Targeting therapy to the neuromuscular junction: Proof of concept

Introduction: The site of pathology in myasthenia gravis (MG) is the neuromuscular junction (NMJ). Our goal was to determine the ability to direct complement inhibition to the NMJ. Methods: A single-chain antibody directed against the alpha subunit of the acetylcholine receptor was synthesized (scFv-35) and coupled to decay-accelerating factor (DAF, scFv-35-DAF). scFv-35-DAF was tested in a passive model of experimentally acquired MG. Results: Administration of scFv-35-DAF to mice deficient in intrinsic complement inhibitors produced no weakness despite confirmation of its localization to the NMJ and no evidence of tissue destruction related to complement activation. Rats with experimentally acquired MG treated with scFV-35-DAF showed less weakness and a reduction of complement deposition. Conclusions: We demonstrate a method to effectively target a therapeutic agent to the NMJ. Muscle Nerve 49: 749–756, 201

Guidelines for pre-clinical assessment of the acetylcholine receptor-specific passive transfer myasthenia gravis model - recommendations for methods and experimental designs.

Antibodies against the muscle acetylcholine receptor (AChR) are the most common cause of myasthenia gravis (MG). Passive transfer of AChR antibodies from MG patients into animals reproduces key features of human disease, including antigenic modulation of the AChR, complement-mediated damage of the neuromuscular junction, and muscle weakness. Similarly, AChR antibodies generated by active immunization in experimental autoimmune MG models can subsequently be passively transferred to other animals and induce weakness. The passive transfer model is useful to test therapeutic strategies aimed at the effector mechanism of the autoantibodies. Here we summarize published and unpublished experience using the AChR passive transfer MG model in mice, rats and rhesus monkeys, and give recommendations for the design of preclinical studies in order to facilitate translation of positive and negative results to improve MG therapies

Differential mRNA expression in ectopic germinal centers of myasthenia gravis thymus

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder resulting in weakness of voluntary muscles. It is caused by antibodies directed against proteins present at the post-synaptic surface of neuromuscular junction (NMJ). A characteristic pathology of patients with early onset MG is thymic hyperplasia with ectopic germinal centers (GC). However, mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. In order to determine the central mechanisms involved in the pathology, thymus samples from MG patients were assessed by histology and grouped based on appearance of GC compared to samples without them. We assessed the differential mRNA expression profiles between the two groups by GeneChip® Human Transcriptome Array 2.0. Partek Genomic Suite 6.6 and Transcript Analysis Console 2.0 programs were used for further analysis. Forty eight annotated mRNA transcripts were identified that were differentially expressed between the two groups with greater than 1.5 fold difference in expression (ANOVA p\u3c0.05). We verified their expression by RT-PCR. We identified Regulator of G protein Signaling 13 or RGS13 that is known to be expressed in GC B-cells and regulate responsiveness to chemokine signaling. Upregulation of RGS13 was found to be associated with specimens having GC. We verified its expression in GC by immunohistochemistry. Gene ontology (GO) enrichment analysis and Ingenuity Pathway Analysis (IPA) core analysis of differentially expressed genes indicate involvement of immune response regulation and cell proliferation pathways, indicating their importance in GC formation and regulation

The Role of Osteopontin and Its Gene on Glucocorticoid Response in Myasthenia Gravis

Biomarkers that assess treatment response for patients with the autoimmune disorder, myasthenia gravis (MG), have not been evaluated to a significant extent. We hypothesized the pro-inflammatory cytokine, osteopontin (OPN), may be associated with variability of response to glucocorticoids (GCs) in patients with MG. A cohort of 250 MG patients treated with standardized protocol of GCs was recruited, and plasma OPN and polymorphisms of its gene, secreted phosphoprotein 1 (SPP1), were evaluated. Mean OPN levels were higher in patients compared to healthy controls. Carriers of rs11728697*T allele (allele definition: one of two or more alternative forms of a gene) were more frequent in the poorly GC responsive group compared to the GC responsive group indicating an association of rs11728697*T allele with GC non-responsiveness. One risk haplotype (AGTACT) was identified associated with GC non-responsiveness compared with GC responsive MG group. Genetic variations of SPP1 were found associated with the response to GC among MG patients

Fueling the fire of B cell activation

Antigen activated naïve B cells undergoing germinal center responses have distinct metabolic requirements

Alpha-fetoprotein inhibition of lymphocyte proliferation in vitro cell culture related to myasthenia gravis

BACKGROUND Autoimmune myasthenia gravis (MG) is a disorder of the neuromuscular junction caused in the majority of patients by autoantibodies directed against the postsynaptic nicotinic acetylcholine receptor (AChR). The prevalence of myasthenia gravis in the United States is estimated at 14 to 20 per 100,000 population and women are more often affected than men. Cholinesterase inhibitors, corticosteroids and immunosuppressant are commonly used classes of drugs to treat MG patients but these drugs do not prevent disease progression and are associated with serious side effects. Alpha-fetoprotein (AFP), a serum protein produced by the yolk sac and the fetal liver, is present in high amounts during pregnancy. The clinical remission of myasthenia gravis during the second half of pregnancy may be attributed to the immunosuppressive effect of AFP. METHOD The peripheral blood lymphocytes (PBLs) were obtained from MG patients and healthy controls (HCs) by using density gradient media (Ficoll). PBLs were cultured 5 days after the proliferation dye combination, and then the effect of PHA, hAChR and AFP on the proliferation of lymphocytes were investigated by using flow cytometry. RESULTS Both PHA and hAChR treatment can promote lymphocytes’ proliferation. The human AChR selectively induced MG patient’s lymphocytes proliferation, but not affect the cell growth of the healthy control. AFP treatment can significantly inhibit lymphocytes proliferation in a dose dependent manner without any effect on the healthy control. CONCLUSION AFP inhibits the peripheral lymphocyte proliferation of MG patients by in vitro cell culture. However, AFP did not affect proliferation of PBLs in healthy control samples. The immunosuppressive effect of AFP demonstrated AFP might be a potential therapeutic reagent for autoimmune myasthenia gravis patients

The presence of survivin on B cells from myasthenia gravis patients and the potential of an antibody to a modified survivin peptide to alleviate weakness in an animal model

Myasthenia gravis (MG) is an autoimmune disease in which Abs target neuromuscular junction proteins, in particular the acetylcholine receptor. We previously identified the antiapoptotic protein survivin in the autoreactive B cells and plasma cells of MG patients. To further define the role of survivin in MG, we have assessed PBMCs from 29 patients with MG and 15 controls. We confirmed the increased expression of survivin in CD20+ lymphocytes from MG patients compared with controls. Furthermore, the CD20+ population of cells from MG patients contained a higher percentage of extracellular survivin compared with controls. The analysis of CD4+ cells showed an increased percentage of intracellular survivin in MG patients compared with controls, whereas the extracellular survivin CD4+ percentage was unaffected. In an experimental mouse model of MG, we assessed the therapeutic potential of an Ab raised to a modified survivin peptide but cross-reactive to survivin. Ab treatment reduced disease severity, lowered acetylcholine receptor-specific Abs, and decreased CD19+ survivin+ splenocytes. The ability to target survivin through Ab recognition of autoreactive cells offers the potential for a highly specific therapeutic agent for MG