom Struys,1 prof . dr . Ivo Lambrichts,1 prof . dr . Veerle Baekelandt,2 prof . dr . Piet

Stinissen,1 prof . dr . Jerome Hendriks,1 dr . Helena Slaets,1 prof . dr . Niels Hellings1

Affiliation 1Department of Immunology, Biomedical Research Institute, Hasselt University,

Diepenbeek, Belgium; 2Department of Neuroscience, Laboratory for Neurobiology

and Gene Therapy, KU Leuven, Kapucijnenvoer 33, Leuven, Belgium; 3Department

of Neurosciences and Department of Pharmaceutical and Pharmacological Sciences,

Leuven Viral Vector Core, KU Leuven, Kapucijnenvoer 33, Leuven, Belgium

Multiple sclerosis (MS) is a chronic disabling disease of the central nervous system (CNS), in which destruction of myelin sheaths leads to disturbed axonal conduction . Available MS therapies modulate the immune response, but are unable to prevent neuro-logical decline . Therefore, great efforts are made to develop therapies that limit demyeli-nation and axonal degeneration . Oncostatin M (OSM), a member of the interleukin (IL)-6 cytokine family, is produced in lesions of MS patients and stimulates neuronal survival . In this study, we reveal that the OSM receptor (OSMR) was robustly upregulated during de- and remyelination . OSMR knock-out (KO) mice displayed exacerbated demyelination (p<0 .001) and complete abrogation of remyelination (p<0 .001) in the cuprizone model . In contrast, CNS-targeted OSM treatment largely prevented demyelination (p<0 .001) and potently re-established remyelination (p<0 .05) of the chronically demyelinated CNS . Tissue inhibitor of metalloproteinase-1 (TIMP-1) produced by astrocytes was found to be one of the determining factors involved in OSM mediated de- and remyelination . A strong increase in TIMP-1 mRNA was observed in the brain of wild type (WT) mice after acute de- and remyelination, while this upregulation was completely absent in OSMR KO mice . This study reveals a previously uncharacterized and protective role for OSM during demyelination and the potential to re-establish remyelination . Therefore, it indicates that OSM is a promising therapeutic candidate to limit CNS damage in demyelinating diseases including MS . For future research we focus on the repair promoting role of OSM-induced astrocytic TIMP-1 in CNS damage .

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PeRSISteNt PReSeNCe of GALeCtIN-4 IN MS LeSIoNS DISRUPtS the fINe-tUNING of ReMyeLINAtIoNAuthors Charlotte G.h.M. de Jong1, Mirjana Stancic1, Tineke Pinxterhuis1, Jack van Horssen2,

Anne-Marie van Dam3, Hans-Joachim Gabius4, Dick Hoekstra1, and Wia Baron1

Affiliation 1Department of Cell Biology, University Medical Center Groningen, University of

Groningen, Groningen, The Netherlands, 2Department Molecular Cell Biology and

Immunology, VU University Medical Center, Amsterdam, The Netherlands 3Department

Anatomy and Neurosciences, VU University Medical Center, Amsterdam, The

Netherlands 4Ludwig Maximilian University of Munich, Institute of Physiological

Chemistry, Munich, Germany

Neuron-derived molecules are potent regulators of oligodendrocyte (OLG) differentiation and myelination during brain development and in demyelinating diseases, such as mul-tiple sclerosis (MS) . Previously, we have identified galectin-4 as a novel negative soluble neuronal regulator in the timing of developmental myelination, preventing premature OLG differentiation . In the present study we investigated whether galectin-4 is a similar regulatory signal for the timing of remyelination following demyelination and whether ga-lectin-4 plays a role in MS pathology . Our findings revealed that galectin-4 is transiently expressed in axons of corpus callosum upon cuprizone-induced demyelination, but not in axons at the relapse phase in cr-EAE, while galectin-4 is permanently expressed by axons in MS lesions . Remarkably, activated microglia/macrophages also harbor galec-tin-4, localizing in MS lesions and in inflammatory infiltrates at the relapse phase in cr-EAE . In vitro analysis showed that galectin-4 is expressed in IL-4 activated bone-mar-row derived macrophages (BMDMs) and microglia and hardly in LPS/IFNγ activated BMDMs and microglia . In contrast to neurons, activated macrophages and microglia are not able to secrete galectin-4, but may promote OLG differentiation by scavenging neuronal galectin-4 . Hence, similar to developmental myelination (re)expressed neuronal galectin-4 during demyelination is likely involved in the timing of OLG differentiation and the persistent presence of galectin-4 may disrupt to the fine-tuning of remyelination in MS lesions . Our findings may provide novel strategies for improving remyelination in demyelinated MS lesions .

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DySReGULAtIoN of the MACRoPhAGe MIGRAtIoN INhIBItoRy fACtoR PAthWAy IN B CeLLS DURING eARLy DeVeLoPMeNt of MULtIPLe SCLeRoSISAuthors Liza Rijvers1 MSc; Roos M . van der Vuurst de Vries2 MD; Jamie van Langelaar1 MSc;

Maeva Stéphant1 MD; Marie-José Melief1 BSc; Annet F . Wierenga-Wolf1 BSc; Jeanet M .

Hogervorst1 BSc; Fred C .G .J . Sweep3 MD, PhD; Anneke Geurts-Moespot3 BSc, Rogier Q .

Hintzen1,2 MD, PhD; and Marvin M . van Luijn1 PhD .

Affiliation 1Departments of Immunology and 2Neurology, MS Center ErasMS, Erasmus University

Medical Center, Rotterdam, the Netherlands . 3Department of Chemical Endocrinology,

Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands .

BackgroundB-cell depletion therapies demonstrate a key role for B cells in multiple sclerosis (MS) . It is believed that early in MS, autoreactive B cells survive peripheral tolerance and migrate into the brain to mediate inflammation . A novel mechanism involved in B-cell survival and migration is the macrophage migration inhibitory factor (MIF) pathway . In mice, MIF binds to surface receptors CXCR4 and CD74 and is crucial for EAE induction . However, the regulation of the MIF pathway in human B cells remains elusive . Hence, we aimed to elucidate the MIF pathway in human B cells during MS development .

MethodsMIF receptor, protein and mRNA levels were determined in resting and in vitro activated B cells using flow cytometry, ELISA and RT-qPCR, respectively . We studied blood B cells of untreated relapsing-remitting MS (RRMS) patients (n=40), age- and gender-matched healthy controls (n=40), as well as clinically isolated syndrome (CIS, MS pre-phrase, n=42) patients who developed MS within 1 year (high-risk) and remained CIS for >5 years (low-risk) .

ResultsThe CXCR4/CD74 surface ratio was strongly elevated in RRMS compared to controls, as well as in high-risk compared to low-risk CIS patients . Serum/plasma MIF levels were not different and did not correlate with these changes in CXCR4/CD74 . When comparing different immune subsets of controls, MIF mRNA levels were significantly higher in B cells than in T cells, monocytes and dendritic cells . Interestingly, in B cells from CIS and RRMS patients, MIF expression was markedly lower than in controls, and correlated with CXCR4/CD74 ratios . After stimulation, B cells from CIS and RRMS patients showed an increased capacity to express MIF mRNA compared to controls .

ConclusionThese data demonstrate that the MIF pathway is differentially regulated in peripheral B cells in early MS . Functional studies are ongoing to explore how these alterations in the MIF pathway affect B-cell function in MS .

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AGe-ASSoCIAteD B CeLLS WIth PRoINfLAMMAtoRy ChARACteRIStICS ARe exPANDeD IN A PRoPoRtIoN of MS PAtIeNtSAuthors Judith fraussen, dr .1, Nele Claes, dr .1, Marjan Vanheusden1, Niels Hellings, Prof . dr .1,

Piet Stinissen, Prof . dr .1, Bart Van Wijmeersch, Prof . dr .1,2, Raymond Hupperts, Prof .

dr .3,4, Veerle Somers, Prof . dr .1

Affiliation 1Hasselt University, Biomedical Research Institute and Transnationale Universiteit

Limburg, School of Life Sciences, Diepenbeek, Belgium; 2Rehabilitation & MS-

Center, Overpelt, Belgium; 3Department of Neuroscience, School of Mental Health and

Neuroscience, Maastricht University, Maastricht, The Netherlands; 4Department of

Neurology, Academic MS Center Limburg, Zuyderland Medisch Centrum, Sittard, The

Netherlands

Immune aging occurs in the elderly and in a proportion of MS patients . Recently, IgD-CD27- (DN) and CD21-CD11c+ (CD21low) B cells were described as age-associated B cells with proinflammatory characteristics . This study investigated the prevalence and functional characteristics of DN and CD21low B cells in MS patients .Using flow cytometry, frequencies of age-associated B cells and their surface immu-noglobulin (Ig)M and IgG expression were measured in the peripheral blood of 64 MS patients and 85 healthy controls (HC) . DN B cell cytokine production was analyzed for 13/47 MS patients and 11/31 HC by intracellular flow cytometry . Expression of costimu-latory (CD80, CD86) and antigen presentation MHC-II molecules was determined on DN and CD21low B cells of 47 MS patients and 31 HC . DN and CD21low B cell frequencies were compared in paired peripheral blood and cerebrospinal fluid (CSF) samples of 6 MS patients .

We demonstrated a higher proportion of MS patients younger than 60 years with periph-eral expansions of DN (8/41) and CD21low (9/41) B cells compared to age-matched HC (1/33 and 2/33, respectively) . The majority of DN B cells had an IgG+ memory phenotype, while CD21low B cells consisted of a mixed population of naive and memory cells . DN B cells showed similar (MS patients) or increased (HC) MHC-II expression as class-switched memory (CSM) B cells and intermediate costimulatory molecule expres-sion between naive and CSM B cells . Moreover, DN B cells produced proinflammatory and cytotoxic cytokines following ex vivo stimulation . In the CSF, DN and CD21low B cell frequencies were even further increased .

In conclusion, a proportion of MS patients showed increased peripheral expansions of age-associated B cells . DN and CD21low B cell frequencies were further increased in MS CSF . These cells could contribute to inflammation by induction of T cell responses and the production of proinflammatory cytokines .

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IDeNtIfICAtIoN AND DeSIGN of DeNDRItIC CeLLS WIth BLooD-BRAIN BARRIeR CRoSSING CAPACIty: MoVING tARGetS to tReAt MULtIPLe SCLeRoSISAuthors Maxime De Laere1, Carmelita Sousa1, Megha Meena1, Wai Ping Lee1, Judith

Derdelinckx1,2, Barbara Willekens2, Zwi Berneman1,3, Nathalie Cools1

Affiliation 1Laboratory of Experimental Hematology, Vaccine & Infectious Disease Institute

(VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp,

B-2610 Wilrijk, Belgium; 2Department of Neurology, Antwerp University Hospital,

B-2560 Edegem, Belgium; 3Center for Cell Therapy an Regenerative Medicine, Antwerp

University Hospital, B-2650 Edegem, Belgium

BackgroundMultiple sclerosis (MS) is characterized by massive infiltration of leukocytes, including dendritic cells (DC), through the blood-brain barrier (BBB) into the central nervous system (CNS) . However, mechanisms underlying DC transmigration remain elusive . Here, we aim to implement an in vitro BBB model to study the mechanisms underlying transmigration of DC from MS patients and healthy controls (HC) as well as of in vitro generated tolero-genic (tol)DC .

MethodsAn in vitro BBB model was optimized and validated by RT-qPCR and by assessing barrier function . This model was used to study CCR5-mediated migration of DC isolated from the blood of MS patients and HC as well as of tolDC . Additionally, CCR7-mediated migration of tolDC was assessed in conventional transwell assays .

ResultsBBB cocultures form functional barriers after 10 days of culture . Pro-inflammatory stim-ulation with TNF-α and/or IFN-γ leads to barrier activation, evident at the functional and molecular level . Migration assays have been conducted with DC from 49 MS patients and 9 HC . Preliminary results indicate significantly enhanced CCR5-driven migration of DC from chronic-progressive MS patients and a similar trend for DC from relapsing-remitting MS patients as compared to those of HC . In contrast, we found reduced CCR7-mediated and similar CCR5-mediated migratory potential of tolDC as compared to in vitro generated control DC . Additionally, chemokine receptor expression correlated with migratory capacity .

ConclusionDC transmigratory capacity is enhanced in MS and identifying the underlying mechanisms may aid in developing and optimizing innovative therapeutic strategies . Indeed, although tolDC display (trans)migratory capacity, optimization of their migration potential could boost their clinical efficacy . The potential to modulate transmigratory capacity of tolDC will be explored in the future by upregulating chemokine receptor expression through mRNA electroporation .

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tReAtMeNtS AND theRAPIeSthursday 14:00h – 15:00h

hIGh DoSe ChoLeCALCIfeRoL (VItAMIN D3) oIL AS ADD-oN theRAPy IN SUBJeCtS WIth ReLAPSING-ReMIttING MULtIPLe SCLeRoSIS ReCeIVING SUBCUtANeoUS INteRfeRoN BetA-1AAuthors Joost Smolders1,2*, Raymond Hupperts1*, Reinhold Vieth3, Trygve Holmøy4, Kurt

Marhardt5, Myriam Schluep6, Joep Killestein7, Frederik Barkhof7, Luigi ME Grimaldi8#,

Manolo Beelke9#

Affiliation 1Department of Neurology, Orbis, Zuyderland Medical Centre Sittard, Maastricht

University Medical Centre, The Netherlands; 2Department of Neurology, Canisius

Wilhelmina Ziekenhuis, Nijmegen, The Netherlands; 3Department of Nutritional

Sciences, University of Toronto, Toronto, Canada; 4Akershus University Hospital and

University of Oslo, Oslo, Norway; 5Merck GmbH, Vienna, Austria; 6Service de Neurologie,

Centre Hospitalier Universitaire Vaudois, Lausanne, Suisse; 7Radiology and Image

Analysis Centre & Nuclear Medicine, VU University Medical Centre, Amsterdam, The

Netherlands; 8Fondazione Istituto San Raffaele G . Giglio di Cefalù, Cefalù, Italy; 9Worldwide Clinical Trials GmbH, Germering, Germany .

*co-first authors contributed equally. #co-final authors contributed equally.

BackgroundThe SOLAR study (NCT01285401) is the largest randomized double-blind placebo controlled study of vitamin D3 as add-on therapy in MS . This study investigated effects of cholecalciferol as add-on therapy to subcutaneous interferon (scIFN) β-1a .

MethodsThis was a prospective, randomized, double-blind, multicenter, 48-week study to evaluate the efficacy of daily oral cholecalciferol (14,000 IU (350µg) vitamin D3) vs placebo as add-on therapy to 44 µg scIFN β-1a tiw in patients with relapsing remitting MS . The pri-mary endpoint was % of subjects disease-activity-free (DAF) at Week 48 . DAF was the ab-sence of relapses, Expanded Disability Status Scale (EDSS) progression or new combined unique active (CUA) lesions . Secondary endpoints at Week 48 included mean annualized relapse rate (ARR), % subjects free from any EDSS progression and mean number of new CUA lesions per subject .

Results 229 patients were randomized to the cholecalciferol or placebo groups . At Week 48 there were no statistically significant differences in DAF between the cholecalciferol and pla-cebo group (37 .2% vs 35 .3%; p=0 .912) . No differences between groups were observed for mean ARR (0 .28 vs 0 .41; p=0 .165) or % of subjects free from any EDSS progression (71 .7% vs 75 .0%; p=0 .566) . Cholecalciferol was associated with a 32% reduction in the number of new CUA lesions compared with placebo (p=0 .005) . The % of subjects free

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from new T1 hypo-intense lesions was significantly higher with cholecalciferol compared with placebo in patients 18–30 years of age (85 .7% vs 46 .8%; p=0 .006), though not in the overall cohort .

ConclusionsCompared with placebo, cholecalciferol as add-on therapy to scIFN β-1a was not associ-ated with an increased % of subjects DAF at Week 48, although it reduced the number of new CUA lesions in the overall population and increased % of subjects free from new T1 hypo intense lesions in patients 18–30 years of age .

PeDIAtRIC ACUte DISSeMINAteD eNCePhALoMyeLItIS foLLoWeD By ReCURReNt oPtIC NeURItIS (ADeM-oN): DISeASe CoURSe AND tReAtMeNtAuthors yyM Wong MD1, Y . Hacohen MD/PhD3, K Rostasy MD/PhD4, ED van Pelt MD1, IA

Ketelslegers MD/PhD1, C . Hemingway MD/PhD3, E . Wassmer MD5, CE Catsman-

Berrevoets MD/PhD2, RQ Hintzen MD/PhD1, RF Neuteboom MD/PhD2

Affiliation 1Department of Neurology, 2Department of pediatric neurology, Erasmus MC, Rotterdam,

The Netherlands . 3Paediatric Neurology, Great Ormond Street Hospital, London, UK . 4Department of pediatric neurology, Vestische Kinder- und Jugendklinik Datteln, Datteln,

Germany . 5Department of Paediatric Neurology, Birmingham Children’s Hospital, UK

IntroductionAcute disseminated encephalomyelitis (ADEM) is typically a monophasic disease . How-ever, a multiphasic disease course can occur . One of the multiphasic entities is ADEM or multiphasic ADEM (MDEM) followed by monophasic or recurrent optic neuritis (ADEM-ON/MDEM-ON) . Little is known about treatment strategies and response in this specific disease group . We here aim to describe disease course and response to treatment of children diagnosed with ADEM-ON or MDEM-ON .

Methods Patients <18 years were identified from established national demyelination programs in the Netherlands (n=7), UK (n=5) and Germany (n=1) . All patients fulfilled International pediatric MS study group diagnostic criteria for ADEM-ON . Demographic and clinical data for each patient at presentation and follow-up . MOG-IgG in serum was performed with cell-based assays .

Results Thirteen patients were identified with ADEM-ON or MDEM-ON . Median age was 6 .6 years (range 4 .1-16 .0) and 46% were male . All but one were tested seropositive for MOG-IgG (92%) . Number of relapses varied from 1 to 9 per patient with a median follow-up dura-tion of 4 .9 years (IQR 1 .6-10 .2) .

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In 8 patients immunosuppressive therapy was initiated with Azathioprine, Mycophe-nolate Mofetil (MMF) or IvIG pulses combined with an oral prednisone taper . All of these patients were highly responsive to corticosteroids: twenty-four out of the total 45 relapses (53%) occurred when oral tapering of prednisone reached a low dose (<10 mg), or was discontinued shortly before . No relapse occurred while on high dose oral prednisone taper and there was an overall good recovery after intravenous infusion . Four patients continued relapsing, and therefore treatment was switched to Rituximab infusions . After switching, the relapses still occurred, but with a larger interval .

Conclusion Children with ADEM-ON are highly responsive to corticosteroids . They do not all respond to Azathioprine or MMF . In those cases Rituximab is an alternative treatment option . Further studies in international collaboration are needed to optimize treatment regimens for children with this rare variant of ADS .

INCReASeD RISk of fALSe NeGAtIVe JC VIRUS PCR IN CSf of NAtALIzUMAB-ASSoCIAteD PML PAtIeNtS WIth LoW PML LeSIoN VoLUMeAuthors Martijn t. Wijburg,1,2 Iris Kleerekooper,1 Birgit I Witte, Marlieke de Vos,1 Clemens

Warnke,4 Bernard MJ Uitdehaag,2 Frederik Barkhof,1 Joep Killestein,2 Mike P . Wattjes1

Affiliation 1Department of Radiology & Nuclear Medicine, Neuroscience Amsterdam, VUmc MS

Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands . 2Department of Neurology, Neuroscience Amsterdam, VUmc MS Center Amsterdam, VU

University Medical Center, Amsterdam, The Netherlands . 3Department of Epidemiology

and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands 4Department of Neurology, University of Düsseldorf, Düsseldorf, Germany .

Background In addition to the clinical presentation and magnetic resonance imaging (MRI), the detection of JC virus (JCV) by PCR in cerebrospinal fluid (CSF) is considered essential for the diagnosis of natalizumab-associated progressive multifocal leukoencephalopathy (NTZ-PML) .

objective To investigate the relationship between MRI findings and the levels of JCV DNA in the CSF of NTZ-PML patients at diagnosis .

Methods Brain MRI symptomatic and asymptomatic NTZ-PML patients performed within 10 days from the first CSF sample were included . PML lesion volumes were measured, and lesion location, lesion dissemination and signs suggestive of inflammation were scored by three

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raters . The association of imaging data and clinical findings with both qualitative and quantitative JCV PCR results were calculated .

Results 56 NTZ-PML patients were included (14 asymptomatic, 9 JCV PCR negative) . Total PML lesion volume was higher in JCV PCR positive patients (median volume [IQR]: 22 .9 [9 .2-60 .4] vs . 6 .7 [4 .9-14 .7] ml, p=0 .008) . There was a positive correlation between PML lesion volume and JC viral load (Spearman’s correlation coefficient: 0 .322, p=0 .031) . Furthermore, total PML lesion volume was higher in symptomatic patients at PML diag-nosis compared to asymptomatic patients (median volume [IQR]: 28 .9 [10 .1-60 .6] vs . 9 .2 [3 .8-18 .3] ml, p=0 .005) . No association was found between PML lesion dissemina-tion, lesion location, signs of inflammation, or presence of PML symptoms and JCV PCR results .

Conclusion There is a significant correlation between PML lesion volume on MRI and the presence/ level of JCV DNA in CSF . In patients with low lesion volumes, there is an increased risk for false negative JCV PCR, which may have considerable implications for diagnostic purposes .

DISeASe CoURSe AfteR CIS IN ChILDReN VeRSUS ADULtS: A PRoSPeCtIVe CohoRt StUDyAuthors Roos M van der Vuurst de Vries*, E Daniëlle van Pelt*, Julia Y Mescheriakova, Yu Yi

M Wong, Immy A Ketelslegers, Dorine AM Siepman, Coriene E Catsman, Rinze F

Neuteboom, Rogier Q Hintzen

*These authors contributed equally to the manuscript.

Affiliation Department of Neurology, MS Centre ErasMS, Erasmus MC, Rotterdam, The Netherlands

BackgroundClinically Isolated Syndrome (CIS) is a first demyelinating event of the central nervous system (CNS) and can be a single event . After CIS a chronic disease course with ongo-ing inflammation and relapses might occur, resulting in a diagnosis of multiple sclerosis (MS) . Whether children and adults with CIS have the same disease course has not been prospectively explored yet .

Methods Patients with CIS, whose age ranged from 1-50 years, were prospectively followed . We divided the patients in three different age groups: patients 1-10, 11-17, and 18-50 years old . Among these groups demographic data, disease course, time to MS diagnosis and annualized relapse rates (ARR) were compared .

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Results We included 383 CIS patients, of whom 218 (56 .9%) were diagnosed with MS . 11-17 year-old children had the highest rate of MS conversion (83 .5% versus 50 .0% in the other age groups together, p<0 .01) and the shortest time to MS diagnosis (median time 2 .6 months (IQR: 0 .6-6 .0) versus 8 .2 months (IQR: 1 .9-28 .2) in the other age groups together, p<0 .01) . ARR corrected for follow-up were higher in children <18 years old than in adults ≥18 years old with MS (mean ARR 0 .65 vs 0 .43, P<0 .01) .

Conclusion Children with CIS tend to have a more inflammatory disease course appearing from the high relapse rate in all children, and the highest rate of MS conversion in 11-17 year-old children . This supports early initiation of first-line disease modifying therapy (DMT) in children, perhaps even at first event in children at high risk for MS in line with current clinical practice in adults .

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UNDeRLyING GeNetIC AND BIoLoGICAL PRoCeSSeS (II)friday 10:15h – 11:00h

PRefeReNtIAL ReCRUItMeNt of hIGhLy INfLAMMAtoRy th17.1 CeLLS to hUMAN BRAIN AS UNDeRLyING MeChANISM of eARLy MULtIPLe SCLeRoSISAuthors Jamie van Langelaar1,5, Roos van der Vuurst de Vries, MD2,5, Malou Janssen1,2,5, Isis

Spilt1,5, Annet Wierenga-Wolf1,5, Marie-José Melief1,5, Gijsbert van Nierop3,5, Georges

Verjans, PhD3,6, Erik Lubberts, PhD4, Rogier Hintzen, MD, PhD1,2,5 and Marvin van Luijn,

PhD1,5

Affiliation 1Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, 2Department of Neurology, Erasmus MC, University Medical Center, Rotterdam, 3Department of Viroscience, Erasmus MC, University Medical Center, Rotterdam, 4Department of Rheumatology, Erasmus MC, University Medical Center, Rotterdam, 5MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, 6Research

Center for Emerging Infections and Zoonosis, University of Veterinary Medicine,

Hannover

BackgroundIn early multiple sclerosis (MS), refinement of existing T cell-directed treatment is highly warranted to increase efficacy and prevent serious complications . The earliest clinical presentation of MS is termed clinically isolated syndrome (CIS) . CIS patients showing impaired CD4+ T-cell quiescence rapidly develop MS . Th17 cells are often considered as IL-17-producing cells, but certain subsets also produce IFN-γ and GM-CSF . This study aimed to determine which Th17 subsets recruit to the human brain to mediate local inflammation during early MS .

MethodsClassical (CCR6+CXCR3-CCR4+) and Th1-like (CCR6+CXCR3+; Th17 .1, CCR4-; Th17 DP, CCR4+) Th17 memory subsets were analyzed in blood, cerebrospinal fluid (CSF) and brain tissues of CIS/MS patients and healthy controls . Co-expression of IL-17A, IFN-γ and GM-CSF was determined by intracellular FACS after PMA/ionomycin stimulation of sorted Th subsets . Brain tissue-resident T cells were isolated and cultured from freshly autopsied material (Netherlands Brain Bank) .

ResultsHighly activated IFN-γ/GM-CSF double-producing (Th1-like) Th17, but not IL-17A-pro-ducing (classical) Th17 effector memory cells were reduced in untreated RRMS compared to matched healthy control blood (n=40; p<0 .001) . Similar results were found between CIS patients who developed clinically definite MS within 1 year and who remained CIS for more than 5 years (n=33; p<0 .01), suggesting preferential recruitment to the brain . Indeed, Th1-like Th17 cells were highly enriched in MS CSF and brain, and locally

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co-produced IFN-γ and GM-CSF stronger than CCR6+ CD8+ T cells as well as their equivalents in blood . Of all Th subsets, Th17 .1 cells revealed the highest VLA-4 expres-sion (n=19) and selectively accumulated in blood of MS patients following natalizumab (anti-VLA-4 mAb) treatment (p<0 .01) . In patients who still experienced acute relapses after treatment, Th17 .1 cells did not accumulate in blood .

ConclusionThis study demonstrates that highly pathogenic Th17 .1, but not Th17 cells contribute to disease onset and response to natalizumab treatment in early MS .

IMPAIReD INfLAMMAtoRy SIGNALING IN MACRoPhAGeS fRoM MS PAtIeNtSAuthors elien Wouters, Winde Jorissen, Tim Vanmierlo, Veerle Somers, Piet Stinissen, Niels

Hellings, Jeroen Bogie, Jerome Hendriks

Affiliation Biomed, Hasselt University, Diepenbeek, Belgium

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease of the central nervous system (CNS) in which macrophages play a crucial role . Apart from being important mediators of CNS inflammation, demyelination, and axonal degeneration, monocyte-de-rived macrophages (MDMs) are indispensable for CNS repair in MS . In particular, we and others found that myelin uptake skews macrophages towards a less-inflammatory, reparative phenotype by activation of lipid sensing nuclear receptors, such as liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) . In this study we set out to define if and how the inflammatory environment in MS patients changes the phenotype of myelin-phagocytosing MDMs . We show that MDMs from MS patients display a reduced ability to suppress an inflammatory phenotype upon myelin internalization compared to MDMs of healthy individuals . This inflammatory phenotype of myelin-phago-cytosing MDMs of MS patients is accompanied by a decreased expression of the nuclear receptor PPARγ . In contrast, a decrease in PPARα, PPARδ, LXRα or LXRβ expression was not observed . By using qPCR and a luciferase ligand binding reporter assay, we further show that myelin potently ligates and activates PPARγ . Interestingly, the decline in PPARγ expression could be mimicked by exposing MDMs of healthy individuals to inflammatory stimuli typically associated with MS . Taken together these findings indicate that in MS the protective phenotype of myelin-phagocytosing macrophages is impaired, and that a reduced expression of PPARγ may underlie this compromised phenotype .

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NeURoNAL exCItAtIoN / INhIBItIoN BALANCe IN MULtIPLe SCLeRoSISAuthors Svenja kiljan MSc, Neeke Plat MSc, John G .J .M . Bol BSc, Yvon Galis-de Graaf BSc,

Jeroen J .G . Geurts prof, Geert J . Schenk PhD

Affiliation Anatomy & Neuroscience, VU university medical center

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease of the central nervous system . Cognitive deficits are highly prevalent in MS and are correlated with (functional) magnetic resonance imaging (MRI) measures such as functional connectivity and atrophy . In MS, MRI-measured atrophy can be explained by loss of neurons and axons . Both loss of excitatory and inhibitory neurons and processes have been described in MS cortex . An imbalance in neuronal excitation and inhibition could be a substrate for altered synchronization patterns measured with fMRI . Also, such an imbalance might induce cell death or process loss . Therefore we investigated whether disparate loss of interneurons and excitatory neurons occurs in normal appearing gray matter (NAGM) in MS . Formalin fixed and paraffin embedded tissue blocks of the cingulate gyrus (CGI) of 6 MS patients and 5 controls were collected and cut at 10µm thickness . A fluorescent double staining for interneurons and neurons was performed . Using stereo investigator software lesion borders (based on myelin presence) were outlined on the fluorescently double stained sec-tions . Numbers of interneurons, neurons and their ratio were quantified following random systematic sampling in regions of interest within myelinated deep cortical layers using the optical fractionator probe on the stereo investigator . ANOVA was used to perform a between group analysis followed by Bonferroni correction . A probability of p<0 .05 was considered statistically significant . We found that the percentage of interneurons against total number of neurons was significantly lower in NAGM in MS cortex compared to control subjects . We found a trend towards significance in the absolute number of interneurons, which was lower in MS subjects . Total neuron counts did not differ between MS and controls . In MS disparate loss of excitatory and inhibitory neurons may occur in myelinated deep cortical layers . Specifically interneurons may be vulnerable for MS pathology .

SeLeCtIVe ChANGeS IN UNCoNVeNtIoNAL CD8+ t-CeLL SUBSetS CULtUReD fRoM MULtIPLe SCLeRoSIS BRAIN tISSUeAuthors Malou Janssen1,2, Gijsbert P . van Nierop1,3, Georges M .G .M . Verjans PhD3, Marie-José

Melief2, Marvin M . van Luijn PhD2, Rogier Q . Hintzen MD, PhD1,2

Affiliation 1Dept . Neurology, MS Center ErasMS, Erasmus MC, University Medical Centre,

Rotterdam, 2Dept . Immunology, MS Center ErasMS, Erasmus MC, University Medical

Centre, Rotterdam, 3Viroscience, Erasmus MC

BackgroundCharacterization of tissue resident CD8+ T cells in multiple sclerosis (MS) brain is highly warranted to better understand their role in pathology . Not only cytotoxic, but also regu-

30

latory CD8+ T cells have been described in autoimmune diseases . Here, we studied the presence of unconventional CD8+ T-cell subsets in human brain using T-cell lines (TCL) generated from MS normal-appearing white matter (NAWM), white matter lesions and non-demented control (NDC) white matter .

MethodsTwo different 12-color flow cytometry panels were used, enabling detection of various CD8+ T-cell subsets like mucosal associated invariant T-cells (MAIT) and natural killer T-cells (NKT) . Lymphocytes were isolated from brain single-cell suspensions using density gradients, and subjected to T-cell specific stimulation to generate TCL .

ResultsIn total, 34 brain-TCL were measured by flow cytometry, of which 6 NDC and 28 MS (5 NAWM , nineteen white matter lesions and four non-staged white matter) . In MS brain-TCL, a significantly lower proportion of CD3+ cells showed an NKT-cell pheno-type (CD3+CD56+CD16+) compared to NDC brain-TCL (p=0 .001) . No difference was observed between NAWM-TCL and lesion-TCL . In all brain-TCL, the vast majority of NKT cells were CD8+ (75 .8%), in sharp contrast to what has been described for blood NKT . An increased proportion of NKT in MS-TCL was CD4+CD8+ (p=0 .0249) . Further-more, more MS-NKT expressed tissue-resident and maturation marker CD103 compared to NDC-NKT (p=0 .0046), whereas CD103 expression on classical CD3+CD56- T cells did not differ . MAIT, recently described to be present in MS brain, were only found in substantial numbers for a few MS-TCL (3/28) . Finally, more CD8+, but not CD4+ T cells, were found to be negative for CD28 in MS-TCL (p=0 .0130) .

ConclusionThese data demonstrate that the distribution of unconventional CD8+ T-cell subsets is altered in MS-compared to NDC brain-TCL . Their described regulatory function indicates enhanced immune pathology by CD8+ T cells in MS brain .

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IMAGING AND BIoMARkeRS friday 11:30h – 12:15h

NetWoRk ChANGeS UNDeRLyING CoGNItIVe IMPAIRMeNt IN PAtIeNtS WIth AND WIthoUt AtRoPhyAuthors A.J.C. eijlers, K .A . Meijer, Q . Geest van, J .J .G . Geurts and M .M . Schoonheim

Affiliation Department of Anatomy & Neurosciences, Amsterdam Neuroscience, MS Center

Amsterdam, VU University Medical Center, Amsterdam, the Netherlands .

BackgroundGrey matter (GM) atrophy is common in multiple sclerosis (MS) and is strongly related to cognitive impairment . Interestingly, not all patients with substantial GM atrophy develop cognitive impairment and not all patients without atrophy remain cognitively preserved . The objective of this study was to determine what underlies cognitive impairment in the absence and presence of GM atrophy .

MethodsA total of 330 MS patients, part of the Amsterdam MS Cohort, and 96 matched healthy controls (HC) underwent volumetric, diffusion-tensor and functional MRI as well as an extensive neuropsychological evaluation . Cognitive impairment was defined as Z<-1 .5 compared to HC on at least two cognitive domains . Similarly, GM atrophy was defined as Z<-1 .5 below controls on both cortical and deep GM volumes . In patients without and with atrophy, cognitively impaired (CI) subgroups were compared to cognitively preserved (CP) subgroups on demographics, white matter tract integrity and functional network changes, measured with eigenvector centrality .

ResultsRegardless of atrophy status, CI patients showed increased centrality of the posterior cingulate cortex compared to CP patients . A lower level of education was only able to distinguish CI from CP in patients without atrophy (p<0 .001), while a lower WM integrity (in 38% of of the WM) distinguished CI from CP in patients with atrophy Lesion volumes could not distinguish CI from CP in any atrophy group .

ConclusionAn increased centrality of the posterior cingulate cortex distinguished the CI from CP pa-tients in both groups, while effects of education and WM integrity loss depended on the presence or absence of atrophy . These findings therefore indicate that functional network centrality may provide a highly sensitive marker of cognitive impairment . Future longitudi-nal studies are needed to further elucidate the causal chain of events leading to network dysfunction and thus cognitive impairment .

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SoLUBLe CD27 LeVeL IN CSf IS A PRoGNoStIC BIoMARkeR IN CLINICALLy ISoLAteD SyNDRoMeAuthors RM van der Vuurst de Vries, JY Mescheriakova, TF Runia, N Jafari, TAM Siepman,

RQ Hintzen

Affiliation MS Centre ErasMS, Neurology, Erasmus Medical Centre, Rotterdam, The Netherlands

Background and objective Several studies found the T-cell activation marker soluble CD27 (sCD27) increased in var-ious autoimmune diseases including multiple sclerosis (MS) . The objective of this study was to investigate whether CSF sCD27 levels, in patients with clinically isolated syndrome (CIS), predict a subsequent diagnosis of MS and are associated with a higher relapse rate .

Methods In this prospective study sCD27 levels were determined in CSF of 77 CIS patients using a commercially available ELISA . CIS patients underwent a lumbar puncture and MRI scan within 6 months after first onset of symptoms . Cox regression analysis was used to calcu-late univariate and multivariate hazard ratios (HR) for MS diagnosis . Association between sCD27 levels and annualized relapse rate (ARR) was assessed using a negative binomial regression model .

Results CIS patients had higher sCD27 levels in CSF than symptomatic controls (SCs) . The ge-ometric mean (95% CI) was 31 .3 u/mL (24 .0-40 .9) vs 4 .67 u/mL (2 .90-7 .50) (p<0 .01) . During a mean follow-up of 54 .8 months, 39 out of 77 (50 .6%) CIS patients were diag-nosed with MS . In a model adjusted for MRI measurements, IgG index, and oligoclonal bands, sCD27 levels were associated with a diagnosis of MS (HR for MS diagnosis: 2 .4 per 100 units/mL increase in sCD27 levels (p=0 .007)) . Additionally MS patients with high sCD27 levels (>46 .3 units/mL(median)) at time of CIS had a 4 .7 times higher ARR than patients with low sCD27 levels (p=0 .02) .

ConclusionsSoluble CD27, measured in CSF of CIS patients, predicts MS diagnosis and a high relapse rate . Therefore, sCD27 is an activation molecule directly related to the immuno-pathology of the disease, and is a potential clinical marker to help in treatment decisions after a first attack of suspected MS .

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ADVANCeD GLyCAtIoN eNDPRoDUCtS IN the BRAIN of MULtIPLe SCLeRoSIS PAtIeNtSAuthors Suzan Wetzels1,2, Kristiaan Wouters2*, Tim Vanmierlo1*, Jean Scheijen2, Marjo van de

Waarenburg2, Casper Schalkwijk2*, Jerome Hendriks1*

Affiliation 1Department of Immunology, BIOMED, Hasselt University, 3590 Diepenbeek, Belgium, 2Department of Internal Medicine, Maastricht University, 6229 Maastricht, the

Netherlands + CARIM

* Authors contributed equally

In multiple sclerosis (MS), the pro-inflammatory activation of innate immune cells induces a metabolic shift towards glycolysis . Moreover, it is known that astrocytes mainly rely on glycolysis to provide neurons with energy . Glycolysis can lead to the formation of reactive dicarbonyl byproducts (e .g . MGO and GO) that are potent glycating agents involved in the formation of advanced glycation endproducts (AGEs) . AGEs are known to induce pro-inflammatory effects . Glyoxalase 1 (Glo1) is the rate-limiting enzyme that neutralizes MGO and GO, thereby preventing the formation of AGEs . In this study, we hypothesize that AGEs are formed as a result of increased glycolysis, potentially contrib-uting to the disease pathology . By using UPLC-MS/MS, we determined AGEs (CML, CEL, MG-H1), MGO and GO in the plasma, spinal cord, and brain of eleven mice subjected to experimental autoimmune encephalomyelitis (EAE), an animal model for MS . We revealed that AGEs (CML: 64,33±1,432 vs 135,5±5,471 p<0,0001) and MGO (3406±227,7 vs 4505±153,7 p=0,0007) are significantly increased in the spinal cord and brain of EAE mice compared to controls . In line, Glo1 activity was decreased in the CNS of EAE mice . To confirm the presence of AGEs in human active MS lesions, fluorescence immuno-histochemistry was used to stain for AGEs combined with various cell markers such as Iba, GFAP, and neurofilament to localize AGEs in macrophages, astrocytes, and neurons respectively . These staining revealed that AGEs are accumulating in GFAP+ astrocytes in active MS lesions . However, when we measured AGEs in the CSF of MS patients (N=22) and compared levels with controls (N=29), we found a decrease of CML (71,50±5,718 vs 49,98±2,423 p=0,033), MG-H1 (109,8±8,863 vs 67,41±5,536 p=0,001), and GO (442,8±33,03 vs 321,5±32,53 p=0,014) in MS patients . Our data indicate that AGEs and their precursors accumulate in the CNS during MS potentially depending on the activity state of the disease .

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PReDICtING PeDIAtRIC MULtIPLe SCLeRoSIS IN MRI-CRIteRIA NeGAtIVe PAtIeNtS: BANDS Do AIDAuthors yyM Wong1, LH Verhey3, ED van Pelt1, J O’Mahony3, IA Ketelslegers1, RF Neuteboom2, CE

Catsman-Berrevoets2, DL Arnold4, A Bar-Or4, BL Banwell5, RQ Hintzen1, on behalf of the

Dutch Pediatric MS Study Group and the Canadian Pediatric Demyelinating Disease Network .

Affiliation 1Department of Neurology, 2Department of Pediatric Neurology, Erasmus MC, Rotterdam,

The Netherlands . 3Neuroscience & Mental Health, Research Institute, The Hospital for Sick

Children, Toronto, Canada 4Department of Neurology, Montreal Neurological Institute and

Hospital, Montreal, Canada 5Division of Neurology, The Children’s Hospital of Philadelphia,

Philadelphia, USA

BackgroundA first demyelinating event in children might be the first presentation of paediatric-onset multiple sclerosis (POMS) . The Verhey criteria showed high predictive value for POMS in acquired demyelinating syndromes (ADS) . However, in clinical practice one is likely to encounter patients who, at clinical onset, do not fulfill McDonald 2010 criteria and are in addition negative for the Verhey criteria . Our aim is to investigate whether OCB may be of additional diagnostic value for predicting clinically definite MS (CDMS) in this group of MRI-criteria negative pediatric ADS patients .

Methods Children in the Canadian and Dutch prospective ADS databases were eligible for this study if the baseline MRI and OCB result were both available . Baseline scans were scored for McD2010 criteria (dissemination in time and space) and for the Verhey criteria (pe-riventricular lesion and T1 hypointense lesions) . Patients were classified as MRI-criteria negative if the patient was negative for McD2010 and in addition scored 0 or 1 on the Verhey criteria (score 0 = neither T1 nor periventricular lesion; score 1 = one of T1 or periventricular, but not both) . Patients who had a follow-up duration of two years were included, unless the patient was diagnosed with CDMS in this period .

Results ADS patients were included who were negative for both MRI criteria (n=113) . Fourteen developed CDMS, and 86% were OCB positive . OCB positivity was found in 25/113 of the ADS patients of whom 12/25 (48%) developed CDMS, in contrast to 2/88 (2%) of patients without OCB . The negative predictive value of OCB was high (97%) . In a Cox pro-portional Hazard model, OCB showed a HR of 11 .6 (95% CI 2 .5 to 53 .5; p=0 .002) after correcting for onset age and the presence of T2 hyperintense lesions at baseline .

ConclusionOCB can aid in prediction of POMS in patients who are negative for McD2010 and Verhey criteria . This is most appreciated in patients with a negative OCB status . These patients have a very low risk of MS diagnosis .

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PoSteR WALk A

A-01exPANSIoN of CytotoxIC CD4+CD28NULL t CeLLS IS ASSoCIAteD WIth WoRSe DISeASe PRoGNoSIS IN MULtIPLe SCLeRoSISAuthors Peeters, L.M.1*, Vanheusden, M .1*, Somers V .1, Van Wijmeersch, B .1,2, Lamers, I .2,

Stinissen P .1, Broux, B .1, Hellings, N .1

Affiliation 1Hasselt University, Biomedical Research Institute and Transnationale Universiteit

Limburg, School of Life Sciences, Agoralaan C, 3590 Diepenbeek, Belgium, 2MS & revalidation center Overpelt, Boemerangstraat 2, 3900 Overpelt, Belgium

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), characterized by inflammation, demyelination and axonal loss . The leading hypothesis states that peripheral autoreactive T cells are activated after encountering self-antigen after which they migrate across the blood brain barrier into the CNS . We recently identified CD4+CD-28null T cells as autoreactive and cytotoxic effector memory T cells which have the ability to migrate to sites of inflammation, possibly contributing to local tissue damage . Based on this, we hypothesize that the presence of CD4+CD28null T cells confers a worse clinical progres-sion . To estimate prognosis, evoked potentials (EP) were measured in 83 MS patients . EP abnormalities were previously reported to predict worse disability progression . Patients were scored for motoric (MEP), visual (VEP) and sensoric (SEP) evoked potentials, after which a global EP score was calculated . The higher the score, the more abnormalities . CD4+CD28null T cell percentages were measured by flow cytometry and expansion was defined as ≥2% of all CD4+ T cells (N = 61 MS patients without expansion; N = 22 MS patients with expansion) . Patients displaying CD4+CD28null T cell expansions had a higher global EP score compared to patients without expansions . These results suggest that CD4+CD28null T cells induce a worse disease progression in MS . Screening for CD4+CD28null T cell expansion may there-fore prove useful to predict clinical progression in the future .

A-02tRANSCRIPtIoNAL PRofILING of PRIMARy hUMAN MICRoGLIA IN NoRMAL APPeARING MULtIPLe SCLeRoSIS tISSUeAuthors M. van der Poel MSc1, T . Ulas Dr3, SSM Miedema MSc1, M .R . Mizee Dr1, J .L . Schultze

Prof3, J . Hamann Dr2, I . Huitinga Dr1

Affiliation 1Department of Neuroimmunology, Netherlands Institute for Neuroscience, Amsterdam, 2Department of Experimental Immunology, Academic Medical Center, Amsterdam, 3Genomics and Immunoregulation, LIMES-Institute Bonn, Germany

Microglial cells are the resident macrophages of the central nervous system (CNS) and play an important role in both tissue homeostasis in the healthy brain and regulation of

Abstracts poster presentations

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the innate immune response during brain injury or inflammation . We previously showed that microglia isolated from normal appearing white matter (NAWM) of multiple sclerosis (MS) brains are in an alerted but immune-suppressed state in that they show altered responses to dexamethasone and differences in Fc-gamma receptor expression, but lack responsiveness to lipopolysaccharide . We hypothesize that microglia in NAWM con-tribute to MS lesion formation and that alerted microglia need a second hit to become fully activated to start the demyelination process in MS lesions . We propose that alerted immune-suppressed microglia are more sensitive and will adapt a highly activated phenotype after a second immune challenge, thereby contributing to MS lesion forma-tion . We isolated primary microglia from 11 MS brain donors and 11 control donors that came to autopsy and studied gene expression by RNA sequencing . A rapid method was used to isolate human microglia acutely, based on Percoll gradient separation followed by enzymatic digestion and CD11b-conjugated magnetic bead cell sorting . Subsequent-ly, extensive flowcytometric analysis of current and upcoming autopsies was performed to validate important innate immune and neuroprotective surface markers . First results show clear differences between microglia isolated from control and MS normal appearing white and grey matter, as well as between grey and white matter microglia on transcrip-tional level .

A-03LIVeR x ReCePtoR ACtIVAtIoN IN MS LeSIoNSAuthors Jo Mailleux1, Tim Vanmierlo1 ,Jeroen Bogie1, Elien Wouters1, Piet Stinissen1, Dieter

Lütjohann2, Jerome Hendriks*1, Jack van Horssen*3

Affiliation 1Hasselt University, Biomedical Research Institute, School of Life Sciences, Diepenbeek,

Belgium, 2Laboratory of special lipid diagnostics, Center for Internal Medicine, University

of Bonn, 3VU medical center, Molecular Cell Biology and Immunology, Amsterdam, The

Netherlands

*These authors contributed equally to this work

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) . It is characterized by infiltrating macrophages that actively phagocytose myelin, the protec-tive insulating layer around axons . Myelin contains high amounts cholesterol . Clearance of myelin debris by macrophages leads to processing and accumulation of cholesterol, turning macrophages into foam cells . Liver X receptors α and β (LXRs) are sterol sensors controlling cholesterol homeostasis . Previous work from our institute has demonstrated that myelin-derived lipids activate LXRs in primary murine macrophages . Therefore, we hypothesize that LXRs are activated in phagocytes that contain myelin in active MS lesions .

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MethodsIn this study we used real-time quantitative PCR (qPCR) and immunohistochemistry (IHC) to determine the expression of LXRs and their response genes ABCA1, ABCG1 and APOE in human phagocytes after myelin phagocytosis and in MS lesions . In addition, we used gas chromatographic/mass spectrometric analysis (GC/MS) to determine LXR-activating oxysterols present and formed in myelin and myelin-incubated cells respectively .

ResultsMyelin ingestion induced the LXR response genes ABCA1 and ABCG1 in human mono-cyte-derived macrophages, demonstrating myelin activates LXRs in human phagocytes . In active MS lesions, we found that both ABCA1 and APOE gene expression and protein lev-els are highly upregulated in MHCII-positive macrophages and microglia, indicating LXRs are activated . Moreover, we found that the LXR ligand 27OHC is significantly increased in human monocyte-derived macrophages after five days of myelin uptake .

ConclusionsWe demonstrated that LXRs are activated in phagocytes active MS lesions . In addition, we have shown that the LXR ligand 27OH-cholesterol is generated in human mono-cyte-derived macrophages after myelin ingestion, suggesting that this oxysterol is in part responsible for LXR activation in macrophages in MS lesions .

A-04CLASS-SWItCheD MeMoRy B CeLLS SeLeCtIVeLy ACCUMULAte AND CoRReLAte WIth eBV LoAD IN NAtALIzUMAB-tReAteD MULtIPLe SCLeRoSIS BLooDAuthors Jamie van Langelaar1*, Liza Rijvers1*, Malou Janssen1,2, Annet F . Wierenga-Wolf1, Marie-

José Melief1, Gijsbert P . van Nierop3, Andrew Bell4 PhD, Menno C . van Zelm5 PhD,

Rogier Q . Hintzen1,2§ MD, PhD and Marvin M . van Luijn1§ PhD

Affiliation 1Departments of Immunology, 2Neurology and 3Viroscience, MS Center ErasMS, Erasmus

MC, University Medical Center, Rotterdam, The Netherlands . 4Institute of Cancer

and Genomic Sciences, University of Birmingham, United Kingdom . 5Department of

Immunology and Pathology, Monash University, Melbourne, Australia .

*,§ Shared authorships

BackgroundThe Epstein-Barr virus (EBV) is considered as an important causative trigger in MS . It is proposed that EBV infects autoreactive B cells, some of which escape control by EBV-spe-cific CD8+ T cells and infiltrate MS brain to mediate local inflammation . EBV establishes lifelong latency in IgM+ and Ig class-switched (CS) memory B-cells . Which EBV-infected memory B-cell subsets are involved in this pathogenic event is poorly understood . To elu-cidate this, we used peripheral blood of MS patients treated with natalizumab (anti-VLA-4

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monoclonal antibody), a highly effective MS treatment that prevents trafficking of pathogenic leukocytes across the blood-brain barrier . This allowed us to study pathogenic EBV-infected memory B cells in the blood .

MethodsThirteen-color flow cytometry was used to assess changes in naive and both CS and non-CS memory B-cell subsets in MS blood pre- and 6, 12 and 24 months post-treatment . In addition, total B cells were sorted from the same patients to determine EBV copy numbers using a highly sensitive qPCR assay .

ResultsWe found that the frequencies of naive mature B cells were reduced, and CD27+, but not CD27- CS memory B cells were increased after natalizumab treatment (p<0 .0001, p<0 .0001) . The accumulation of CD27+IgG+, but not IgA+ memory subsets negatively correlated with the presence of CD8+ T cells in blood (r=-0 .6066, p<0 .05) . No correla-tions were found between percentages of memory B-cell subsets, regulatory and follicular CD4+ T cells . Although EBV load did not differ between patients and controls, a group with high (µ= 5313 .14) and low (µ=413 .6) EBV load in B cells could be distinguished in treated patients . CD27-IgG+ memory B-cells, not affected by natalizumab treatment, showed a significant percentage increase in the high compared to the low EBV load group (p<0 .01) .

ConclusionThis study implies that EBV-infected CS memory B cells are differentially controlled by CD8+ T cells in MS .

A-05GRey MAtteR oPCS ARe MoRe IMMAtURe thAN WhIte MAtteR oPCS; CoNSeqUeNCeS foR ReMyeLINAtIoNAuthors Dennis Lentferink, Wia Baron

Affiliation Department of Cell Biology, University Medical Center Groningen, University of

Groningen, Groningen, The Netherlands

In the chronic demyelinating disease multiple sclerosis (MS) remyelination ultimately fails despite the presence of oligodendrocyte progenitor cells (OPCs) in most lesions, resulting in neurodegeneration and disease progression . Strikingly, remyelination appears more pronounced in grey matter lesions as compared to white matter MS lesions . Simi-larly, upon toxin-induced demyelination, spontaneous remyelination occurs faster in grey than in white matter lesions . Here, we investigated whether this difference in regional remyelination efficiency may be attributed to intrinsic differences between grey and white

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matter OPCs per se . Our preliminary results showed that grey matter OPCs have increased mRNA levels of hes5, a transcription factor that represses myelin gene expression, while white matter OPCs are more enriched in genes involved in oligodendrocyte differentiation and myelin formation (ikbkap, lpar1 and nkx6-2) . In addition, differences in the extent and the morphology of the produced myelin membranes of grey and white matter oligo-dendrocytes are observed . Hence, regional OPCs seem to carry a memory and display their own intrinsic identity . Grey matter OPCs appear to be more immature and may therefore be better responsive to demyelination and subsequent remyelination . We are currently ex-ploring the molecular background of the difference in remyelination capacity of grey and white matter OPCs . This may lead to a better understanding of the process of remyelina-tion, and may open therapeutic avenues to enhance remyelination in MS lesions .

A-06MoLeCULAR ReGULAtIoN of the BLooD-BRAIN BARRIeR PheNotyPe. the RoLe of eNDotheLIAL to MeSeNChyMAL tRANSItIoNAuthors C. Derada troletti1, B . Van Het Hof1, V . Porrini2, L . Faggi2, SMA . Van der Pol1, R . Fontijn1,

G . Kooij1, M . Pizzi2, A . Horrevorst1, H .E de Vries1

Affiliation 1Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical

Center, Amsterdam, The Netherlands . 2Division of Pharmacology, Department of

Molecular and Translational Medicine, National Institute of Neuroscience, University of

Brescia, Italy

BackgroundMultiple sclerosis (MS) is a progressive neurodegenerative disorder of the central nervous system (CNS) characterized by brain inflammation . An early event in MS is the im-pairment function of the blood-brain barrier (BBB) which consists of specialized brain endothelial cells (BECs) . The damage of the BBB may be due to a loss of structure and proper functions of endothelial cells, a phenomenon witnessed in pathological events in other tissue linked to cancer and fibrosis . In those cases endothelial cells change de-differentiate into mesenchymal cells, in a process known as endothelial to mesenchy-mal transition (EndoMT) . Our hypothesis is that the process of EndoMT could play an important role in the alteration of the BBB phenotype .

MethodsBECs were stimulated with 10ng/ml of TGF-β and IL-1β . Gene and protein expression were analyzed . ChIP-qPCR was performed on EndoMT-induced BECs in order to investigate spe-cific epigenetic changes . Immunocytochemistry was preformed to evaluate the subcellular distributions of Claudin-5 and VE-Cadherin . Immunohistochemical analyses of post-mor-tem brain tissues from non-neurological control and different MS lesions were performed in order to evaluate the expression of Snail1 and Snail2 in the brain vasculature .

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ResultsOur results show that TGFβ+IL1β treatment of BECs can induce EndoMT . Indeed, we observed an upregulation of Snail1 and Snail2 transcription factors and decrease expres-sion level of specific endothelial markers and increase of mesenchymal genes . Moreover, knockdown of Snail1 and Snail2 was sufficient to partially prevent EndoMT . Furthermore, our preliminary ChIP-qPCR results show an increase level of H3K9-18 acetylation in the promotor of Snail1 and Snail2 and a decrease acetylation level in the promotor of Clau-din-5 . Finally, immunohistochemical analyses of post-mortem brain tissues show Snail1 and Snail2 expression in the brain vasculature .

ConclusionsOur findings suggest that human BECs can undergo EndoMT and de-differentiate into mesenchymal cells . Interestingly, we found that this de-differentiation was associated with changes in histones acetylation level .

A-07the RoLe of the ChoRoID PLexUS IN MULtIPLe SCLeRoSISAuthors Sabela Rodríguez-Lorenzo1, David Francisco2, Remy Bruggmann2, Gijs Kooij1, HE de Vries1

Affiliation 1Department of Molecular Cell Biology and Immunology, VUmc MS center Amsterdam,

Neuroscience Campus Amsterdam, VUmc, Netherlands; 2Interfaculty Bioinformatics

Unit, University of Bern and Swiss Institute of Bioinformatics, Bern, Switzerland

The choroid plexus (CP) are highly vascularised projections of the brain ventricles that act as a barrier between the blood and the cerebrospinal fluid (CSF) . Their best known function is to produce CSF, but increasing evidence indicates they are also involved in immune homeostasis of the brain . CP are surrounded by a layer of epithelial cells that separates the CP stroma from the CSF, thus forming the blood-CSF-barrier (BCSFB) and regulating immune cell entry into the CNS . Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS characterized by inflammation and neurodegeneration . We hypothesise that, due to its strategic functions and location, a dysregulation of the CP could contribute to MS pathology . To reveal alterations in the CP associated with MS, we will perform RNA-sequencing from human post-mortem CP derived from non-neurological controls and MS patients . Data will be analysed aiming to find differentially expressed genes and pathways . Results will be validated at the mRNA and protein level . At a later stage, functional studies will be performed in the HIBCPP cell line, an in vitro model of the human BCSFB .Differentially expressed genes and pathways are expected to be found between MS and control CP, particularly those involved in the trafficking of cells and molecules through the BSCFB, and those regulating immune homeostasis of the CNS-resident immune cells . This study will shed light on the specific roles of the choroid plexus in MS, being, to the best of our knowledge, the first transcriptomic study of human choroid plexus in MS .

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A-08hIGh AND SeLeCtIVe ABUNDANCe of MAtURe PLASMA CeLL SUBPoPULAtIoNS IN MULtIPLe SCLeRoSIS LePtoMeNINGeSAuthors Malou Janssen1,2, Gijsbert P . van Nierop1,3, Georges M .G .M . Verjans PhD3, Marvin M . van

Luijn PhD2, Rogier Q . Hintzen MD, PhD1,2

Affiliation 1Dept . Neurology, MS Center ErasMS, Erasmus MC, University Medical Centre,

Rotterdam; 2Dept . Immunology, MS Center ErasMS, Erasmus MC, University Medical

Centre, Rotterdam; 3Viroscience, Erasmus MC

BackgroundB cells play a key role in multiple sclerosis (MS) brain pathology . Ectopic B-cell follicles have been described in ~40% of leptomeningeal structures in progressive MS, but which B-cell subsets are involved remains elusive . Here we studied B cells at this particular site of disease activity with respect to other MS and control brain compartments .

MethodsDonor material was obtained from the Netherlands Brain Bank; post-mortem delay was always <12 hours, enabling collection of viable cells in 8 end-stage MS patients and 7 non-demented controls (NDC) . B-cell populations from white matter (WM), leptomeninges (LM), cerebrospinal fluid (CSF) and peripheral blood (PB) were assessed directly ex vivo by 13-colour flow cytometry using subset- and maturation-defining markers .

ResultsOverall, we found an over two-fold increase of viable CD19+ cells within the CD45+ fraction in MS compared to NDC (p =0 .0056) . Of all CD19+ cells, higher proportions had a CD27hiCD38hi plasma cell (PC)-like phenotype in MS (p=0 .024), especially in LM (28 .8%) and CSF (29 .2%) . CD19+CD45- PC were selectively found in 3 MS patients in brain tissue (1/3), CSF (2/3) and LM (3/3), but not in PB and NDC . In these compart-ments, more B cells showed a regulatory CD24+CD27high phenotype . Finally, CXCR4 was upregulated (p=0 .014) and HLA-DR was downregulated (p=0 .0035) on PC from MS LM compared to MS PB and NDC LM respectively, suggesting locally increased numbers of mature PC .

ConclusionsThis study is the first to show characteristics of B cells present in LM compared to other paired body compartments directly ex vivo from 15 brain donors . The increased proportion of mature PC subsets in MS LM implies that local antibody production plays an important role during pathogenesis for at least a subgroup of MS patients .

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A-09DIStINCt ChoLeSteRoL effLUx IN GRey AND WhIte MAtteR AStRoCyteS: IMPLICAtIoNS foR ReMyeLINAtIoNAuthors Inge Werkman, Jo Mailleux, Jerome Hendriks, Wia Baron

Affiliation Department of Cell Biology, University Medical Center Groningen, University of

Groningen, Groningen, The Netherlands

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system which eventually results in axonal loss mainly due to failure of remyelination . Striking-ly, remyelination is far more efficient in grey matter (GM) lesions as compared to white matter (WM) lesions, which may be a reflection of distinct local extrinsic factors . Indeed, our in vitro experiments show that myelination is significantly enhanced in myelinating cultures seeded on a feeding layer of GM astrocytes compared to a WM astrocyte layer, indicating that regional difference in astrocytes may be responsible for the observed difference in remyelination efficiency . Here, we aim to reveal underlying mechanism . Oligodendrocytes require high amount of cholesterol for the formation of myelin, which is among others supplied be supplied by astrocytes . Therefore we hypothesize that the failure of WM astrocytes to deliver the necessary cholesterol to oligodendrocytes impairs remyelination in WM MS lesions . The efflux of cholesterol is elevated in GM astrocytes as compared to WM astrocytes . Interestingly, a mixture of the pro-inflammatory cytokines TNFα, IFNγ and IL-1β decreased the efflux of cholesterol from both GM and WM astrocytes, although more from GM astrocytes, whereas the TLR4 agonist LPS decreased cholesterol efflux only from GM astrocytes . Thus, cholesterol efflux from GM astrocytes seems more modifiable . Moreover, the expression of cholesterol transporter ABCA1 and the nuclear receptor Liver X receptor β (LXRβ), which controls cholesterol efflux, are increased in GM astrocytes as compared to WM astrocytes . Preliminary immune-histochemical analysis revealed that in WM MS lesions LXRβ is localized to the plasma membrane and not, as in GM astrocytes, present in the nucleus, indicating that cholesterol export may be impaired in WM astrocytes . Hence, an increased supply of astrocyte-derived cholesterol in GM lesions may potentiate remyelination in GM lesions .

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PoSteR WALk B

B-01CoLLeCtIN SUB-fAMILy MeMBeR 12 IS A NoVeL ReCePtoR INVoLVeD IN the UPtAke of MyeLIN By PhAGoCyteSAuthors Jeroen Bogie1, Jo Mailleux1, Elien Wouters1, Winde Jorissen1, Jasmine Vanmol1, Kristiaan

Wouters2,3, Niels Hellings1, Jack Van Horsen4, Tim Vanmierlo1, Jerome Hendriks1*

Affiliation 1Biomedical Research Institute, Hasselt University / Transnational University Limburg,

School of Life Sciences, Diepenbeek, Belgium; 2Cardiovascular Research Institute

Maastricht (CARIM), Maastricht University Medical Centre (MUMC), Maastricht, The

Netherlands; 3Department of Internal Medicine, Maastricht University Medical Centre

(MUMC), Maastricht, The Netherlands; 4Department of Molecular Cell Biology and

Immunology, VU University Medical Center, Amsterdam, The Netherlands

Multiple sclerosis (MS) is characterized as a chronic, inflammatory, neurodegenerative disease of the central nervous system (CNS) . Macrophage- and microglia-mediated myelin destruction is considered to be the primary effector mechanism in MS . Previous studies defined that complement-receptor 3, scavenger receptors (SRA) I/II, and Fcγ receptors, facilitate the clearance of myelin by macrophages and microglia . However, considering the complexity of myelin, it is unlikely that solely these receptors are involved in the uptake of myelin in MS lesions . By using genome wide gene expression analysis, we previously found that collectin sub-family member 12 (COLEC12), also called collectin placenta 1, was one of the most potently induced genes in macrophages after uptake of myelin . COLEC12 is structurally related to scavenger receptor class A (SRA) due to its collagen-like domain . However, COLEC12 also contains a C-type lectin/carbohydrate recognition domain (C-type CRD), typically found on C-type lectin receptors . In this study, we sought to determine whether myelin internalization increases the surface expression of COLEC12 on both peripheral and CNS-resident phagocytes, if it is involved in internaliza-tion of myelin, and at what level and cell type it is expressed in MS lesions . We show that myelin uptake increases the cell surface expression of COLEC12 on mouse and human peripheral phagocytes in vitro . Interestingly, this increase was not found on primary mouse microglia . In active MS lesions, an increased COLEC12 immunoreactivity was observed on parenchymal and perivascular phagocytes . Finally, we show that COLEC12 facilitates myelin clearance . Collectively, our data indicate that COLEC12 likely plays a key role in MS lesion progression by mediating myelin uptake .

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B-02DySReGULAteD MMP7 LeVeLS MAy CoNtRIBUte to the INABILIty to CLeAR ReMyeLINAtIoN-IMPAIRING fIBRoNeCtIN IN MS LeSIoNSAuthors Peng Wang, Rianne P . Gorter, Jenny C . de Jonge, Dick Hoekstra, Wia Baron

Affiliation Department of Cell Biology, University Medical Center Groningen, University of

Groningen, the Netherlands

Upon central nervous system demyelination at normal physiological conditions, transient expression of fibronectin precedes successful remyelination . However, in the chronic demyelinating disease multiple sclerosis (MS), aggregates of fibronectin are persistently present, which contribute to remyelination failure . Accordingly, the ability to effective-ly remove fibronectin (aggregates) would constitute an effective strategy for promoting remyelination in MS . Matrix metalloproteinases (MMPs) are enzymes that are known to remodel extracellular matrix components, including fibronectin . Here, we examined the ability of MMP3, MMP7 and MMP9 to degrade fibronectin (aggregates), and addressed the question why fibronectin aggregates remain in MS lesions . While MMP7 and MMP9 were able to degrade dimeric fibronectin, only MMP7 degraded fibronectin aggregates . MMP7 and MMP3, known to activate both proMMP7 and proMMP9, were upregulated upon toxin-induced demyelination, indicating the potential for fibronectin clearance . In contrast, in active MS lesions proMMP3 levels were increased, whereas proMMP7 expression levels were substantially decreased in both active and inactive MS lesions as compared to control white matter . Microglia and macrophages were the main cellular source of proMMP7 . More specifically, proMMP7 expression was increased in IL-4 activated macrophages as compared to control and IFNγ/LPS activated macrophages, while only IL-4 activated mac-rophages secrete significant levels of proMMP7, but not MMP3 . Also, conditioned medium of IL-4 activated macrophages most efficiently degraded fibronectin (aggregates) upon MMP-activating conditions . These findings suggest that the absence of sufficient levels of MMP7 may contribute to the persistent presence of fibronectin (aggregates) in MS lesions, and hence remyelination failure . Given that macrophage activation is disturbed in MS lesions, upregulating MMP7 expression levels, by skewing macrophages towards the IL-4 phenotype seems to be an attractive approach to promote remyelination in MS lesions .

B-03MRNA eLeCtRoPoRAtIoN IS AN effeCtIVe tooL to INDUCe LoNG-teRM MyeLIN exPReSSIoN AND PReSeNtAtIoN By hUMAN toLeRoGeNIC DeNDRItIC CeLLSAuthors Judith Derdelinckx1,2, Wai-Ping Lee1, Maxime De Laere1, Christel Bocken3, Patrick Cras2,

Barbara Willekens2, Zwi N . Berneman1,4, Niels Hellings3, Nathalie Cools1

Affiliation 1Laboratory of Experimental Hematology, Vaxinfectio, Faculty of Medicine and Health

Sciences, University of Antwerp, Belgium . 2Antwerp University Hospital, Division of

Neurology, Antwerp, Belgium . 3Department of Immunology, Biomedical Research

Institute, Hasselt University, Diepenbeek, Belgium .

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Background In the pursuit of re-establishing tolerance in autoimmune disorders such as multiple sclerosis (MS), the use of antigen-loaded tolerogenic dendritic cells (tolDC) is a promising disease antigen-specific strategy . For antigen loading of tolDC, electroporation with mRNA encoding full-length proteins may offer the potential to prevent epitope spreading by allowing protein processing and presentation of myelin-derived epitopes in a non-HLA-re-stricted manner .

objectiveTo generate myelin antigen-expressing human tolDC using mRNA electroporation .

MethodsMonocyte-derived, 1-alpha,25-dihydroxyvitamin D3-treated tolDC were electroporated with mRNA encoding myelin oligodendrocyte glycoprotein (MOG) or myelin basic protein (MBP) . mRNA and protein levels were analyzed in function of time following electro-poration using qRT-PCR and Western blot, respectively . Interferon (IFN)-γ release by a MBP-specific T cell clone in co-culture with mature DC (mDC) electroporated with MBP mRNA was quantified using ELISA .

ResultsMOG protein expression was detectable until at least 72 hours after mRNA electropora-tion as evidenced by Western blot . MBP mRNA-electroporated mDC were able to induce IFN-γ production by a MBP-reactive T cell clone in an antigen-specific manner, demon-strating their capability to present antigen . Importantly, no difference in the expression of costimulatory markers CD80 and CD86 by electroporated immature and matured tolDC (itolDC and tolDC) nor in their capacity to induce T cell hyporesponsiveness in an alloge-neic mixed lymphocyte reaction was found following mRNA electroporation .

ConclusionOur results demonstrate that mRNA electroporation of tolDC effectively induces MOG expression without affecting their tolerogenic properties . Moreover, mDC electroporated with MBP-encoding mRNA are capable to present MBP peptides to T cells . The modulato-ry effect of mRNA-electroporated tolDC on autologous myelin-specific T cell responses is currently being evaluated . Ultimately, therapeutic vaccination with tolDC electroporated with mRNA encoding full-length myelin-derived proteins may lead to a more effective therapy for MS by induction of T cell tolerance to a wider range of naturally-processed myelin-derived antigens .

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B-04VItAMIN D3 SUPPLeMeNtAtIoN ReDUCeS ANtIBoDy tIteRS AGAINSt the ePSteIN-BARR VIRUS NUCLeAR ANtIGeN 1 (eBNA-1) IN ReLAPSING ReMIttING MULtIPLe SCLeRoSISAuthors Linda Rolf MD1,2*, Anne-Hilde Muris MD1,2, Amandine Matthias PhD3, Renaud Du

Pasquier MD,PhD3, Inga Koneczny PhD1, Giulio Disanto MD,PhD4, Jens Kuhle MD,PhD5,

Jan Damoiseaux PhD6, Joost Smolders MD,PhD2,7, Raymond Hupperts MD,PhD1,2

Affiliation 1School for Mental Health and Neuroscience, Maastricht University Medical Center,

Maastricht, the Netherlands; 2Academic MS Center Limburg, Zuyderland Medical Center,

Sittard, the Netherlands; 3Laboratory of neuroimmunology, University Medical Center

Vaudois (CHUV), Lausanne, Switzerland; 4Neurocenter of Southern Switzerland (NSI),

Ospedale Civico, Lugano, Switzerland; 5Department of Neurology, University Hospital

Basel, Basel, Switzerland; 6Central Diagnostic Laboratory, Maastricht University Medical

Center, Maastricht, the Netherlands; 7Department of Neurology, Canisius Wilhelmina

Hospital, Nijmegen, the Netherlands .

Background In search for the trigger of multiple sclerosis (MS) two environmental risk factors have consistently been identified: Epstein-Barr virus (EBV) infection and vitamin D insufficien-cy . Moreover, there may be an interaction between these risk factors . The objective of this study was to investigate the effect of high dose vitamin D3 supplementation on antibody levels against the EBV nuclear antigen 1 (EBNA-1) in patients with relapsing remitting (RR-) MS, and to assess potential targets of vitamin D in order to gain more insight into the underlying mechanism .

MethodsThis study was part of the SOLAR(IUM) trial (NCT01285401), a randomized controlled trial in which relapsing remitting (RR) MS patients received either high dose vitamin D3 (n=30) supplementation (14000 IU/day) or placebo (n=23) during 48 weeks . Pre- and post-supplementation 25(OH)D levels, anti-EBNA-1, anti-viral capsid antigen (VCA) and anti-cytomegalovirus (CMV) immunoglobulin G ( IgG) levels were measured in blood . Fur-thermore, the effect of vitamin D3 supplementation on the EBV viral load and the amount of EBV-specific CD8+ cytotoxic T cells was explored, as well as a direct effect of vitamin D on anti-EBNA-1 IgG secreting B cells in vitro .

ResultsThe median antibody level against EBNA-1, but not VCA and CMV, significantly reduced in the vitamin D3 supplemented arm (526 (368 – 1683) - 455 (380 – 1148) U/mL) compared to the placebo arm (432 (351 – 1280) - 429 (297 – 1290 U/mL; p=0 .023) . The proportion of EBV-specific CD8+ T cells was not affected . Results of further explora-tive analyses will be presented .

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Conclusion Our results show that high dose vitamin D3 supplementation selectively reduces anti-EB-NA-1 IgG levels in patients with RRMS . This observation may either support an interac-tion between vitamin D and EBV in MS or reflect a better immune regulation by vitamin D in general .

B-05fAtIGUe PeRSIStS AfteR A fIRSt AttACk of SUSPeCteD MULtIPLe SCLeRoSISAuthors RM van der Vuurst de Vries1, JJA van den Dorpel1, JY Mescheriakova1, TF Runia1,

N Jafari1, TA Siepman1, D Rizopoulos2, EW Steyerberg3, RQ Hintzen1

Affiliation 1Department of Neurology, MS Centre ErasMS, Erasmus MC, Rotterdam, The

Netherlands; 2Department of Biostatistics, Erasmus MC, Rotterdam, The Netherlands, 3Department of Public Health, Erasmus MC, Rotterdam, The Netherlands

Background and objective Fatigue is a common symptom in MS, reported by more than 75% of MS patients . In an earlier study we showed that fatigue is not only a common symptom in patients at time of CIS (46%), but also predicts a subsequent diagnosis of CDMS and is associated with a shorter time to a next attack after CIS . Furthermore, in another study, fatigue in patients with MS has been found predictive of disease progression . The aim of this study was to explore the longitudinal course of fatigue during follow-up after CIS .

Methods In this study 235 CIS patients, aged between 18 and 50 years, were prospectively fol-lowed . CIS patients with comorbidities other than depression, which are likely to cause fatigue, were excluded from the analysis . Patients filled in the Krupp’s Fatigue Severity Scale (FSS) at baseline and annually . When they reached CDMS diagnosis, EDSS was also obtained annually . Mixed effects models were used to analyse the longitudinal FSS measurements and to evaluate the association between the baseline FSS during fol-low-up .

ResultsIn this prospective study 89 out of 235 CIS patients were diagnosed with CDMS (mean follow up 53 .1 months (SD ±29 .5)) . Fatigue at baseline was validated as an independent predictor for CDMS (HR 2 .9, 95% CI:1 .8-4 .9) . At time of CIS, patients who were diag-nosed with CDMS during follow-up had a significantly higher FSS score than monophasic CIS patients . We found a nonlinear average longitudinal evolution of FSS in time and this evolution was not altered after CDMS diagnosis (p=0 .44) . However, we found a significant increase of FSS score by 0 .86 units when patients experienced a second attack after CIS (p=0 .01) .

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Conclusions Fatigue, which is often present at time of CIS, persists over time and may increase after a second attack .

B-06IMMUNe ReGULAtIoN By DIMethyL fUMARAte (DMf) IN ReLAPSING-ReMIttING MS PAtIeNtSAuthors Montes Diaz Gwendoline1, Fraussen Judith1, Van Wijmeersch Bart1,2, Hupperts

Raymond3,4, Somers Veerle1

Affiliation 1Hasselt University, Biomedical Research Institute and Transnationale Universiteit

Limburg, Diepenbeek, Belgium, 2Rehabilitation & MS-Center, Overpelt, Belgium, 3Department of Neuroscience, School of Mental Health and Neuroscience, Maastricht

University, Maastricht, The Netherlands

BackgroundDimethyl fumarate (DMF, Tecfidera®) is a new oral therapy for relapsing-remitting MS (RRMS) with a yet unknown working mechanism . The aim of this study is to explore whether DMF contributes to the restoration of immune homeostasis in RRMS patients .

Methods Peripheral blood is collected from 20 RRMS patients before, after 3 months and after 1 year of DMF treatment . Of these RRMS patients, 10 are treatment-naïve and 10 are previously treated with interferon (IFN)-β or glatiramer acetate . T and B cell subtype dis-tribution and T helper cell cytokine expression, including IFN-γ, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-17, IL-4 and IL-10 are analyzed by flow cytometry . In vitro addition of DMF to B cell cultures is used to analyze direct effects on B cell apoptosis (annexin V) and expression of costimulatory molecules (CD40, CD80, CD86), antigen presentation MHCII molecules and B cell activating factor receptor (BAFFR) .

ResultsEight RRMS patients have completed the follow-up . After 12 months of DMF treatment, a decrease in pro-inflammatory T cell subsets, including memory and effector memory CD4+ and CD8+ T cells, was evident while naive CD4+ and CD8+ T cells increased . A decreased percentage of IFN-γ, GM-CSF and IL-17 positive T helper cells was detected . In the B cell population, class-switched memory B cells were decreased, although not significant, while transitional and naive B cells were increased . In vitro analysis demon-strated DMF induced apoptosis of B cells in a direct and concentration dependent man-ner . Further, DMF decreased expression of the costimulatory molecules CD40 and CD80, MHCII and the survival marker BAFFR on B cells .

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ConclusionPreliminary longitudinal results indicate a redistribution of T and B cell subtypes in DMF-treated RRMS patients . DMF can work by inducing apoptosis of B cells and chang-ing the expression of functional B cell markers, thereby influencing B cell function .

B-07hoW Do MS PheNotyPeS DIffeR IN teRMS of CoRtICAL AND DeeP GRey MAtteR fUNCtIoNAL CoNNeCtIVIty?Authors k.A. Meijer, A .J .C . Eijlers, J .J .G . Geurts, M .M . Schoonheim

Affiliation Department of Anatomy and Neurosciences, VUmc MS Center Amsterdam, Amsterdam

Neuroscience, VU University Medical Center, Amsterdam, The Netherlands

objectiveThe role of functional connectivity changes for the clinical and cognitive deterioration across different stages of MS remains unclear . Therefore, the present study investigated functional connectivity changes within and between cortical and DGM areas across differ-ent MS phenotypes .

MethodA cohort of 121 early RRMS, 122 late RRMS, 53 SPMS patients and 96 healthy controls underwent MRI and neuropsychological testing . Functional connectivity changes were investigated by looking at (1) within-DGM connectivity, (2) within-cortex connectivity and (3) DGM-cortex connectivity . Subsequently, a post-hoc regional analysis was performed, in order to identify which regions were most responsible statistically for the measured connectivity changes .

ResultsIncreased levels of within-DGM connectivity and DGM-cortex connectivity were observed in SPMS and late RRMS compared to early RRMS . Within-cortex connectivity was only decreased in SPMS relative to early RRMS . Post hoc regional analysis revealed that the putamen and pallidum were statistically responsible for the observed increased DGM-cor-tex connectivity, while increased within-DGM connectivity was not driven by one specific region but by all DGM regions . Decreased within-cortex connectivity was driven by tempo-ral, parietal and frontal regions .

ConclusionAlong the disease course, the interaction within DGM regions and between DGM and cortical regions increased . Decreased within-cortex connectivity was unique to SPMS . These functional network changes could help us to understand the clinical and cognitive deterioration in SPMS .

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B-08hIGh LeVeLS of the t-CeLL ACtIVAtIoN MARkeR SCD27 IN CSf ARe ASSoCIAteD WIth MS IN ChILDhooD ACqUIReD DeMyeLINAtING SyNDRoMeSAuthors yyM Wong1, Roos van der Vuurst de Vries1, ED van Pelt1, IA Ketelslegers1, CE Catsman-

Berrevoets2, RF Neuteboom2, RQ Hintzen1

Affiliation 1Department of Neurology, 2Department of pediatric neurology, Erasmus MC, Rotterdam,

The Netherlands .

IntroductionIt is hard to predict a future diagnosis of multiple sclerosis (MS) at the onset of acquired demyelinating syndromes (ADS) in childhood . MRI lesion type and CSF oligoclonal bands (OCB) contribute to prediction, but additional markers remain needed . In adult MS, ele-vated levels of soluble CD27 (sCD27), that is released upon T-cell activation, are found in CSF . This marker appears an accurate biomarker for MS-related neuro-inflammation . We here examined whether quantification of CSF sCD27 can distinguish a monophasic versus multiphasic disease course after pediatric ADS .

MethodsChildren <18 years old included in the Dutch Nationwide ADS study were eligible if a lumbar puncture was performed within 6 months after onset . International consensus criteria were applied for diagnosis of pediatric ADS . sCD27 levels were determined by a commercially available ELISA (Sanquin, Amsterdam) . Parametric and non-parametric tests were used when appropriate . Cox proportional Hazard model was used for univariate and multivariate analysis .

ResultsSixty-seven children were included (monophasic acute disseminated encephalomyelitis (ADEM) n=26, clinically isolated syndrome (CIS) n=12, MS n=29) . Thirty-nine patients were female (58%) . Median age of onset was 10 .9 years . A multiphasic disease course was observed in 23 patients (34%) with a median follow-up duration of 3 .0 years .Multiphasic and monophasic patients had significantly different sCD27 levels (p<0 .00) with a geometric mean of 56 .7 (95% CI 39 .5-81 .6 u/ml) vs 16 .9 (95% CI 11 .4-25 .0 u/ml) respectively . Levels of sCD27 were higher in MS patients compared to ADEM and CIS patients, geometric mean: 56 .1 u/ml, 17 .7 u/ml and 9 .8 u/ml respectively (p-value <0 .00) .

Conclusion sCD27 levels in CSF of pediatric ADS patients is associated with a multiphasic disease course, with the highest levels in MS patients . This seems to be a promising new bio-marker for prediction of MS at ADS onset .

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B-09ReStING-StAte fLexIBLe BRAIN CoNNeCtIVIty AND ItS ReLAtIoNShIP WIth CoGNItIVe fUNCtIoNINGAuthors quinten van Geest, MSc; Hanneke E . Hulst, PhD; Jeroen J .G . Geurts, PhD; Linda Douw, PhD

Affiliation Department of Anatomy and Neurosciences, MS Center Amsterdam, VU University

Medical Center Amsterdam, The Netherlands

BackgroundThe brain is not a static network of functionally connected areas, but displays high levels of flexible connectivity (flexibility), resulting in complex human behavior such as cogni-tion . To date, cognitive decline in MS has often been investigated using static functional network measures, such as resting-state (RS) functional connectivity (FC) analysis . In-vestigating flexibility of RS networks (e .g . frontoparietal network (PFN) and default mode network (DMN)) or between brain regions (hippocampus-posterior cingulate cortex (PCC)) that are highly relevant for specific cognitive function may provide better understanding of cognitive decline in MS . Currently, stability and robustness of the new measure of flexibil-ity are not sufficiently clear, although essential to know before this novel method can be applied to study cognitive decline in MS .

AimTo investigate whether RS flexibility is reproducible over a period of 1 .5 years in healthy subjects (HS), and to study its relationship with cognition .

MethodsTwenty-two HS (mean age 44 .5±9 .0 years) underwent neuropsychological testing (ex-tended version of the BRB-N, including tests for verbal, visuospatial, short-term memory, working memory, information processing speed and verbal fluency) and structural and RS functional magnetic resonance imaging on two occasions (mean follow-up time 1 .5±0 .6 years) . Using two different atlases, we obtained time series from brain regions and net-works . Pearson correlations between atlas regions were calculated for each RS volume . The standard deviation of each connection was calculated over 43 sliding windows (length 60s, shift 11s) and normalized for its average FC (i .e . coefficient of variation), resulting in individual flexibility matrices of brain regions and networks . Finally, flexibility measures were correlated to cognition .

ResultsWhole-brain flexibility was consistent across time points (t=0 .43, p=0 .68), but did not correlate with overall cognitive functioning . More specifically, flexibility between FPN and DMN did not correlate with working memory or information processing speed . However, flexibility between the hippocampus and PCC correlated with visuospatial memory (Spear-man’s ρ=0 .55, p=0 .01) .

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ConclusionRS flexibility is reproducible over time in HS . Higher levels of hippocampus/PCC flexibility were related to better visuospatial memory . Currently, we are applying this novel method to MS, to better understand cognitive decline from a dynamic network perspective .

B-10SURVIVAL AND fUNCtIoNALIty of hUMAN INDUCeD PLURIPoteNt SteM CeLL-DeRIVeD oLIGoDeNDRoCyteS IN A NoN-hUMAN PRIMAte MoDeL foR MULtIPLe SCLeRoSISAuthors Arun thiruvalluvan1, Marcin Czepiel1, Yolanda Kap3, Ietje Mantingh-Otter1, Ilia

Vainchtein1, Jeroen Kuipers2, Marjolein Bijlard2, Wia Baron2, Ben Giepmans2, Wolfgang

Brück4, Bert ’t Hart1,3, Erik Boddeke1, Sjef Copray1*

Affiliation 1Department of Neuroscience, University Medical Centre Groningen, University of

Groningen, 9713AV Groningen, the Netherlands . 2Department of Cell Biology, University

Medical Centre Groningen, University of Groningen, 9713AV Groningen, the Netherlands . 3Department of Immunobiology, Biomedical Primate Research Centre, 2288JC Rijswijk,

The Netherlands . 4Department of Neuropathology, University Medical Centre Göttingen,

37075 Göttingen, Germany .

Background and objectiveFast remyelination by endogenous oligodendrocyte precursor cells (OPCs) is essential to prevent axonal and subsequent retrograde neuronal degeneration in demyelinating lesions in multiple sclerosis (MS) . In chronic lesions, however, the remyelination capacity of OPCs becomes insufficient . Cell therapy with exogenous remyelinating cells may be a strategy to replace the failing endogenous OPCs .

MethodHere, we differentiated human induced pluripotent stem cells iPSCs (hiPSCs) into OPCs and validated their proper functionality in-vitro as well as in-vivo in mouse models for MS . Functional hiPSC-derived OPCs were injected in nonhuman primate (marmoset) model for progressive MS .

ResultsInjected hiPSC-derived OPCs in a nonhuman primate (marmoset) model for progressive MS: the grafted OPCs specifically migrated towards the MS-like lesions in the corpus callosum where they myelinated denuded axons .

ConclusionHiPSC-derived OPCs may become a first therapeutical tool to address demyelination and neurodegeneration in the progressive forms of MS .

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the speed dates are meant for MS researchers (PhD students and post-docs) to stimulate collaborations between different institutes and disciplines. every participant has three speed dates and will write a short research proposal with the best matching speed date partner. only PhD students and post-docs that are present on both the thursday and the friday can take part.

ProgramThursday 13:15h – 14.00h - Speed datesEvery participant has 3 speed dates of each 7 minutes . After 7 minutes the horn will give the signal that the time is up and the participants proceed to their next speed date . Speed date partners share the research topics they are involved in and discuss possibili-ties for a joint research proposal .

Thursday until Friday 09:30h - write research proposalWith the speed date partner you have the best match, you will write a short research proposal . For this you use the MS-speed date form, that you can fill in with pen . Remem-ber to hand in your proposal by 9:30h on Friday morning in the designated box at the registration desk (one copy) .

Friday 11.00h - announcement nomineesThe three nominated speed date couples will be announced .

Friday 14:30h - presentations nomineesThe three nominated speed date couples will have 5 minutes per couple to present their research proposal (elevator pitch, no slides) .

Friday 14:50h - award ceremonyThe speed date couple with the best research proposal and presentation will be rewarded with the opportunity to write a full research proposal for a € 15 .000,- research grant .

Guidelines- Only one research proposal per person;- A multidisciplinary approach is preferred;- Feasibility should be taken into account .

the jury consists of- Wia Baron - Anne-Marie van Dam- Jeffrey Bajramovic

Instructions for MS Speed date session

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