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Cell surface complement regulators moderate experimental myasthenia gravis pathology.

ABSTRACT: Intrinsic mouse complement regulators influence the severity of passively induced experimental acquired myasthenia gravis (EAMG). To assess the potential influence of CD59b in the absence of CD59a background, we used the mCD59ab(-/-) mouse model to re-evaluate mCD59 in protecting the neuromuscular junction (NMJ).EAMG was induced with monoclonal antibody to the acetylcholine receptor (AChR) in Daf1(-/-) , CD59ab(-/-) , Daf1(-/-) CD59ab(-/-) , and wild-type C57Bl/6 mice. Animals were monitored throughout the experiment. Diaphragms were analyzed for NMJ injury.Daf1(-/-) CD59ab(-/-) mice required euthanasia 24 hours after disease induction because of severe weakness. Histological assessment demonstrated reduced AChR density, simplification of synaptic folds, and disrupted mitochondria. CD59ab-deficient mice demonstrated mild weakness and reduction in weight after 24 hours. In contrast, Daf1(-/-) had more severe weakness at 60 hours. The NMJ of EAMG-induced Daf1(-/-) and CD59ab(-/-) mice demonstrated similar AChR density.NMJs of CD59 and DAF mice are protected from complement-mediated injury of passive EAMG.

SUBMITTER: Kusner LL

PROVIDER: S-EPMC4308955 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Cell surface complement regulators moderate experimental myasthenia gravis pathology.

Kusner Linda L LL Halperin Jose A JA Kaminski Henry J HJ

Muscle & nerve 20121005 1

<h4>Introduction</h4>Intrinsic mouse complement regulators influence the severity of passively induced experimental acquired myasthenia gravis (EAMG). To assess the potential influence of CD59b in the absence of CD59a background, we used the mCD59ab(-/-) mouse model to re-evaluate mCD59 in protecting the neuromuscular junction (NMJ).<h4>Methods</h4>EAMG was induced with monoclonal antibody to the acetylcholine receptor (AChR) in Daf1(-/-) , CD59ab(-/-) , Daf1(-/-) CD59ab(-/-) , and wild-type C57 ...[more]

PMID: 23042232

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Project description:Background and objectivesAutoantibodies targeting the acetylcholine receptor (AChR), found in patients with myasthenia gravis (MG), mediate pathology through 3 mechanisms: complement-directed tissue damage, blocking of the acetylcholine binding site, and internalization of the AChR. Clinical assays, used to diagnose and monitor patients, measure only autoantibody binding. Consequently, they are limited in providing association with disease burden, understanding of mechanistic heterogeneity, and monitoring therapeutic response. The objective of this study was to develop a cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation.MethodsAn HEK293T cell line-modified using CRISPR/Cas9 genome editing to disrupt expression of the complement regulator genes (CD46, CD55, and CD59)-was used to measure AChR autoantibody-mediated MAC formation through flow cytometry.ResultsSerum samples (n = 155) from 96 clinically confirmed AChR MG patients, representing a wide range of disease burden and autoantibody titer, were tested along with 32 healthy donor (HD) samples. AChR autoantibodies were detected in 139 of the 155 (89.7%) MG samples through a cell-based assay. Of the 139 AChR-positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), whereas MAC formation was undetectable in the HD group or AChR-positive samples with low autoantibody levels. MAC formation was positively associated with autoantibody binding in most patient samples; ratios (mean fluorescence intensity) of MAC formation to AChR autoantibody binding ranged between 0.27 and 48, with a median of 0.79 and an interquartile range of 0.43 (0.58-1.1). However, the distribution of ratios was asymmetric and included extreme values; 16 samples were beyond the 10-90 percentile, with high MAC to low AChR autoantibody binding ratio or the reverse. Correlation between MAC formation and clinical disease scores suggested a modest positive association (rho = 0.34, p = 0.0023), which included a subset of outliers that did not follow this pattern. MAC formation did not associate with exposure to immunotherapy, thymectomy, or MG subtypes defined by age-of-onset.DiscussionA novel assay for evaluating AChR autoantibody-mediated complement activity was developed. A subset of patients that lacks association between MAC formation and autoantibody binding or disease burden was identified. The assay may provide a better understanding of the heterogeneous autoantibody molecular pathology and identify patients expected to benefit from complement inhibitor therapy.

Project description:Myasthenia gravis (MG) is an autoimmune disease affecting the neuromuscular junction, whose clinical hallmark is muscle weakness and early fatigability. Azathioprine (AZA) is commonly used in Myasthenia Gravis therapy. AZA is a purine antagonist, which inhibits the cell cycle in the resting and DNA synthesis phases. It is usually used as an immunosuppressant to block T- and B-cell proliferation. AZA, bioconverted to 6-mercaptopurine by glutathione S-transferase (GST), can be metabolized either through the hypoxanthine phosphoribosyl transferase pathway to active 6-thioguanine nucleotides (6-TGN) or through the thiopurine S-methyltransferase (TPMT) pathway to inactive methyl-thiopurine metabolites. The incorporation of active 6-TGNs into DNA, causing breaks in DNA strands resulting in interference with RNA production and thereby protein synthesis, is responsible for the drug effect. The response to AZA is determined by which metabolic pathway is being favored. While balanced use of both HPRT and TPMT pathways results in responsiveness to AZA, hyperactivity of TPMT skews the balance towards the TPMT pathway and results in unresponsiveness to AZA with no pharmacological effect. In contrast, low TPMT activity skews the balance towards the HPRT pathway, resulting in increasing side-effects due to the accumulation of 6-TGNs. Current treatments for MG therapy are often inadequate because less than 40% of patients achieve complete remission with available drugs. This reflects the lack of drugs acting on target sites for which there is strong evidence of pathogenicity and the inability to identify responder patients. No criteria for responsiveness are available, exposing patients to unpredictable failures and unpredictable side effects. These individuals are at particular risk for adverse drug reaction (ADRs) or therapeutic failure. Genetic profiling with the Affymetrix drug metabolizing enzymes and transports genotyping array offers the ability to determine 1,931 variants and 225 genes involved in drug metabolism and disposition. Accordingly, the study of well-known drug-metabolizing genes can be involved in the specific metabolic pathway of a drug, which is more likely to define genotype-phenotype association and thereby genotype profiles relevant to drug response that can be applied as predictive biomarkers for pharmacological treatment.

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Cell surface complement regulators moderate experimental myasthenia gravis pathology.

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Cell surface complement regulators moderate experimental myasthenia gravis pathology.

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