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Aquaporin-4 autoantibodies in Neuromyelitis Optica: AQP4 isoform-dependent sensitivity and specificity.

ABSTRACT: Neuromyelitis Optica (NMO) is an autoimmune demyelinating disease, characterized by the presence of autoantibody (NMO-IgG) to Aquaporin-4 (AQP4). NMO-IgG identification supports NMO diagnosis and several diagnostic tests have been developed, but their sensitivity is too variable, and some assay show low sensitivity. This impairs correct diagnosis of NMO. By cell based assay (CBA) we here evaluate the efficacy of different strategies to express AQP4 in mammalian cells in terms of: a) AQP4 translation initiation signals; b) AQP4 isoforms (M1 and M23) and fluorescent tag position; c) NMO serum concentration and AQP4 degradation. Our results demonstrate that when using AQP4-M1, the nucleotide in position -3 of the AUG greatly affects the AQP4-M1/M23 protein ratio, NMO-IgG binding, and consequently test sensitivity. Test sensitivity was highest with M23 expressing cells (97.5%) and only 27.5% with AQP4-M1. The fluorescent tag added to the N-terminus of AQP4-M23 considerably affected the NMO-IgG binding, and test sensitivity, due to disruption of AQP4 suprastructures. Furthermore, sera used at high concentration resulted in AQP4 degradation which affected test sensitivity. To further evaluate the reliability of the M23 based CBA test, samples of one NMO patient collected during about 2 years clinical follow-up were tested. The results of serum titer correlated with disease activity and treatment response. In conclusion, we provide a molecular explanation for the contrasting CBA test data reported and suggest the use of M23 with a C-terminus fluorescent tag as the proper test for NMO diagnosis.

SUBMITTER: Pisani F

PROVIDER: S-EPMC3829826 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Aquaporin-4 autoantibodies in Neuromyelitis Optica: AQP4 isoform-dependent sensitivity and specificity.

Pisani Francesco F Sparaneo Angelo A Tortorella Carla C Ruggieri Maddalena M Trojano Maria M Mola Maria Grazia MG Nicchia Grazia Paola GP Frigeri Antonio A Svelto Maria M

PloS one 20131115 11

Neuromyelitis Optica (NMO) is an autoimmune demyelinating disease, characterized by the presence of autoantibody (NMO-IgG) to Aquaporin-4 (AQP4). NMO-IgG identification supports NMO diagnosis and several diagnostic tests have been developed, but their sensitivity is too variable, and some assay show low sensitivity. This impairs correct diagnosis of NMO. By cell based assay (CBA) we here evaluate the efficacy of different strategies to express AQP4 in mammalian cells in terms of: a) AQP4 transla ...[more]

PMID: 24260168

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Project description:BackgroundAquaporin-4-immunoglobulin G (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (herein called NMO) is an autoimmune disease of the central nervous system in which AQP4-IgG binding to AQP4 on astrocytes results in complement-dependent astrocyte injury and secondary inflammation, demyelination, and neuron loss. We previously reported evidence for a complement bystander mechanism for early oligodendrocyte injury in NMO. Herein, we tested the hypothesis that complement bystander injury, which involves diffusion to nearby cells of activated soluble complement components from complement-injured astrocytes, is a general phenomenon that may contribute to neuronal injury in NMO.MethodsPrimary cocultures of rat astrocytes and cortical neurons were established to study complement-dependent cell death after exposure to AQP4-IgG and complement. In animal experiments, AQP4-IgG was delivered to adult rats by intracerebral injection. Cell cultures and rat brain were studied by immunofluorescence.ResultsIn primary astrocyte-neuron cocultures, addition of AQP4-IgG and complement resulted in death of neurons nearby astrocytes. Deposition of complement membrane attack complex C5b-9 was seen on neurons nearby astrocytes, whereas C1q, the initiating protein in the complement pathway, was seen only on astrocytes. Neuron death was not seen with a complement inhibitor, with C1q- or C6-depleted complement, in pure neuron cultures exposed to AQP4-IgG and complement or in cocultures exposed to an astrocyte toxin. Intracerebral injection in rats of AQP4-IgG and a fixable dead cell fluorescent marker produced death of neurons near astrocytes, with C5b-9 deposition. Neuron death was not seen in rats receiving a complement inhibitor or in AQP4-IgG-injected AQP4 knockout rats.ConclusionThese results support a novel mechanism for early neuron injury in NMO and provide evidence that complement bystander injury may be a general phenomenon for brain cell injury following AQP4-IgG-targeted astrocyte death.

Project description:Neuromyelitis optica spectrum disorders (NMOSD) are central nervous system inflammatory disorders causing significant morbidities and mortality. The majority of NMOSD patients have autoimmunity against aquaporin-4 (AQP4), evidenced by seropositivity for autoantibodies against aquaporin-4 (AQP4-IgG). AQP4-IgG is pathogenic with neuroinflammation initiated upon binding of AQP4-IgG to astrocytic AQP4. Complement activation contributes to astrocytic cytotoxicity, neuroinflammation, and tissue necrosis in NMOSD, but the role of complement-independent mechanisms is uncertain. We studied the complement-independent pathogenic effects of AQP4-IgG by passive transfer of IgG from NMOSD patients to mice with breached blood-brain barrier (BBB). Mice, pretreated with bacterial proteins, received daily intraperitoneal injections of IgG purified from AQP4-IgG-seropositive NMOSD patients [IgG(AQP4+)], or IgG from AQP4-IgG-seronegative patients [IgG(AQP4-)] or healthy subjects [IgG(Healthy)] for 8 days. Motor function was tested by walking across narrow beams, and spinal cords were collected for immunofluorescent analysis. We found that human IgG infiltrated into cord parenchyma of mice with breached BBB without deposition of complement activation products. Spinal cord of mice that received IgG(AQP4+) demonstrated loss of AQP4 and glial fibrillary acidic protein (suggestive of astrocyte loss), decrease in excitatory amino acid transporter 2, microglial/macrophage activation, neutrophil infiltration, patchy demyelination, and loss in axonal integrity. Mice that received IgG(AQP4+) required longer time with more paw slips to walk across narrow beams indicative of motor slowing and incoordination. Our findings suggest that AQP4-IgG induces complement-independent cord pathologies, including astrocytopathy, neuroinflammation, demyelination, and axonal injuries/loss, which are associated with subtle motor impairments. These complement-independent pathophysiologies likely contribute to early NMOSD lesion development.

Project description:ImportanceImmunoglobulin G autoantibodies for aquaporin 4 (AQP4-IgG) serve as diagnostic biomarkers for neuromyelitis optica spectrum disorder (NMOSD), and the most sensitive and specific laboratory tests for their detection are cell-based assays (CBAs). Nevertheless, the limited availability of special instruments limits the widespread use of CBAs in routine laboratories.ObjectiveTo validate an enzyme immunodot assay for simple and rapid detection of AQP4-IgG.Design, setting, and participantsThis multicenter case-control study, conducted from May 2020 to February 2023, involved 4 medical centers (3 in China and 1 in Korea). The study included patients with AQP4-IgG-positive NMOSD, patients with other immune-related diseases, and healthy control individuals. Participants were excluded if they did not agree to participate or if their serum sample had turbidity.ExposuresSerum AQP4 antibodies measured with immunodot assay.Main outcomes and measuresThe main outcome was performance of the immunodot assay compared with the gold standard CBA for detecting AQP4-IgG. To examine generalizability, cross-validation in Korea and at a second site in China, validation of patients with other immune-related diseases, and follow-up validation of the original cohort were performed.ResultsA total of 836 serum samples were collected; 400 were included in the diagnostic study and 436 in the validation sets. In a head-to-head diagnostic study involving 200 patients with NMOSD with AQP4-IgG (mean [SD] age, 43.1 [13.5] years; 188 [94%] female) and 200 healthy controls, use of an immunodot assay demonstrated antibody detection performance comparable to that of the gold standard (κ = 98.0%). The validation sets included 47 patients with NMOSD and 26 patients with other autoimmune diseases from Korea, 31 patients with NMOSD at a second site in China, 275 patients with other diseases, and 57 patients with NMOSD at follow-up. In the validation study, of 436 cases, 2 (<1%) were false positive and none were false negative. The CBA identified 332 AQP4-IgG-positive samples and 504 negative samples (200 [40%] in controls and 304 [60%] in patients with other diseases); 2 of the positive cases (<1%) were false negative and 4 of the negative cases (<1%) were false positive. The overall sensitivity of the immunodot assay was 99.4% (95% CI, 97.8%-99.9%), and the specificity was 99.2% (95% CI, 98.0%-99.8%).Conclusions and relevanceThis case-control study found that the immunodot assay was comparable to CBA for detecting AQP4-IgG. With its time- and cost-efficient characteristics, the immunodot assay may be a practical option for AQP4-IgG detection.

Dataset Information

Aquaporin-4 autoantibodies in Neuromyelitis Optica: AQP4 isoform-dependent sensitivity and specificity.

Publications

Aquaporin-4 autoantibodies in Neuromyelitis Optica: AQP4 isoform-dependent sensitivity and specificity.

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