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Autoantibody-associated demyelinating diseases of the central nervous system such as myelin oligodendrocyte glycoprotein-antibody associated disease (MOGAD) and aquaporin 4-antibody positive neuromyelitis optica spectrum disorders (AQP4+ NMOSD) are rare diseases but can cause severe disability. In both diseases, associated neuroinflammation is accompanied by blood and cerebrospinal fluid cytokine and chemokine signatures, which were shown to be distinct from those observed in patients with multiple sclerosis (MS). In this study, we aimed to confirm and extend these findings by analyzing a larger number of serum cytokines, chemokines and related molecules in patients with MOGAD or AQP4+ NMOSD in comparison to MS, to better understand the pathophysiology and to identify biomarkers potentially useful in clinical practice for diagnostic and treatment purposes. A total of 65 serum cytokines, chemokines and related molecules like growth factors and soluble receptors were measured by Procartaplex multiplex immunoassays in 40 MOGAD, 40 AQP4+ NMOSD and 54 MS patients at baseline. Furthermore, follow-up samples of 25 AQP4+ NMOSD and 40 MOGAD patients were measured after 6-12 months. Selected analytes were validated in a subgroup of samples using other bead-based assays and ELISA. At baseline, 36 analytes in MOGAD and 30 in AQP4+ NMOSD were significantly increased compared to MS. K-means cluster analysis of all significantly altered molecules revealed three distinct groups: Cluster I, including 12 MOGAD, 2 AQP4+ NMOSD and 3 MS patients, had a specific association with 11 IL-6/IL-17A associated cytokines. In this cluster, 9/17 (53%) patients were children. Cluster II with 13 MOGAD, 24 AQP4+ NMOSD and 1 MS patient was associated with 31 upregulated analytes. Cluster III contained 15 MOGAD, 14 AQP4+ NMOSD and 50 MS patients. In cluster II and III the majority were adults (82% and 92%). Most measured analytes remained stable over time. Validation of selected cytokines and chemokines using other analytical methods revealed moderate to high correlation coefficients, but absolute values differed between assays. In conclusion, these results obtained by bead-based multiplex assays highlight a significant association of biomarkers of peripheral inflammation in patients with antibody-associated demyelinating diseases in comparison with MS., Competing Interests: AB is an employee of VASCage – Research Centre on Vascular Ageing and Stroke and has participated in meetings sponsored by or received travel funding from Novartis, Sanofi Genenzyme, Merck, Almirall and Biogen. KB has participated in meetings sponsored by and received travel funding from Roche, Biogen and Teva. HH has participated in meetings sponsored by, received speaker honoraria or travel funding from Bayer, Biogen, Celgene, Merck, Novartis, Sanofi-Genzyme, Siemens and Teva, and received honoraria for consulting Biogen, Celgene, Novartis and Teva. FD has participated in meetings sponsored by or received honoraria for acting as an advisor/speaker for Alexion, Almirall, Biogen, Celgene, Janssen, Merck, Novartis, Roche and Sanofi-Genzyme. His institution received scientific grants from Biogen and Sanofi-Genzyme.TB has participated in meetings sponsored by and received honoraria lectures, advisory boards, consultations from pharmaceutical companies marketing treatments for MS: Allergan, Bayer, Biogen, Bionorica, BMS/Celgene, GSK, GW/Jazz Pharma, Horizon, Janssen-Cilag, MedDay, Merck, Novartis, Octapharma, Roche, Sandoz, Sanofi-Genzyme, Teva and UCB. His institution has received financial support in the past 12 months by unrestricted research grants Biogen, Bayer, BMS/Celgene, Merck, Novartis, Roche, Sanofi-Genzyme, Teva and for participation in clinical trials in multiple sclerosis sponsored by Alexion, Bayer, Biogen, Merck, Novartis, Octapharma, Roche, Sanofi-Genzyme, Teva. RH has received honoraria for lectures from Novartis and Biogen. KR has served as consultant for the PARADIGM Study/Novartis without payment. SM received speaker honoraria from Novartis and Biogen. MR was supported by a research support from Euroimmun and Roche. The University Hospital and Medical University of Innsbruck Austria, employer of MR receives payments for antibody assays MOG, AQP4, and other autoantibodies and for MOG and AQP4 antibody validation experiments organized by Euroimmun Lübeck, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.​ The authors declare that this study received funding from Roche Austria. The funder was involved in the study design and critical revision of the article for important intellectual content., (Copyright © 2022 Bauer, Rudzki, Berek, Dinoto, Lechner, Wendel, Hegen, Deisenhammer, Berger, Höftberger, Rostasy, Mariotto and Reindl.)

Background: Neuromyelitis optica spectrum disorders (NMOSD) are severe inflammatory demyelinating disorders of the central nervous system mainly characterized by recurrent episodes of uni- or bilateral optic neuritis (ON), transverse myelitis (TM) and brainstem syndromes (BS). The majority of adult patients has serum antibodies directed against the water channel protein aquaporin 4 (AQP4-abs). In pediatric patients, AQP4-abs are less, while antibodies against myelin oligodendrocyte glycoprotein (MOG-abs) are more frequently detectable than in adults. Some children with NMOSD have neither AQP4- nor MOG-ab (double-seronegative). Objective: Evaluation of epidemiological data regarding incidence and prevalence of pediatric NMOSD in Germany and Austria. Methods: We recruited pediatric NMOSD patients between 1 March 2017 and 28 February 2019 with five different tools: (1) ESPED (Surveillance Unit for Rare Pediatric Disorders in Germany), (2) ESNEK (Surveillance for Rare Neurological Disorders during Childhood), (3) pediatric neurology working group within the Austrian Society of Pediatrics and Adolescent Medicine, (4) BIOMARKER Study and (5) NEMOS (Neuromyelitis optica Study Group). We requested data regarding clinical symptoms, antibody status, therapy regimen and response via a standardized questionnaire. Results: During the 2-year recruitment period, 46 (both incidental and prevalent) patients with a suspected diagnosis of NMOSD were brought to our attention. Twenty-two of these patients did not fulfill the inclusion criteria. Of the remaining 24 children, 22 had a median age at onset of 11 (range 3–17) years and 16/22 were female (72.7%) (no data in two patients). Sixteen of 24 patients were AQP4-ab positive (67%), 4/24 MOG-ab positive (16.7%), three children were double-seronegative and in one patient no antibody testing was done. We calculated an incidence rate of 0.022 per 100,000 person-years for Germany, while there was no incidental case in Austria during the recruitment period. The prevalence rate was 0.147 and 0.267 per 100,000 persons in Germany and Austria, respectively. Conclusion: Pediatric NMOSD, with and without associated antibodies, are very rare even considering the different limitations of our study. An unexpected finding was that a considerable proportion of patients was tested neither for AQP4- nor MOG-abs during diagnostic work-up, which should prompt to establish and disseminate appropriate guidelines. [ABSTRACT FROM AUTHOR]

Objective: Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD).Methods: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1).Results: Results of tests on 92 controls identified 12assays as highly specific (0-1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5-100%) of all 21 assays. The specificities (85.8-100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples.Conclusions: The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology. [ABSTRACT FROM AUTHOR]

Background: The discovery of a highly specific antibody against the aquaporin-4 (AQP4) water channel (AQP4-IgG) unified the spectrum of neuromyelitis optica spectrum disorders (NMOSD), which are considered to be antibody-mediated autoimmune diseases. The AQP4 water channel is located on astrocytic end-feet processes and consists of six transmembrane helical domains forming three extracellular loops A, C, and E in which defined amino acids were already proven to be critical for AQP4-IgG binding. However, the clinical relevance of these findings is unclear. Therefore, we have characterized the epitope specificity of AQP4-IgG-positive NMOSD patients.Methods: We established a cell-based flow cytometry assay for the quantitative detection of AQP4-IgG-positive serum samples. Human embryonic kidney (HEK) cells were transiently transfected with an EmGFP-tagged AQP4-M23, AQP4-M1, or six AQP4-M23 extracellular loop mutants including two mutations in loop A (serial AA substitution, insertion of a myc-tag), two in loop C (N153Q, insertion of a myc-tag), and two in loop E (H230G, insertion of a myc-tag). Fourty-seven baseline and 49 follow-up serum samples and six paired cerebrospinal fluid (CSF) baseline samples of 47 AQP4-IgG-positive Austrian NMOSD patients were then tested for their binding capability to AQP4-M1 and AQP4-M23 isoforms and these six extracellular loop mutants.Results: Overall, we could identify two broad patterns of antibody recognition based on differential sensitivity to mutations in extracellular loop A. Pattern A was characterized by reduced binding to the two mutations in loop A, whereas pattern B had only partial or no reduced binding to these mutations. These two patterns were not associated with significant differences in demographic and clinical parameters or serum titers in this retrospective study. Interestingly, we found a change of AQP4-IgG epitope recognition pattern in seven of 20 NMOSD patients with available follow-up samples. Moreover, we found different binding patterns in five of six paired CSF versus serum samples, with a predominance of pattern A in CSF.Conclusions: Our study demonstrates that AQP4-IgG in sera of NMOSD patients show distinct patterns of antibody recognition. The clinical and diagnostic relevance of these findings have to be addressed in prospective studies. [ABSTRACT FROM AUTHOR]

In neuromyelitis optica (NMO), astrocytes become targets for pathogenic aquaporin 4 (AQP4)-specific antibodies which gain access to the central nervous system (CNS) in the course of inflammatory processes. Since these antibodies belong to a T cell-dependent subgroup of immunoglobulins, and since NMO lesions contain activated CD4 T cells, the question arose whether AQP4-specific T cells might not only provide T cell help for antibody production, but also play an important role in the induction of NMO lesions. We show here that highly pathogenic, AQP4-peptide-specific T cells exist in Lewis rats, which recognize AQP4 as their specific antigen and cause severe panencephalitis. These T cells are re-activated behind the blood-brain barrier and deeply infiltrate the CNS parenchyma of the optic nerves, the brain, and the spinal cord, while T cells with other AQP4-peptide specificities are essentially confined to the meninges. Although AQP4-specific T cells are found throughout the entire neuraxis, they have NMO-typical 'hotspots' for infiltration, i.e. periventricular and periaqueductal regions, hypothalamus, medulla, the dorsal horns of spinal cord, and the optic nerves. Most remarkably, together with NMO-IgG, they initiate large astrocyte-destructive lesions which are located predominantly in spinal cord gray matter. We conclude that the processing of AQP4 by antigen presenting cells in Lewis rats produces a highly encephalitogenic AQP4 epitope (AQP4), that T cells specific for this epitope are found in the immune repertoire of normal Lewis rats and can be readily expanded, and that AQP4-specific T cells produce NMO-like lesions in the presence of NMO-IgG. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the frequency and implications of antibodies to myelin oligodendrocyte glycoprotein (MOG-ab) and aquaporin 4 antibodies (AQP4-ab) in adult patients with suspected limited forms of neuromyelitis optica (NMO). The study is investigated by cell-based assays, Mann-Whitney U test, and Chi-square test. Results show that MOG-ab is commonly detected in the serum of patients with NMO, longitudinally extensive myelitis (LETM), and optic neuritis (ON).

The recent detection of aquaporin-4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG) autoantibodies in acquired inflammatory demyelinating diseases, such as neuromyelitis optica, or acute disseminated encephalomyelitis, and multiple sclerosis, in children strongly indicates that B-cell-dependent mechanisms contribute to the pathogenesis. This review aims to give an overview of the role of autoantibodies in inflammatory demyelinating pediatric diseases, with a focus on antibodies to AQP4 and MOG. [ABSTRACT FROM AUTHOR]

Background: A new autoantibody (termed NMO-IgG, or AQP4-Ab) has recently been described in patients with neuromyelitis optica (NMO) and its formes frustes, longitudinally extensive transverse myelitis (LETM) and recurrent optic neuritis (rON). However, AQP4-Ab has been found also in patients with co-existing rheumatic diseases such as systemic lupus erythematosus (SLE) or Sjögren’s syndrome (SS), conditions which are characterized by broad, polyspecific B cell activation.Objectives: In this study, we aimed at evaluating the syndrome specificity and frequency of AQP4-Ab in patients with rheumatic diseases and neurological symptoms.Methods: For this purpose, serum samples from 109 neurological patients with established connective tissue disorders (CTD) (n = 54), possible CTD (n = 42), or vasculitis (n = 13) were analysed for the presence of AQP4-Ab by a cell-based assay employing recombinant human AQP4.Results: AQP4-Ab was detectable in 31/40 (78%) patients with CTD and NMO spectrum disorders (median titre, 1:1000) but in none of the samples obtained from patients with CTD or vasculitis and neurological disorders other than NMO, LETM, or rON (n = 69).Conclusion: The high syndrome specificity of the antibody for neuromyelitis optica spectrum disorders (NMOSDs) in patients with CTD supports the concept of AQP4-Ab being involved in the pathogenesis of these neurological conditions, and argues against AQP4-Ab simply being part of the polyclonal B cell activation generally associated with rheumatic diseases. Moreover, the finding that AQP4-Ab is present in patients with CTD and co-existing NMOSD with approximately the same frequency as in patients without CTD strengthens the case of CTD and AQP4-Ab positive NMOSD representing two co-existing yet distinct entities in the majority of patients. [ABSTRACT FROM AUTHOR]

Inflammatory lesions in the central nervous system of patients with neuromyelitis optica are characterized by infiltration of T cells and deposition of aquaporin-4-specific antibodies and complement on astrocytes at the glia limitans. Although the contribution of aquaporin-4-specific autoantibodies to the disease process has been recently elucidated, a potential role of aquaporin-4-specific T cells in lesion formation is unresolved. To address this issue, we raised aquaporin-4-specific T cell lines in Lewis rats and characterized their pathogenic potential in the presence and absence of aquaporin-4-specific autoantibodies of neuromyelitis optica patients. We show that aquaporin-4-specific T cells induce brain inflammation with particular targeting of the astrocytic glia limitans and permit the entry of pathogenic anti-aquaporin-4-specific antibodies to induce NMO-like lesions in spinal cord and brain. In addition, transfer of aquaporin-4-specific T cells provoked mild (subclinical) myositis and interstitial nephritis. We further show that the expression of the conformational epitope, recognized by NMO patient-derived aquaporin-4-specific antibodies is induced in kidney cells by the pro-inflammatory cytokine gamma-interferon. Our data provide further support for the view that NMO lesions may be induced by a complex interplay of T cell mediated and humoral immune responses against aquaporin-4. [ABSTRACT FROM AUTHOR]

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Background: Neuromyelitis optica (NMO), a severe demyelinating disease, represents itself with optic neuritis and longitudinally extensive transverse myelitis. Serum NMO-IgG autoantibodies (Abs), a specific finding in NMO patients, target the water channel protein aquaporin-4 (AQP4), which is expressed as a long (M-1) or a short (M-23) isoform. Methodology/Principal Findings: The aim of this study was to analyze serum samples from patients with NMO and controls for the presence and epitope specificity of IgG and IgM anti-AQP4 Abs using an immunofluorescence assay with HEK293 cells expressing M-1 or M-23 human AQP4. We included 56 patients with definite NMO (n = 30) and high risk NMO (n = 26), 101 patients with multiple sclerosis, 27 patients with clinically isolated syndromes (CIS), 30 patients with systemic lupus erythematosus (SLE) or Sjögren's syndrome, 29 patients with other neurological diseases and 47 healthy controls. Serum anti-AQP4 M-23 IgG Abs were specifically detected in 29 NMO patients, 17 patients with high risk NMO and two patients with myelitis due to demyelination (CIS) and SLE. In contrast, IgM anti-AQP4 Abs were not only found in some NMO and high risk patients, but also in controls. The sensitivity of the M-23 AQP4 IgG assay was 97% for NMO and 65% for high risk NMO, with a specificity of 100% compared to the controls. Sensitivity with M-1 AQP4 transfected cells was lower for NMO (70%) and high risk NMO (39%). The conformational epitopes of M-23 AQP4 are the primary targets of NMO-IgG Abs, whereas M-1 AQP4 Abs are developed with increasing disease duration and number of relapses. Conclusions: Our results confirm M-23 AQP4-IgG Abs as reliable biomarkers in patients with NMO and high risk syndromes. M-1 and M-23 AQP4-IgG Abs are significantly associated with a higher number of relapses and longer disease duration. [ABSTRACT FROM AUTHOR]

Autoantibodies targeting conformationally intact myelin oligodendrocyte glycoprotein (MOG) are found in different inflammatory diseases of the CNS, but their antigenic epitopes have not been mapped. We expressed mutants of MOG on human HeLa cells and analyzed sera from 111 patients (104 children, 7 adults) who recognized cell-bound human MOG, but had different diseases, including acute disseminated encephalomyelitis (ADEM), one episode of transverse myelitis or optic neuritis, multiple sclerosis (MS), anti-aquaporin-4 (AQP4)-negative neuromyelitis optica (NMO), and chronic relapsing inflammatory optic neuritis (CRION). We obtained insight into the recognition of epitopes in 98 patients. All epitopes identified were located at loops connecting the β strands of MOG. The most frequently recognized MOG epitope was revealed by the P42S mutation positioned in the CC'-loop. Overall, we distinguished seven epitope patterns, including the one mainly recognized by mouse mAbs. In half of the patients, the anti-MOG response was directed to a single epitope. The epitope specificity was not linked to certain disease entities. Longitudinal analysis of 11 patients for up to 5 y indicated constant epitope recognition without evidence for intramolecular epitope spreading. Patients who rapidly lost their anti-MOG IgG still generated a long-lasting IgG response to vaccines, indicating that their loss of anti-MOG reactivity did not reflect a general lack of capacity for long-standing IgG responses. The majority of human anti-MOG Abs did not recognize rodent MOG, which has implications for animal studies. Our findings might assist in future detection of potential mimotopes and pave the way to Ag-specific depletion. [ABSTRACT FROM AUTHOR]

Abstract: Neuromyelitis optica spectrum disorders (NMOSD) are associated with anti-aquaporin-4 autoantibodies (AQP4-IgG). Limited data is available on longitudinal cerebrospinal fluid (CSF) AQP4-IgG and their relation to disease activity and inflammatory parameters. AQP4-IgG titers were measured in matched longitudinal serum and CSF samples of 12 patients with NMOSD by an immunofluorescence assay and correlated with clinical parameters. CSF AQP4-IgG were present in patients with high serum titers and correlated with spinal MRI lesion length and CSF parameters. Clinical improvement was associated with a decrease in CSF, but not serum, AQP4-IgG titers. Thus, CSF AQP4-IgG were associated with clinical activity and neuroinflammation. [Copyright &y& Elsevier]