Neurology: Neuroimmunology & Neuroinflammation
Supplemental DataAppendix e-1Breakdown of diagnoses in patients with BON and/or LETM presenting to collaborating centers (2004-2014)A total of 38 patients presented with bilateral and/or recurrent optic neuritis (BON) to the participating centers from 2004-2014. Of these, eleven patients had a diagnosis of AQP4-antibody-positive NMO/NMOSD, nine had confirmed MOG-antibody-positive NMO/NMOSD, four were double seronegative and had a monophasic illness, six went on to fulfill diagnostic criteria for MS, one patient was diagnosed with ADEM, one with vasculitis, and six patients were lost to follow-up. Within this time frame, a total of 34 patients presented with LETM (2 with sequential LETM and BON, 13 with LETM and unilateral ON, and 19 with isolated LETM). Of these, seventeen patients were AQP4-antibody-positive NMO/NMOSD, one had MOG-antibody-positive NMO, eleven were double seronegative, one had a diagnosis of Japanese opticospinal MS, one was post-vaccination, and three were unclassified.Appendix e-2Cell-based assay for detection of antibodies to cell surface MOG in serum Live cells were harvested using versene (Invitrogen) and washed in phosphate buffered saline (PBS) supplemented with 2% foetal bovine serum (FBS) (PBS/FBS). 50,000 cells were then incubated with serum at a 1:50 dilution in a 96 well V-bottom plate (Corning) for 30 minutes at room temperature as previously describedPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5BbWF0b3VyeTwvQXV0aG9yPjxZZWFyPjIwMTM8L1llYXI+
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ADDIN EN.CITE.DATA 1-5. Cells were then washed three times with 200?l PBS/FBS, and incubated with Alexa Fluor 647-conjugated goat anti-human IgG secondary antibody (Life Technologies) for 30 minutes at room temperature. Cells were washed three times with PBS/FBS and then resuspended in 20?l PBS/FBS before analysis. A total of 10,000 events/well were recorded on a BD LSRII instrument equipped with a high-throughput sampler (BD Biosciences). Data analysis was performed using Flow Jo software (TreeStar, Ashland, OR, USA) and Excel. Binding was expressed as mean fluorescence intensity (MFI). Levels of antibody binding in ZsGreen-positive transfected cells were expressed as MFI. MFI was determined by the subtraction of MFI obtained with HEK293CTL cells from the MFI obtained with HEK293MOG+ cells. A MFI greater than the mean+3xstandard deviations of the control cohort was considered positive. Samples were considered positive if they were above threshold at least two times out of three repeated experiments, and the intra-assay variation is summarized below. Surface expression on transduced cells was analyzed by flow cytometry after staining with a mouse monoclonal anti-human extracellular MOG (Clone 818C5, kind gift of Prof C. Linington) in combination with an Alexa Fluor 647-conjugated appropriate secondary antibody (Life Technologies).The intra-assay variation is summarized as follows:Group3 out of 3 above mean + 3SD threshold2 out of 3 above mean + 3SD threshold1 out of 3 above mean + 3SD threshold0 out of 3 above mean + 3SD thresholdMOG-antibody-positive NMO/NMOSD (n=9)9000MOG-antibody-negative NMO/NMOSD (n=14)00311MS (n=76)10372Controls (n=52)00250Appendix e-3figure e-1: MOG-antibodies are not of the IgM isotype in adults with NMO/NMOSD. Antibody reactivity to MOG was determined by a flow cytometry live cell-based assay and none of the patients demonstrated IgM reactivity. The dotted line on the graph represents the threshold of positivity. MOG-antibody positivity is shown between brackets. A representative experiment out of three experiments is shown. AQP4 Ab = aquaporin 4 antibody; IgM = immunoglobulin M; MFI = mean fluorescence intensity; MOG = myelin oligodendrocyte glycoprotein; MOG antibody = myelin oligodendrocyte glycoprotein antibody; MS = multiple sclerosis; NMO/NMOSD = neuromyelitis optica/ neuromyelitis optica spectrum disorder.Appendix e-4Patient 7 (table 2) is a 22 year-old man who experienced five relapses since disease onset four years prior. Early treatment with injectable β-interferon reflected a provisional diagnosis of MS. Subsequent relapses led to referral to a tertiary center, and revision of diagnosis and management. At this stage his VFSS was already poor with a score of 5. His latest relapse (44 months post disease onset) resulted in a VFSS of 6, and was managed aggressively with pulsed steroids, followed by maintenance immunosuppression with oral prednisone and mycophenolate mofetil, and subsequently rituximab. Despite this, the best VFSS at last follow-up remained at 5. OCT performed at 40 months post disease onset revealed significant RNFL atrophy with measures of 57 ?m (OD) and 79 ?m (OS).Appendix e-5The comparison of visual outcomes in MOG-antibody-positive and MOG-antibody-negative patients1. Specific visual function tests performed in MOG-antibody-positive and antibody-negative patientsMOG-antibody-positive (n=9)aMOG-antibody-negative (n=4)bAcuteFollow-upAcuteFollow-upVisual acuity scores9/99/94/44/4Visual field testing7/95/94/42/4Retinal nerve fiber layer thickness OCT5/95/92/42/4a n=8 BON, n=1 sequential BON and LETMb n=3 BON, n=1 sequential BON and LETM2. Visual acuity scores in MOG-antibody-positive and -negative patients. MOG-antibody-positive (n=9)MOG-antibody-negative (n=4)AcuteFollow-upAcuteFollow-upVisual Functional System Score002021011023501300014001053120630003. Visual field deficit scores in MOG-antibody-positive and -negative patients. MOG-antibody-positive (n=10 affected eyes)cMOG-antibody-negative (n=4 affected eyes)cAcuteFollow-upAcuteFollow-upVisual Field Deficit ScoreNormal1413Mild2401Moderate4220Severe3010c n=5 MOG-antibody-positive and n=2 MOG-antibody-negative patients had both acute and follow-up visual field testing done, hence only these ten and four affected eyes respectively are included in this table.4. Retinal nerve fiber layer thickness in MOG-antibody-positive and -negative patients Retinal nerve fiber layer thickness at acute presentationdRetinal nerve fiber layer thickness at follow-updMOG-antibody-positive (n=3)ODOSODOSPatient 2146 ?m223 ?m77 ?m71 ?mPatient 4119 ?m122 ?m113 ?m111 ?mPatient 6185 ?m248 ?m119 ?m115 ?mMOG-antibody-negative (n=1)100 ?m109 ?m70 ?m71 ?md n=3 MOG-antibody-positive and n=1 MOG-antibody-negative patient had both acute and follow-up optical coherence tomography performed, so only these patients are included in this table. The three MOG-antibody-positive patients included are patients 2, 4, and 6 on table 2.References ADDIN EN.REFLIST 1.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 2013.2.Brilot F, Dale RC, Selter RC, et al. Antibodies to native myelin oligodendrocyte glycoprotein in children with inflammatory demyelinating central nervous system disease. Annals of Neurology 2009;66:833-842.3.Dale RC, Merheb V, Pillai S, et al. Antibodies to surface dopamine-2 receptor in autoimmune movement and psychiatric disorders. Brain 2012;135:3453-3468.4.Mohammad SS, Sinclair K, Pillai S, et al. Herpes simplex encephalitis relapse with chorea is associated with autoantibodies to N-Methyl-D-aspartate receptor or dopamine-2 receptor. Mov Disord 2014;29:117-122.5.Dale RC, Tantsis E, Merheb V, et al. Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte cytoskeleton. Neurol Neuroimmunol Neuroinflammation 2014;1:e12. doi 10.1212/NXI.0000000000000012. ................
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