Antibodies to MOG have ademyelination phenotype and affect oligodendrocyte cytoskeleton ·...

Russell C Dale PhDEsther M Tantsis

MBBSVera Merheb BScRaani-Yogeeta A

Kumaran BMedScNese Sinmaz BScKarrnan Pathmanandavel

MBBSSudarshini Ramanathan

FRACPDavid R Booth PhDLouise A Wienholt PhDKristina Prelog FRACPDamien R Clark FRACPGilles J Guillemin PhDChai K Lim PhDEmily K Mathey PhDFabienne Brilot PhD

Correspondence toDr BrilotFabiennebrilotsydneyeduau

Supplemental dataat Neurologyorgnn

Antibodies to MOG have a demyelinationphenotype and affect oligodendrocytecytoskeleton

ABSTRACT

Objective To examine the clinical features of pediatric CNS demyelination associated with pos-itive myelin oligodendrocyte glycoprotein (MOG) antibodies and to examine the functional effectsof MOG antibody on oligodendrocyte cytoskeleton

Methods WemeasuredMOGantibody using a fluorescence-activated cell sorting live cell-based assayin acute sera of 73 children with CNS demyelination (DEM) (median age 8 years range 13ndash153)followed for a median of 4 years We used MO313 cells to examine immunoglobulin (Ig) G effects onoligodendrocyte cytoskeleton using 3D deconvolution imaging

ResultsMOGantibodieswere found in 3173 patients with DEM (42) but in 024 controls At firstpresentation MOG antibodyndashpositive patients were more likely to have bilateral than unilateral opticneuritis (ON) (910 vs 15 respectively p 5 003) less likely to have brainstem findings (231 vs1642 p5 0005) more likely to have a raised erythrocyte sedimentation rate20 mmh (919 vs321 p 5 005) less likely to have intrathecal oligoclonal bands (016 vs 527 p 5 018) and lesslikely to be homozygous or heterozygous for human leukocyte antigen DRB11501 (318 vs 722p5 046) MOG antibody positivity varied according to clinical phenotype with ON and relapsing ONmost likely to be seropositive Two relapsingMOG antibodyndashpositive patients treated with mycophe-nolate mofetil remain in remission and have become MOG antibody seronegative Oligodendrocytesincubated with purified IgG from MOG antibodyndashpositive patients showed a striking loss of organi-zation of the thin filaments and the microtubule cytoskeleton as evidenced by F-actin and b-tubulinimmunolabelings

Conclusions MOG antibody may define a separate demyelination syndrome which has therapeu-tic implications MOG antibody has functional effects on oligodendrocyte cytoskeleton Neurol

Neuroimmunol Neuroinflammation 20141e12 doi 101212NXI0000000000000012

GLOSSARYADEM 5 acute disseminated encephalomyelitis AQP4 5 aquaporin-4 CIS 5 clinically isolated syndrome DEM 5 demyelinatingdiseasesESR5 erythrocyte sedimentation rate FACS5 fluorescence-activated cell sortingHC5 healthy controlHEK5 humanembryonic kidney HLA 5 human leukocyte antigen Ig 5 immunoglobulin MBP 5 myelin basic protein MMF 5 mycophenolatemofetil MOG 5 myelin oligodendrocyte glycoprotein MS 5 multiple sclerosis NMO 5 neuromyelitis optica ON 5 optic neuritisSNP 5 single nucleotide polymorphism TM 5 transverse myelitis

Recently autoantibodies that bind to cell surface antigens have been shown to be importantdiagnostic biomarkers in autoimmune brain disease including autoimmune encephalitis andautoimmune demyelination1ndash3 Myelin oligodendrocyte glycoprotein (MOG) is a minor com-ponent of myelin proteins but has been the focus of extensive research in demyelinating diseasesMOG is localized on the outermost surface of myelin and has a proposed role in the regulation

These authors contributed equally to the manuscript

From the Neuroimmunology Group (RCD EMT VM R-YAK NS K Pathmanandavel SR FB) Institute for Neuroscience andMuscle Research The Kids Research Institute at the Childrenrsquos Hospital at Westmead Sydney Medical School University of Sydney WestmeadAustralia Institute for Immunology and Allergy Research (DRB) Westmead Millenium Institute for Medical Research University of SydneyWestmead Australia Clinical Immunology (LAW) Royal Prince Alfred Hospital Sydney Medical School Immunology amp Infectious DiseasesUniversity of Sydney Camperdown Australia Department of Radiology (K Prelog) the Childrenrsquos Hospital at Westmead Australia Departmentof Paediatric Neurology (DRC) Womenrsquos and Childrenrsquos Hospital North Adelaide Australia Neuroinflammation Group (GJG CKL)MND and Neurodegenerative Diseases Research Centre Macquarie University Australian School of Advanced Medicine North Ryde Australiaand Neuroinflammation Group (EKM) Brain and Mind Research Institute University of Sydney Camperdown Australia

Go to Neurologyorgnn for full disclosures Funding information and disclosures deemed relevant by the authors if any are provided at the end ofthe article The Article Processing Charge was paid by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-Noncommercial No Derivative 30 License whichpermits downloading and sharing the work provided it is properly cited The work cannot be changed in any way or used commercially

Neurologyorgnn copy 2014 American Academy of Neurology 1

ordf 2014 American Academy of Neurology Unauthorized reproduction of this article is prohibited

of microtubule stability4 Autoantibodiesagainst MOG (MOG antibodies) have beenshown to mediate demyelination in rodentsin ldquo2-hit modelsrdquo and also in primates5ndash8

The importance of MOG antibodies inhuman demyelinating disease has previouslybeen controversial predominantly due to theuse of antibody assays that denature proteinand alter conformation More recently usingcell-based assays high titer MOG antibody hasbeen unequivocally found in 20ndash40 of chil-dren with acute CNS demyelination9 In partic-ular MOG antibodies have been shown to beassociated with acute disseminated encephalomy-elitis (ADEM) and patients with neuromyelitisoptica (NMO)-like phenotypes who are negativefor NMO immunoglobulin (Ig) G10ndash16 How-ever detailed clinical and radiologic phenotypingassociated with MOG antibodies is still lackingand the role of MOG antibodies as a biomarkerin clinical practice is still not clear

Herein we further define the clinical signif-icance of MOG antibody as a biomarker andshow that MOG antibody can modify themicrotubule network and thin filaments ofoligodendrocytes

METHODS Patients and controls Patients The stored

acute serum (280degC) taken from 73 children during their first

episode of CNS demyelination (DEM) was used for this study

(median age 8 years range 13ndash153 37 females) All sera were

acute and before immune therapy The clinical and radiologic

features of 60 of the patients have been reported previously17

but the serologic investigation of this cohort has not been pre-

viously reported The patients were clinically phenotyped using

2013 consensus criteria18 The first episode of demyelination

was ADEM (n 5 28) transverse myelitis (TM n 5 15) optic

neuritis (ON n 5 15) and other clinically isolated syndrome

(CIS) excluding TM and ON (n 5 15) These other CIS

patients had polyfocal CIS cerebellar CIS brainstem CIS or

hemispheric CIS The acute MRI brain scans (n 5 70) and

MRI spine (n5 30) were reviewed and rated using MRI criteria

blinded to the laboratory findings as previously described using

McDonald KIDMUS Callen and Verhey criteria17 The

patients were followed for a median of 40 years (range 03ndash

137 years) At study census and classification 54 patients had a

monophasic disease (ADEM n 5 24 TM n 5 13 ON n 5 7

ldquoother CISrdquo n 5 10) Nineteen of 73 patients had a relapsing

demyelinating disorder (multiple sclerosis [MS] n5 15 relaps-

ing ON n 5 4) The 15 patients with MS fulfilled criteria by

Krupp et al18 and had 2 or more clinical events

Controls We have previously shown that MOG antibodies

are specific to CNS demyelination10 To generate a control

range for this study 24 pediatric controls with other neurologic

disease including epilepsy cerebral palsy neurometabolic dis-

ease and neurodegenerative disorders were used (median age

11 years range 2ndash14)

Patient and control sera had IgG concentrations measured by

nephelometry (BN ProSpec Siemens Germany) and IgG values

were within the normal range (62ndash144 gL)

CSF samples (n 5 20 controls and n 5 22 demyelinating dis-

orders) were taken at acute presentation at the same time as sera

Standard protocol approvals registrations and patientconsents Ethics approval for this study was granted by the Syd-

ney Childrenrsquos Hospitals Network Human Ethics Committee

(12SCHN395 SSA12SCHN398 08CHW108 09

CHW56 SSA09CHW143) and written informed consent

was obtained from patients

Cloning and expression of human MOG Human full-length

MOG cDNA were cloned from a fetal brain RNA library (gift from

Dr Monkol Lek) Sequence-verified MOG cDNA was subcloned

into pIRES2-ZsGreen 1 lentivirus vector enabling both MOG and

ZSGreen to be coexpressed in cells separately (gift from

Dr Stuart Turville) We used published protocols to transduce

and obtain MOG-expressing human embryonic kidney 293 cells

(HEK293MOG1) and oligodendroglial (MO313MOG1) cells19

Control cells (HEK293Ctl and MO313Ctl cells) were obtained by

transduction by particles with empty pIRES2-ZSGreen vectors We

used HEK293MOG1 cells for the MOG antibody assay to conform to

recent reports and MO313MOG1 cells for functional studies due to

their oligodendrocyte characteristics Transduction in both cell lines

resulted in 75ndash85 MOG-expressing cells in culture

Cell-based assay for detection of antibodies to cell surfaceMOG in serum and CSF We used fluorescence-activated cell

sorting (FACS) analysis to detect antibody binding of patient serum

IgG to surface MOG transduced in HEK293 cells as we have

previously described (appendix e-1 at Neurologyorgnn)1020ndash23

Samples were considered positive if they were above threshold at

least 2 times out of 3 repeated experiments and the intra-assay

variation is summarized in appendix e-1

ELISA assay for detection of antibodies to aquaporin-4antibody in serum Serum samples were tested with an

aquaporin-4 (AQP4) autoantibody ELISA kit according to the

manufacturerrsquos instructions (RSR Limited Pentwyn Cardiff

United Kingdom)

Functional effects on human oligodendroglial cells MO313

cell line is an immortal human-human hybrid cell line that expresses

phenotypic characteristics of primary oligodendrocytes and was

created by fusing a 6-thioguanine-resistant mutant of the human

rhabdomyosarcoma RD (cancer of skeletal muscle) with adult

human oligodendrocytes by a lectin-enhanced polyethylene glycol

procedure24 Transduced human oligodendroglial MO313MOG1

and MO313Ctl cells were cultured as previously described24 and

were immunostained with described protocols (appendix e-1)2223

We purified human IgG or human MOG IgG from human sera

using protein G-agarose and Microcon (Millipore Billerica MA)

and human MOG bound to an activated N-hydroxysuccinimide

agarose column (GE Healthcare Little Chalfont United

Kingdom)2526 We also immunoabsorbed MOG antibodyndashpositive

sera by incubating 6 wells of live HEK293MOG1Ctl cells with MOG

antibodyndashpositive and ndashnegative sera22 In order to visualize effects of

protein Gndashpurified human IgGs on single cells MO313 cells were

seeded at low density and incubated with 6 mg of protein Gndashpurified

human IgGs from patient or control sera and HEK293MOG1-

immunoabsorbed MOG antibodyndashpositive serum for 45 minutes

at room temperature followed by goat anti-human IgG secondary

antibody for 15minutes at room temperature2728 After washing cells

were fixed and permeabilized and cytoskeleton filamentous actin

(F-actin marker of cytoskeleton thin filaments) and b-tubulin

2 Neurology Neuroimmunology amp Neuroinflammation


(marker of cytoskeleton microtubule) were quantified using 3D

deconvolution microscopy (detailed methodology in appendix e-1)29

Human leukocyte antigen DRB11501 genotyping DNA

from patients with DEM (n 5 40) was extracted from saliva using

a saliva-specific DNA extraction kit according to the manufacturerrsquos

instructions (Oragene DNA Genotek Kanata Ontario Canada)

The rs9271366 single nucleotide polymorphism (SNP) that lies on

chromosome 6 at position 32694832 upstream of the allelic variation

(A G) was used to genotype the patients for human leukocyte

antigen (HLA)-DRB1150130 Patients with the allelic A are negative

for HLA-DRB11501 whereas patients with the allelic G are HLA-

DRB11501ndashpositive The SNP rs9271366 is always inherited with

the HLA-DRB11501 allele (complete linkage disequilibrium with

HLA-DRB11501)30 DNA was amplified using PCR (detailed

methodology in appendix e-1) and sequences were analyzed by the

software Sequencher (Gene Codes Corporation Ann Arbor MI)

Statistical analysis Chi-square with Yates correction test was usedto compare MOG antibody positivity between patient and control

groups and between patient subgroups and to compare binary clin-

ical and radiologic features between MOG antibodyndashpositive and ndash

negative groups Wilcoxon 2-sample test was used to compare ages

of patients in 2 groups No adjustment for multiple testing has been

performed The effects of IgG on F-actin and b-tubulin were

normally distributed thus means and SDs were analyzed with

the 2-tailed Student t test A p value less than 005 was

considered significant for the 2-tailed Student t test (MOG

antibody pathogenic effect)

RESULTS Surface MOG IgG antibody in children with

demyelinating diseases In order to align our detectionmethod to recent reports we first detected MOGantibody in serum using FACS assay andHEK cells ex-pressing human MOG1114163132 The mean fluores-cence intensity correlated with antibody concentration(figure 1A) Using the mean plus 3 SDs to establish thethreshold for positivity MOG antibodies were foundin 3173 (42) of the DEM group but in 024 con-trols (figure 1 BndashD p 5 0000) We have tested 57other medical and neurologic controls and all werenegative We also tested MOG antibody in CSF (fig-ure 1E) We had CSF available in 22 patients withDEM of which 5 were positive for MOG antibodyin serum Only 322 CSF samples were positive 2 ofwhich were serum positive and 1 of which was serumnegative (figure 1E) Serologic testing for AQP4 anti-bodies was performed in 64 patients with DEM andwas negative in all (data not shown)

Clinical and investigation findings according to serum

MOG antibody findings First episode We compared theclinical features according to MOG antibody statusduring the first episode of demyelination in detailand findings are presented in table e-1 Patients withpositive MOG antibody were younger (median age67 vs 104 years p5 006) and marginally more likelyto be female (1831 58 vs 1942 45 p 5 040)MOG antibody positivity was negatively associatedwith brainstem signs (231 vs 1642 42 p 5 0005)In patients with ON positive MOG antibody was

more common in patients with bilateral compared tounilateral ON (910 vs 15 respectively p 5 003 (fig-ure 1F) MOG antibodyndashpositive patients were morelikely to have a raised erythrocyte sedimentation rate(ESR) 20 mmh during the first episode than MOGantibodyndashnegative patients (919 vs 321 p 5 005)There was no correlation between age and elevatedESR in MOG antibodyndashpositive patients (figure 1G)Intrathecal oligoclonal bands were not present in theMOG antibodyndashpositive patients and were seen onlyin MOG antibodyndashnegative patients (016 vs 527p 5 018) MOG antibodyndashpositive patients were lesslikely to be homozygous or heterozygous for HLA-DRB11501 than patients with negative MOG anti-body (318 17 vs 722 32 p 5 046) Detailedblinded radiologic analysis of MRI brain (n 5 70) andMRI spine (n5 30) during the first episode showed nosignificant differences according to MOG antibody sta-tus (table e-2) There was no difference in MRI MScriteria usingMcDonald KIDMUS Callen and Verhey(table e-3)

Follow-up diagnosis To compare MOG antibodyfindings we compared the subgroups according to clas-sification at follow-up (figure 1 H and I) In the mon-ophasic subgroups MOG antibodies were positive in1124 (46) patients with ADEM 67 (86) patientswith ON 413 (31) patients with TM and 010(0) patients with ldquoother CISrdquo (defined in Methods)Patients with monophasic ON were more likely to beMOG antibodyndashpositive than the rest of the cohort(67 vs 2566 p 5 004) and patients with ldquootherCISrdquo were less likely to be MOG antibodyndashpositivethan the rest of the cohort (010 vs 3163 p 5

001) In the relapsing patients MOG antibody waspositive in 715 (47) of patients with MS and 34(75) patients with relapsing ON It is interesting thatthe 3 MOG antibodyndashpositive patients with relapsingON were negative for AQP4 antibody whereas theMOG antibodyndashnegative patient with relapsing ONwas not tested for AQP4 antibody due to insufficientserum In the relapsing patients (n 5 19) the MOGantibodyndashpositive patients were younger (median 56years range 26ndash142) than the ndashnegative patients(median 129 years range 25ndash153 p 5 003) TheMOG antibodyndashpositive patients had fewer relapses(22 relapses in 603 patient-years median annualizedrelapse rate 056) than the MOG antibodyndashnegativepatients (26 relapses in 39 patient-years median annu-alized relapse rate 065)

Longitudinal MOG antibody in 2 patients treated with

immune suppression Two recent patients (figure 2 andappendix e-2) with positive MOG antibodies who ful-filled 2013 criteria for MS18 were treated with myco-phenolate mofetil (MMF) which produced clinicalremission no new lesions on follow-up MRI scans

Neurology Neuroimmunology amp Neuroinflammation 3


Figure 1 Distribution of MOG IgG antibody in pediatric demyelinating diseases

(A) Antibody reactivity to myelin oligodendrocyte glycoprotein (MOG) was determined by flow cytometry live cell-based assay (B) Representative example offlow cytometry histograms for oneMOG antibodyndashpositive patient with a very high DMFI and (C) an intermediate DMFI MFI values are noted in the legend (D)Human surface MOG IgG antibody was detected in 3173 sera from patients with demyelinating diseases (DEM) and 024 controls (CTL) Magenta line ongraph represents the positivity threshold MOG antibody positivity is shown between brackets (E) Surface MOG antibody was detected in 322 CSF frompatients with DEM and 020CSF from patients with other neurologic diseases (CTL) Black circles represent patients with positiveMOG antibody in CSF andin serum (F) Distribution and number of MOG antibodyndashpositive patients in optic neuritis (ON) (G) Correlation between erythrocyte sedimentation rate andage in MOG antibodyndashpositive patients (H) Distribution and (I) number of MOG antibodyndashpositive patients in demyelinating diseases at follow-up Ab 5

antibody ADEM5 acute disseminated encephalomyelitis CIS5 clinically isolated syndrome BON5 bilateral optic neuritis HEK5 human embryonic kidneyIg 5 immunoglobulin MFI 5 mean fluorescence intensity MS 5 multiple sclerosis TM 5 transverse myelitis UON 5 unilateral optic neuritis



and normalization of MOG antibodies (figure 2 andappendix e-2) In both patients upon treatment withMMF MOG antibody decreased below the thresholdof positivity (figure 2 A and B)

Loss of cytoskeleton organization by MOG antibody or

purified IgG from children with demyelinating diseases

Human oligodendrocyte MO313MOG1 cells expressedMOG at their surface whereas noMOG expression wasobserved on MO313Ctl cells (figure 3) MO313 cellsalso expressed oligodendrocyte markers such as 29 39-cyclic nucleotide 39-phosphodiesterase (CNPase) galac-tocerebroside (GalC) oligodendrocyte marker O4vimentin and c-series ganglioside-specific antigen(A2B5) (figure 3A) Myelin basic protein (MBP) aspecific marker of mature oligodendrocytes wasobserved only in PMA-differentiated MO313MOG1

cells (figure 3A) suggesting that undifferentiatedMO313 cells are immature oligodendrocytes Using

purified IgG and immunoaffinity-purified MOG IgGfromMOG antibodyndashpositive sera we immunolabeledHEK293MOG1 and HEK293Ctl cells on live cells byFACS (figure 3B) and on fixed MO313MOG1 cellsby immunocytochemistry (figure 3C) and showeda positive immunostaining compared to MOGantibodyndashnegative sera suggesting that proteinGndashpurified IgG includes MOG-specific IgG Due tosmall volumes of pediatric sera we used proteinGndashpurified IgG in pathogenic experiments Next wetreated fixed and live MO313MOG1Ctl cells with IgGfrom MOG antibodyndashpositive and ndashnegative patientswith DEM and healthy controls (HCs) Then all cellswere immunolabeled for b-tubulin (marker ofmicrotubule) or F-actin (marker of thin filaments) Wequantified results and expressed them by the F-actin andb-tubulin relative enrichments in the cytoplasm andperinuclear region over the entire cell All results were

Figure 2 Temporal distribution of MOG antibody in serum of 2 relapsing patients with demyelinating diseases

In both patient A (A) and patient B (B) upon treatment mycophenolate mofetil (MMF) myelin oligodendrocyte glycoprotein (MOG) antibody decreased towithin the healthy control range (below threshold of positivity) and these low titers were associated with remission Representative dot plot out of 3 experi-ments is shown Magenta lines on graphs represent the positivity threshold (obtained with 24 control samples) Black squares represent serum analysis dur-ing acute demyelination episodes and black circles represent sera during remission Type of demyelinating episode is shown on graph (C D) RepresentativeT2 axial MRI scans demonstrate demyelinating lesions during the first acute event and during convalescence Patient A (C) had globular deep white matterlesions on acute scan (left panel) which show residual gliosis on convalescent scan and no new lesions (right panel) Patient B (D) had inflammatory lesions inbasal ganglia and white matter on acute scan (left panel) with complete resolution on convalescent scan and no new lesions (right panel) Ab 5 antibodyADEM5 acute disseminated encephalomyelitis CEREB5 cerebellar episode Ig5 immunoglobulin MFI5mean fluorescence intensity ON5 optic neuritisTM 5 transverse myelitis



normalized using F-actin and b-tubulin in HCIgG-treated fixed cells Organization of themicrotubule and thin filament networks wassimilar in fixed cells incubated with HC (1006 101 and 100 6 226 respectively figure 4A and C) or DEM IgGs (1043 6 113 and858 6 318 respectively figure 4 A and C)

there was clear visualization of ldquostress-fibersrdquo andfilopedia after F-actin immunolabeling (figure 4Aupper panels) and bright mesh-like staining spreadout through the entire cell after b-tubulinimmunolabeling (figure 4A lower panels) Livecells treated by HC IgG displayed a smalldisorganization of F-actin thin filaments (916 144

Figure 3 Human oligodendroglial MO313MOG1 cells express markers of oligodendrocytes and are immunolabeled with protein G- and humanMOG-purified human IgG from MOG antibodyndashpositive DEM patients

(A) Immunocytochemistry on fixed permeabilized cells showed that MO313MOG1 cells expressed oligodendrocyte markers (B) Protein G- and human MOG-immunopurified IgG from MOG antibodyndashpositive serum immunolabeled live HEK293MOG1 cells but did not immunolabel HEK293Ctl cells compared to MOGantibodyndashnegative serum Binding to cells was determined by flow cytometry Mean fluorescence intensity (MFI) values are shown in legends (C) ProteinG- and human MOG-purified IgG from MOG antibodyndashpositive serum also immunolabeled fixed unpermeabilized MO313MOG1 cells compared to MOGantibodyndashnegative serum Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy) Nuclei stainedwith 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm A2B55 c-series ganglioside-specific antigen A2B5 Ab5 antibody CNPase529 39-cyclic nucleotide39-phosphodiesterase DEM 5 demyelinating diseases GalC 5 galactocerebroside HEK 5 human embryonic kidney Ig 5 immunoglobulin MBP 5 myelinbasic protein MOG 5 myelin oligodendrocyte glycoprotein O4 5 oligodendrocyte marker O4



Figure 4 Purified IgG from DEM patients induces loss of cytoskeleton organization in live human oligodendroglial MO313MOG1 cells

F-actin (upper images) and b-tubulin (lower images) immunolabelings in human fixed (A) or live (B) oligodendroglial MO313MOG1 cells incubated with purifiedhealthy control (HC) IgG or MOG antibodyndashpositive DEM IgG Representative data are shown (volume projection of entire Z-stack acquired using deconvo-lution microscopy) Nuclei stained with 4rsquo6-diamidino-2-phenylindole (DAPI) Bar 10 mm Dotted lines on right images represent contour of cells as deter-mined by differential interference contrast images (not shown) (C) Quantification of change in distribution of thin filament (F-actin upper scatter plot) andmicrotubule network (b-tubulin lower scatter plot) organization using 3D deconvolution microscopy F-actin and b-tubulin were majorly affected in live cellsincubated with DEM IgG Forty different cells (1 cell5 1 diamond) from 2 HC and 2 DEM patients out of 3 independent experiments are shown Results areexpressed as ratio of the relative enrichment (RE) between cytoplasm and nucleus and values shown are percentages relative to HC IgG FIX (100) Redbars represent mean Note that there also was a loss of organization of the thin filaments after HC IgG incubation on live cells compared to fixed conditionsbut to a lesser extent than after DEM IgG (D) Cell viability assay by flow cytometry on live human oligodendroglial MO313MOG1 cells incubated 45 minuteswith 6 mg of purified HC IgG or DEM IgG No difference in cell death was observed Dead cell percentages are noted in the legend 7AAD 5 7-amino-actinomycin D DEM 5 demyelinating diseases Ig 5 immunoglobulin MOG 5 myelin oligodendrocyte glycoprotein



p 00001 figure 4 B and C upper images)and undisturbed microtubular b-tubulin network(1074 6 321 figure 4 B and C) similar to thatobserved in fixed DEM and HC IgG-treated cells(figure 4A upper panels) Following treatment withDEM IgG on live MO313MOG1 cells we observed astriking loss of organization in both F-actin (7246 117 p 00001 figure 4 B and C) andb-tubulin networks (522 6 131 p 00001figure 4 B and C) in otherwise healthy-lookingundividing cells visualized by differential interferencecontrast imaging (figure 4B dotted line and data notshown) and analyzed by FACS by which there was nodifference in viability between live MO313MOG1 livecells treated with DEM IgG or HC IgG for45 minutes (figure 4D 8726 06 vs 8726 211) or10 hours (data not shown) No loss of cytoskeletonorganization was observed when live MO313Ctl cellswere incubated with HC IgG and MOG antibodyndashpositive DEM IgG nor when live MO313MOG1 cellswere incubated with MOG antibodyndashnegative DEMIgG (figure e-1 and appendix e-3) In additionsuccessful immunoabsorption of MOG antibodyndashpositive serum on HEK293MOG1 cells led to nochange in cytoskeleton organization whenimmunoabsorbed serum was incubated on liveMO313MOG1 cells (figure e-1 and appendix e-3)Overall after incubation with MOG antibodyndashpositive DEM IgG both F-actin and b-tubulinimmunolabelings appeared to be enriched withina perinuclear region in the center of the cellssurrounding the nucleus

DISCUSSION As in this study recent reports haveconsistently found that serum MOG antibody is pre-sent in a significant proportion of children with CNSdemyelinating disease whereas AQP4 antibodies arerare9 We believe that the clinical and investigationfindings in this report support the evolving conceptthat MOG antibodies may define a separate autoim-mune demyelinating syndrome that may be distinctfrom classic adult-onset MS MOG antibodies arepresent in up to 50 of children with ADEM as inthis report Proebstel et al33 found that ADEMMOGantibody seropositive children became seronegative inthe recovery period whereas children with relapsingMS typically remained seropositive More recentlyMOG antibodies have been found in children withON and in some patients with NMO phenotypenegative for NMO IgG1431 In this report we havestrengthened this association with ON and found thatpatients with bilateral rather than unilateral ON weremore likely to be MOG antibodyndashpositive BilateralON is a common ON phenotype in children but israre in adults in whom unilateral ON is the moretypical demyelinating event of MS In contrast

patients with brainstem signs and CIS other thanON and TM were rarely MOG antibodyndashpositiveAn elevated ESR was more common in MOGantibodyndashpositive patients An elevated ESR isatypical of MS and should raise suspicion of anautoimmune disorder or alternate inflammatorymimic of MS We showed that elevation of ESR inMOG antibodyndashpositive patients was independent ofage Intrathecal oligoclonal bands are one of the classicbiomarkers of MS and were absent in all MOGantibodyndashpositive patients and were present only inthe MOG antibodyndashnegative patients Only 17 ofthe MOG antibodyndashpositive patients had 1 or bothHLA-DRB11501 alleles the main MS susceptibilityallele which is the same as the previously reportedpercentage in the ldquonormalrdquo healthy population inAustralia34 whereas the MOG antibodyndashnegativepatients were more likely to have 1 or more HLA-DRB11501 allele This negative association wasnot statistically significant and larger cohorts arerequired to test whether this correlation is a realrather than a suspected phenomenon In contrastradiologic features did not appear to differentiateMOG antibodyndashpositive from ndashnegative patients inthis childhood cohort

The presence of MOG antibodies is unlikely tohave therapeutic implications in the first demyelinat-ing event but is more likely to be therapeuticallyimportant in the relapsing patient Although 2 recentpatients fulfilled criteria for MS the presence ofMOG antibodies and elevated ESR led us to treatthem more like NMO patients with the immune sup-pression agent MMF rather than conventional MSdisease-modifying therapies Both patients had noclinical or radiologic relapse for 12 months and 36months respectively and both became MOG anti-body seronegative Further studies are required tounderstand the longitudinal course of patients withMOG antibodyndashassociated relapsing disease and thetreatment approach required

Cell-based assays have been essential to detectMOG antibody with a high specificity9 especiallywhen the full-length MOG is expressed in cells ratherthan the C-terminal truncated MOG35 Our experi-ence with neuronal antigens informed us that FACSis sensitive and quantitative10132022333637 We detectedCSF MOG antibody in only 3 of 22 DEM samples ofwhich 5 were positive in serum This suggests CSF isless sensitive than serum for MOG antibody detectionwhich could be due to a lack of sensitivity or couldrepresent a genuine absence of intrathecal Ig synthesisin these patients Supporting this concept none of thepatients with positive MOG antibody had intrathecaloligoclonal bands

The pathogenic function of MOG antibody is stillunclear but over the recent years the detection of



MOG antibody against the conformational nativeform of the antigen has enabled pathogenic studiesusing native MOG antibody from patient seraMOG antibodies have been shown to be of theIgG1 isotype which can activate complement1316

and cell-mediated cytotoxicity10 and induce experi-mental autoimmune encephalomyelitis in mice1538

Although the function of MOG is still unclearMOG has been proposed to stabilize cytoskeletal mi-crotubules4 The monoclonal MOG antibody 818C5has been shown to induce a rapid b-tubulin dephos-phorylation and invoke a rapid retraction of oligoden-drocyte processes2728 and to alter the cytoskeletalstructure and microtubular polymerization afterlong-term MOG antibody exposure in culturedmurine oligodendrocytes39 This effect on b-tubulinoccurred after MOG redistributed over internal MBPdomains independently of F-actin In our cell modelMBP is only expressed after oligodendrocyte differ-entiation with phorbol ester and F-actin was clearlydisorganized after incubation with patient Igs sug-gesting that there is an MBP-independent microtu-bular disorganization that would also involve F-actinin our immature oligodendrocyte cell model We alsoobserved a fast effect after short incubation times thatmay be due to high expression of surface MOG intransduced cells Despite the significant alteration incytoskeleton no cell death occurred as previouslyobserved in animal models Our findings add to thepathogenic potential of MOG antibody and it isconceivable that different pathogenic effects may bepresent in different patients Recent studies haveshown that different patients have different epitopesinvolved in antibody binding that may influencepathogenic properties of MOG antibody40

Taken together our findings suggest thatMOG anti-body may define an autoimmune demyelinating syn-drome that is clinically and potentially pathologicallyseparate from ldquoclassic MSrdquo MOG antibody fulfills cri-teria as a cell surface autoantibody-associated syndromeand may prove to be a valuable biomarker with thera-peutic implications in patients with relapsing demyelin-ating disorders

AUTHOR CONTRIBUTIONSStudy design and conceptualization RCD and FB Drafting of manuscript

RCD and FB Acquisition analysis and interpretation of results RCD

EMT VM R-YAK NS SR KP LAW DRB K Prelog

DRC GJG CKL EKM FB Statistical analysis RCD EMT

FB Critical revisions of manuscript RCD EMT VM NS SR

KP DRB LAW K Prelog DRC GJG CKL EKM FB

ACKNOWLEDGMENTThe authors thank all the patients and family members who provided

samples for the study The authors thank Dr Maggie Wang for use of

the Flow Cytometry Core Facility of the Westmead Millennium Institute

(Australia) The authors thank Dr Hong Yu for use of the Imaging Core

Facility at the Westmead Research Hub

STUDY FUNDINGSupported by the Star Scientific Foundation (Australia) Trish Multiple

Sclerosis Research Foundation Multiple Sclerosis Research Australia

MS Angels Melbourne (Australia) and the Petre Foundation (Australia)

DISCLOSURERC Dale has received research funding from the Star Scientific Foundation

the Trish Multiple Sclerosis Research Foundation and Multiple Sclerosis

Research Australia NHMRC and the Petre Foundation has received hon-

oraria from Biogen Idec and is on advisory boards for Queensland Child-

renrsquos Medical Institute Research and MSARD EM Tantsis has received

a scholarship from the Australian Postgraduate Award program V Merheb

and R-YA Kumaran report no disclosures N Sinmaz has received a schol-

arship from the Australian Postgraduate Award program K Pathmanandavel

has received a scholarship from the Petre Foundation (Australia)

S Ramanathan has received a scholarship from the National Health and

Medical Research Council (Australia) DR Booth has received funding from

the National Health and Medical Research Council (Australia) the Australian

Research Council Multiple Sclerosis Research Australia and the Leukemia

Foundation has received honoraria and funding from Biogen Idec Merck

Serono Sanofi-Aventis Genzyme and Pfizer and is an editor for PLOS

ONE LA Wienholt K Prelog and DR Clark report no disclosures

GJ Guillemin is the editor-in-chief for the International Journal for Trypto-

phan Research and has received research support from the Australian Research

Council CK Lim has received research funding from Multiple Sclerosis

Research Australia and Trish MS Research Foundation EK Mathey has

received funding from the National Health and Medical Research Council

(Australia) and the Rebecca L Cooper Medical Research Foundation

(Australia) F Brilot has received research funding from the Star Scientific

Foundation the Trish Multiple Sclerosis Research Foundation Multiple

Sclerosis Research Australia MS Angels Melbourne (Australia) and the Petre

Foundation and is an associate editor for the Journal of Visualized Experiments

Go to Neurologyorgnn for full disclosures

Received February 25 2014 Accepted in final form April 16 2014

REFERENCES1 Papadopoulos MC Verkman AS Aquaporin 4 and neu-

romyelitis optica Lancet Neurol 201211535ndash544

2 Vincent A Bien CG Irani SR Waters P Autoantibodies

associated with diseases of the CNS new developments

and future challenges Lancet Neurol 201110759ndash772

3 Wingerchuk DM Lennon VA Lucchinetti CF

Pittock SJ Weinshenker BG The spectrum of neuromy-

elitis optica Lancet Neurol 20076805ndash815

4 Johns TG Bernard CC The structure and function of

myelin oligodendrocyte glycoprotein J Neurochem

1999721ndash9

5 Linington C Bradl M Lassmann H Brunner C Vass K

Augmentation of demyelination in rat acute allergic

encephalomyelitis by circulating mouse monoclonal anti-

bodies directed against a myelinoligodendrocyte glycopro-

tein Am J Pathol 1988130443ndash454

6 Pollinger B Krishnamoorthy G Berer K et al Spontaneous

relapsing-remitting EAE in the SJLJ mouse MOG-reactive

transgenic T cells recruit endogenous MOG-specific B cells

J Exp Med 20092061303ndash1316

7 Schluesener HJ Sobel RA Linington C Weiner HL A

monoclonal antibody against a myelin oligodendrocyte

glycoprotein induces relapses and demyelination in central

nervous system autoimmune disease J Immunol 1987

1394016ndash4021

8 von Budingen HC Hauser SL Fuhrmann A Nabavi CB

Lee JI Genain CP Molecular characterization of antibody

specificities against myelinoligodendrocyte glycoprotein

in autoimmune demyelination Proc Natl Acad Sci U S A

2002998207ndash8212



9 Reindl M Di Pauli F Rostasy K Berger T The spectrum

of MOG autoantibody-associated demyelinating diseases

Nat Rev Neurol 20139455ndash461

10 Brilot F Dale RC Selter RC et al Antibodies to native

myelin oligodendrocyte glycoprotein in children with

inflammatory demyelinating central nervous system dis-

ease Ann Neurol 200966833ndash842

11 Di Pauli F Mader S Rostasy K et al Temporal dynamics

of anti-MOG antibodies in CNS demyelinating diseases

Clin Immunol 2011138247ndash254

12 Lalive PH Hausler MG Maurey H et al Highly reactive

anti-myelin oligodendrocyte glycoprotein antibodies dif-

ferentiate demyelinating diseases from viral encephalitis

in children Mult Scler 201117297ndash302

13 McLaughlin KA Chitnis T Newcombe J et al Age-

dependent B cell autoimmunity to a myelin surface anti-

gen in pediatric multiple sclerosis J Immunol 2009183

4067ndash4076

14 Rostasy K Mader S Schanda K et al Anti-myelin oligo-

dendrocyte glycoprotein antibodies in pediatric patients

with optic neuritis Arch Neurol 201269752ndash756

15 Zhou D Srivastava R Nessler S et al Identification of a

pathogenic antibody response to native myelin oligoden-

drocyte glycoprotein in multiple sclerosis Proc Natl Acad

Sci U S A 200610319057ndash19062

16 Mader S Gredler V Schanda K et al Complement activating

antibodies to myelin oligodendrocyte glycoprotein in neuro-

myelitis optica and related disorders J Neuroinflammation

20118184

17 Tantsis EM Prelog K Brilot F Dale RC Risk of multiple

sclerosis after a first demyelinating syndrome in an

Australian Paediatric cohort clinical radiological features

and application of the McDonald 2010 MRI criteria Mult

Scler 2013191749ndash1759

18 Krupp LB Tardieu M Amato MP et al International

Pediatric Multiple Sclerosis Study Group criteria for pedi-

atric multiple sclerosis and immune-mediated central ner-

vous system demyelinating disorders revisions to the 2007

definitions Mult Scler 2013191261ndash1267

19 Aggarwal A Iemma TL Shih I et al Mobilization of HIV

spread by diaphanous 2 dependent filopodia in infected

dendritic cells PLoS Pathog 20128e1002762

20 Amatoury M Merheb V Langer J Wang XM Dale RC

Brilot F High-throughput flow cytometry cell-based assay

to detect antibodies to N-methyl-D-aspartate receptor or

dopamine-2 receptor in human serum J Vis Exp 2013

81e50935

21 Brilot F Merheb V Ding A Murphy T Dale RC Anti-

body binding to neuronal surface in Sydenham chorea but

not in PANDAS or Tourette syndrome Neurology 2011

761508ndash1513

22 Dale RC Merheb V Pillai S et al Antibodies to surface

dopamine-2 receptor in autoimmune movement and psy-

chiatric disorders Brain 20121353453ndash3468

23 Mohammad SS Sinclair K Pillai S et al Herpes simplex

encephalitis relapse with chorea is associated with autoanti-

bodies to N-Methyl-D-aspartate receptor or dopamine-2

receptor Mov Disord 201429117ndash122

24 Buntinx M Vanderlocht J Hellings N et al Characteri-

zation of three human oligodendroglial cell lines as a

model to study oligodendrocyte injury morphology and

oligodendrocyte-specific gene expression J Neurocytol

20033225ndash38

25 Elliott C Lindner M Arthur A et al Functional identi-

fication of pathogenic autoantibody responses in patients

with multiple sclerosis Brain 20121351819ndash1833

26 Mathey EK Derfuss T Storch MK et al Neurofascin as a

novel target for autoantibody-mediated axonal injury


27 Marta CB Montano MB Taylor CM Taylor AL

Bansal R Pfeiffer SE Signaling cascades activated upon

antibody cross-linking of myelin oligodendrocyte glyco-

protein potential implications for multiple sclerosis

J Biol Chem 20052808985ndash8993

28 Marta CB Taylor CM Coetzee T et al Antibody cross-

linking of myelin oligodendrocyte glycoprotein leads to its

rapid repartitioning into detergent-insoluble fractions and

altered protein phosphorylation and cell morphology

J Neurosci 2003235461ndash5471

29 Brilot F Strowig T Roberts SM Arrey F Munz C NK

cell survival mediated through the regulatory synapse with

human DCs requires IL-15Ralpha J Clin Invest 2007

1173316ndash3329

30 Australia and New Zealand Multiple Sclerosis Genetics

Consortium (ANZgene) Genome-wide association study

identifies new multiple sclerosis susceptibility loci on chro-

mosomes 12 and 20 Nat Genet 200941824ndash830

31 Kitley J Woodhall MWaters P et al Myelin-oligodendrocyte

glycoprotein antibodies in adults with a neuromyelitis optica

phenotype Neurology 2012791273ndash1277

32 Rostasy K Mader S Hennes EM et al Persisting myelin

oligodendrocyte glycoprotein antibodies in aquaporin-4

antibody negative pediatric neuromyelitis optica Mult


33 Probstel AK Dornmair K Bittner R et al Antibodies to

MOG are transient in childhood acute disseminated

encephalomyelitis Neurology 201177580ndash588

34 Stewart GJ Teutsch SM Castle M Heard RN

Bennetts BH HLA-DR -DQA1 and -DQB1 associations

in Australian multiple sclerosis patients Eur J Immunogenet

19972481ndash92

35 Waters P Woodhall M Hacohen Y et al Antibodies to

myelin oligodendrocyte glycoprotein (MOG) in children

and adults with demyelinating disorders (NMO) Ann

Neurol 201374S18ndashS19

36 OrsquoConnor KC McLaughlin KA De Jager PL et al Self-

antigen tetramers discriminate between myelin autoantibodies

to native or denatured protein Nat Med 200713211ndash217

37 Waters PJ McKeon A Leite MI et al Serologic diagnosis

of NMO a multicenter comparison of aquaporin-4-IgG

assays Neurology 201278665ndash671

38 Lalive PH Menge T Delarasse C et al Antibodies to

native myelin oligodendrocyte glycoprotein are serologic

markers of early inflammation in multiple sclerosis Proc

Natl Acad Sci U S A 20061032280ndash2285

39 Dyer CA Matthieu JM Antibodies to myelinoligodendrocyte-

specific protein and myelinoligodendrocyte glycoprotein

signal distinct changes in the organization of cultured

oligodendroglial membrane sheets J Neurochem 1994

62777ndash787

40 Mayer MC Breithaupt C Reindl M et al Distinction

and temporal stability of conformational epitopes on mye-

lin oligodendrocyte glycoprotein recognized by patients

with different inflammatory central nervous system dis-

eases J Immunol 20131913594ndash3604



DOI 101212NXI000000000000001220141 Neurol Neuroimmunol Neuroinflamm

Russell C Dale Esther M Tantsis Vera Merheb et al cytoskeleton

Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte

This information is current as of May 22 2014

2014 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

ServicesUpdated Information amp

httpnnneurologyorgcontent11e12fullhtmlincluding high resolution figures can be found at

Supplementary Material httpnnneurologyorgcontentsuppl2014052211e12DC1

Supplementary material can be found at

References httpnnneurologyorgcontent11e12fullhtmlref-list-1

This article cites 40 articles 10 of which you can access for free at

Citations httpnnneurologyorgcontent11e12fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

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httpnnneurologyorgcgicollectionoptic_neuritisOptic neuritis see Neuro-ophthalmologyOptic Nerve

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of microtubule stability4 Autoantibodiesagainst MOG (MOG antibodies) have beenshown to mediate demyelination in rodentsin ldquo2-hit modelsrdquo and also in primates5ndash8

The importance of MOG antibodies inhuman demyelinating disease has previouslybeen controversial predominantly due to theuse of antibody assays that denature proteinand alter conformation More recently usingcell-based assays high titer MOG antibody hasbeen unequivocally found in 20ndash40 of chil-dren with acute CNS demyelination9 In partic-ular MOG antibodies have been shown to beassociated with acute disseminated encephalomy-elitis (ADEM) and patients with neuromyelitisoptica (NMO)-like phenotypes who are negativefor NMO immunoglobulin (Ig) G10ndash16 How-ever detailed clinical and radiologic phenotypingassociated with MOG antibodies is still lackingand the role of MOG antibodies as a biomarkerin clinical practice is still not clear

Herein we further define the clinical signif-icance of MOG antibody as a biomarker andshow that MOG antibody can modify themicrotubule network and thin filaments ofoligodendrocytes

METHODS Patients and controls Patients The stored

acute serum (280degC) taken from 73 children during their first

episode of CNS demyelination (DEM) was used for this study

(median age 8 years range 13ndash153 37 females) All sera were

acute and before immune therapy The clinical and radiologic

features of 60 of the patients have been reported previously17

but the serologic investigation of this cohort has not been pre-

viously reported The patients were clinically phenotyped using

2013 consensus criteria18 The first episode of demyelination

was ADEM (n 5 28) transverse myelitis (TM n 5 15) optic

neuritis (ON n 5 15) and other clinically isolated syndrome

(CIS) excluding TM and ON (n 5 15) These other CIS

patients had polyfocal CIS cerebellar CIS brainstem CIS or

hemispheric CIS The acute MRI brain scans (n 5 70) and

MRI spine (n5 30) were reviewed and rated using MRI criteria

blinded to the laboratory findings as previously described using

McDonald KIDMUS Callen and Verhey criteria17 The

patients were followed for a median of 40 years (range 03ndash

137 years) At study census and classification 54 patients had a

monophasic disease (ADEM n 5 24 TM n 5 13 ON n 5 7

ldquoother CISrdquo n 5 10) Nineteen of 73 patients had a relapsing

demyelinating disorder (multiple sclerosis [MS] n5 15 relaps-

ing ON n 5 4) The 15 patients with MS fulfilled criteria by

Krupp et al18 and had 2 or more clinical events

Controls We have previously shown that MOG antibodies

are specific to CNS demyelination10 To generate a control

range for this study 24 pediatric controls with other neurologic

disease including epilepsy cerebral palsy neurometabolic dis-

ease and neurodegenerative disorders were used (median age

11 years range 2ndash14)

Patient and control sera had IgG concentrations measured by

nephelometry (BN ProSpec Siemens Germany) and IgG values

were within the normal range (62ndash144 gL)

CSF samples (n 5 20 controls and n 5 22 demyelinating dis-

orders) were taken at acute presentation at the same time as sera

Standard protocol approvals registrations and patientconsents Ethics approval for this study was granted by the Syd-

ney Childrenrsquos Hospitals Network Human Ethics Committee

(12SCHN395 SSA12SCHN398 08CHW108 09

CHW56 SSA09CHW143) and written informed consent

was obtained from patients

Cloning and expression of human MOG Human full-length

MOG cDNA were cloned from a fetal brain RNA library (gift from

Dr Monkol Lek) Sequence-verified MOG cDNA was subcloned

into pIRES2-ZsGreen 1 lentivirus vector enabling both MOG and

ZSGreen to be coexpressed in cells separately (gift from

Dr Stuart Turville) We used published protocols to transduce

and obtain MOG-expressing human embryonic kidney 293 cells

(HEK293MOG1) and oligodendroglial (MO313MOG1) cells19

Control cells (HEK293Ctl and MO313Ctl cells) were obtained by

transduction by particles with empty pIRES2-ZSGreen vectors We

used HEK293MOG1 cells for the MOG antibody assay to conform to

recent reports and MO313MOG1 cells for functional studies due to

their oligodendrocyte characteristics Transduction in both cell lines

resulted in 75ndash85 MOG-expressing cells in culture

Cell-based assay for detection of antibodies to cell surfaceMOG in serum and CSF We used fluorescence-activated cell

sorting (FACS) analysis to detect antibody binding of patient serum

IgG to surface MOG transduced in HEK293 cells as we have

previously described (appendix e-1 at Neurologyorgnn)1020ndash23

Samples were considered positive if they were above threshold at

least 2 times out of 3 repeated experiments and the intra-assay

variation is summarized in appendix e-1

ELISA assay for detection of antibodies to aquaporin-4antibody in serum Serum samples were tested with an

aquaporin-4 (AQP4) autoantibody ELISA kit according to the

manufacturerrsquos instructions (RSR Limited Pentwyn Cardiff

United Kingdom)

Functional effects on human oligodendroglial cells MO313

cell line is an immortal human-human hybrid cell line that expresses

phenotypic characteristics of primary oligodendrocytes and was

created by fusing a 6-thioguanine-resistant mutant of the human

rhabdomyosarcoma RD (cancer of skeletal muscle) with adult

human oligodendrocytes by a lectin-enhanced polyethylene glycol

procedure24 Transduced human oligodendroglial MO313MOG1

and MO313Ctl cells were cultured as previously described24 and

were immunostained with described protocols (appendix e-1)2223

We purified human IgG or human MOG IgG from human sera

using protein G-agarose and Microcon (Millipore Billerica MA)

and human MOG bound to an activated N-hydroxysuccinimide

agarose column (GE Healthcare Little Chalfont United

Kingdom)2526 We also immunoabsorbed MOG antibodyndashpositive

sera by incubating 6 wells of live HEK293MOG1Ctl cells with MOG

antibodyndashpositive and ndashnegative sera22 In order to visualize effects of

protein Gndashpurified human IgGs on single cells MO313 cells were

seeded at low density and incubated with 6 mg of protein Gndashpurified

human IgGs from patient or control sera and HEK293MOG1-

immunoabsorbed MOG antibodyndashpositive serum for 45 minutes

at room temperature followed by goat anti-human IgG secondary

antibody for 15minutes at room temperature2728 After washing cells

were fixed and permeabilized and cytoskeleton filamentous actin

(F-actin marker of cytoskeleton thin filaments) and b-tubulin

































































































































1999721ndash9














1394016ndash4021





2002998207ndash8212




















4067ndash4076







Sci U S A 200610319057ndash19062




20118184


















81e50935




761508ndash1513












20033225ndash38




















1173316ndash3329


















19972481ndash92



















62777ndash787





























Reprints


































































































































1999721ndash9














1394016ndash4021





2002998207ndash8212




















4067ndash4076







Sci U S A 200610319057ndash19062




20118184


















81e50935




761508ndash1513












20033225ndash38




















1173316ndash3329


















19972481ndash92



















62777ndash787





























Reprints





















4067ndash4076







Sci U S A 200610319057ndash19062




20118184


















81e50935




761508ndash1513












20033225ndash38




















1173316ndash3329


















19972481ndash92



















62777ndash787





























Reprints

























Reprints




Antibodies to MOG have ademyelination phenotype and affect oligodendrocyte cytoskeleton ·...

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