Evaluation of a Multiparametric Immunofluorescence Assay for Standardization of Neuromyelitis Optica Serology

Background: Neuromyelitis optica (NMO) is a severely disabling autoimmune disorder of the central nervous system, which predominantly affects the optic nerves and spinal cord. In a majority of cases, NMO is associated with antibodies to aquaporin-4 (AQP4) (termed NMO-IgG). Aims: In this study, we evaluated a new multiparametric indirect immunofluorescence (IIF) assay for NMO serology. Methods: Sera from 20 patients with NMO, 41 patients with multiple sclerosis (MS), 30 healthy subjects, and a commercial anti-AQP4 IgG antibody were tested in a commercial composite immunofluorescence assay ("Neurology Mosaic 17"; Euroimmun, Germany), consisting of five different diagnostic substrates (HEK cells transfected with AQP4, non-transfected HEK cells, primate cerebellum, cerebrum, and optic nerve tissue sections). Results: We identified AQP4 specific and non-specific fluorescence staining patterns and established positivity criteria. Based on these criteria, this kit yielded a high sensitivity (95%) and specificity (100%) for NMO and had a significant positive and negative likelihood ratio (LR+ = ∞, LR- = 0.05). Moreover, a 100% inter- and intra-laboratory reproducibility was found. Conclusions: The biochip mosaic assay tested in this study is a powerful tool for NMO serology, fast to perform, highly sensitive and specific for NMO, reproducible, and suitable for inter-laboratory standardization as required for multi-centre clinical trials

Multicentre comparison of a diagnostic assay: Aquaporin-4 antibodies in neuromyelitis optica

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

Deep Gray Matter Iron Content in Neuromyelitis Optica and Multiple Sclerosis

Background. Neuromyelitis optica (NMO) and multiple sclerosis (MS) are often presenting with overlapping symptoms. The aim of this study was to determine whether and how NMO and MS differ regarding cerebral iron deposits in deep gray matter (DGM) and the correlation between iron deposition and clinical severity as well as to regional atrophy of the DGM. Methods. We analyzed 20 patients with NMO, 40 patients with a relapsing-remitting (RR) form of MS, and 20 healthy controls with 1.5T MRI. Quantitative susceptibility mapping (QSM) was performed to estimate iron concentration in the DGM. Results. Patients with NMO have higher magnetic susceptibility values in the substantia nigra compared to healthy controls. RRMS patients have lower magnetic susceptibility values in the thalamus compared to healthy controls and NMO patients. Atrophy of the thalamus, pulvinar, and putamen is significant both in RRMS compared to NMO patients and healthy controls. A correlation was found between the disability score (EDSS) and magnetic susceptibility in the putamen in RRMS. Conclusions. This study confirms that a disturbed cerebral iron homeostasis in patients with NMO occurs in different structures than in patients with RRMS. Increased magnetic susceptibility in substantia nigra in NMO and decreased magnetic susceptibility within the thalamus in RRMS were the only significant differences in the study sample. We could confirm that iron concentration in the thalami is decreased in RRMS compared to that in the HC group. Positive association was found between putaminal iron and EDSS in RRMS

Natalizumab promotes anti-inflammatory and repair effects in multiple sclerosis.

BackgroundMultiple sclerosis is an inflammatory and degenerative disease of the central nervous system leading to demyelination and axonal loss. Relapsing-remitting multiple sclerosis (RRMS) is commonly treated by anti-inflammatory drugs, where one of the most effective drugs to date is the monoclonal antibody natalizumab.MethodsThe cerebrospinal fluid (CSF) proteome was analyzed in 56 patients with RRMS before and after natalizumab treatment, using label-free mass spectrometry and a subset of the changed proteins were verified by parallel reaction monitoring in a new cohort of 20 patients, confirming the majority of observed changes.ResultsA total of 287 differentially abundant proteins were detected including (i) the decrease of proteins with roles in immunity, such as immunoglobulin heavy constant mu, chitinase-3-like protein 1 and chitotriosidase, (ii) an increase of proteins involved in metabolism, such as lactate dehydrogenase A and B and malate-dehydrogenase cytoplasmic, and (iii) an increase of proteins associated with the central nervous system, including lactadherin and amyloid precursor protein. Comparison with the CSF-PR database provided evidence that natalizumab counters protein changes commonly observed in RRMS. Furthermore, vitamin-D binding protein and apolipoprotein 1 and 2 were unchanged during treatment with natalizumab, implying that these may be involved in disease activity unaffected by natalizumab.ConclusionsOur study revealed that some of the previously suggested biomarkers for MS were affected by the natalizumab treatment while others were not. Proteins not previously suggested as biomarkers were also found affected by the treatment. In sum, the results provide new information on how the natalizumab treatment impacts the CSF proteome of MS patients, and points towards processes affected by the treatment. These findings ought to be explored further to disclose potential novel disease mechanisms and predict treatment responses

Complement activation in patients with neuromyelitis optica

The role of complement has been demonstrated in experimental models of neuromyelitis optica (NMO), however, only few studies have analysed complement components longitudinally in NMO patients. We measured serum or plasma concentrations of anti-C1q antibodies and complement split products C3a and C4a and soluble C5b-9 in patients with NMO, multiple sclerosis and healthy controls. NMO patients had higher levels of C3a and anti-C1q antibodies than healthy controls. C3a levels correlated with disease activity, neurological disability and aquaporin-4 IgG in NMO patients suggesting a role of the alternative pathway of complement in the pathogenesis of NMO and supporting the strategy of therapeutic complement inhibition

Western blot analysis for the detection of serum antibodies recognizing linear Aquaporin-4 epitopes in patients with Neuromyelitis Optica

The current study presents a western blot assay showing good sensitivity (81%) and specificity (97%) for detecting serum antibodies to Aquaporin-4 (AQP4) in patients with Neuromyelitis Optica (NMO). By using western blot, we were able, for the first time, to distinguish serum immunoreactivity against either of the two AQP4 isoforms, showing that antibodies recognizing the linear AQP4-M1 isoform are specific for NMO. Moreover, the high sensitivity and specificity of the western blot assay in detecting serum anti-AQP4 antibodies in NMO patients suggest that such auto-antibodies may be of linear nature, thus recognizing epitopes that are displayed in denatured protein

Myelin oligodendrocyte glycoprotein antibodies are associated with a non-MS course in children

OBJECTIVE: To determine whether myelin oligodendrocyte glycoprotein antibodies (MOG-Abs) were predictive of a demyelination phenotype in children presenting with acquired demyelinating syndrome (ADS). METHOD: Sixty-five children with a first episode of ADS (12 acute disseminated encephalomyelitis, 24 optic neuritis, 18 transverse myelitis, 11 other clinically isolated syndrome) were identified from 2 national demyelination programs in the United Kingdom and France. Acute serum samples were tested for MOG-Abs by cell-based assay. Antibodies were used to predict diagnosis of multiple sclerosis (MS) at 1 year. RESULTS: Twenty-three of 65 (35%) children had MOG-Abs. Antibody-positive and antibody-negative patients were not clinically different at presentation, but identification of MOG-Abs predicted a non-MS course at 1-year follow-up: only 2/23 (9%) MOG-Ab–positive patients were diagnosed with MS compared to 16/42 (38%) MOG-Ab–negative patients (p = 0.019, Fisher exact test). Antibody positivity at outset was a useful predictor for a non-MS disease course, with a positive predictive value of 91% (95% confidence interval [CI] 72–99), negative predictive value of 38% (95% CI 24–54), positive likelihood ratio of 4.02 (CI 1.0–15.4), and odds ratio of 6.5 (CI 1.3–31.3). CONCLUSIONS: MOG-Abs are found at presentation in 35% of patients with childhood ADS, across a range of demyelinating disorders. Antibody positivity can be useful in predicting a non-MS disease course at onset

Blood metabolomic and transcriptomic signatures stratify patient subgroups in multiple sclerosis according to disease severity

Summary: There are no blood-based biomarkers distinguishing patients with relapsing-remitting (RRMS) from secondary progressive multiple sclerosis (SPMS) although evidence supports metabolomic changes according to MS disease severity. Here machine learning analysis of serum metabolomic data stratified patients with RRMS from SPMS with high accuracy and a putative score was developed that stratified MS patient subsets. The top differentially expressed metabolites between SPMS versus patients with RRMS included lipids and fatty acids, metabolites enriched in pathways related to cellular respiration, notably, elevated lactate and glutamine (gluconeogenesis-related) and acetoacetate and bOHbutyrate (ketone bodies), and reduced alanine and pyruvate (glycolysis-related). Serum metabolomic changes were recapitulated in the whole blood transcriptome, whereby differentially expressed genes were also enriched in cellular respiration pathways in patients with SPMS. The final gene-metabolite interaction network demonstrated a potential metabolic shift from glycolysis toward increased gluconeogenesis and ketogenesis in SPMS, indicating metabolic stress which may trigger stress response pathways and subsequent neurodegeneration

Myelin oligodendrocyte glycoprotein antibodies are associated with a non-MS course in children

OBJECTIVE: To determine whether myelin oligodendrocyte glycoprotein antibodies (MOG-Abs) were predictive of a demyelination phenotype in children presenting with acquired demyelinating syndrome (ADS). METHOD: Sixty-five children with a first episode of ADS (12 acute disseminated encephalomyelitis, 24 optic neuritis, 18 transverse myelitis, 11 other clinically isolated syndrome) were identified from 2 national demyelination programs in the United Kingdom and France. Acute serum samples were tested for MOG-Abs by cell-based assay. Antibodies were used to predict diagnosis of multiple sclerosis (MS) at 1 year. RESULTS: Twenty-three of 65 (35%) children had MOG-Abs. Antibody-positive and antibody-negative patients were not clinically different at presentation, but identification of MOG-Abs predicted a non-MS course at 1-year follow-up: only 2/23 (9%) MOG-Ab–positive patients were diagnosed with MS compared to 16/42 (38%) MOG-Ab–negative patients (p = 0.019, Fisher exact test). Antibody positivity at outset was a useful predictor for a non-MS disease course, with a positive predictive value of 91% (95% confidence interval [CI] 72–99), negative predictive value of 38% (95% CI 24–54), positive likelihood ratio of 4.02 (CI 1.0–15.4), and odds ratio of 6.5 (CI 1.3–31.3). CONCLUSIONS: MOG-Abs are found at presentation in 35% of patients with childhood ADS, across a range of demyelinating disorders. Antibody positivity can be useful in predicting a non-MS disease course at onset

Diagnostic sensitivity, specificity and likelihood ratios (LR+, LR−) for NMO calculated for each single substrate of the BioChip, obtained by testing 91 serum samples on the “Neurology Mosaic 17”.

<p>NMO: neuromyelitis optica, LR: likelihood ratio.</p