High-dose prednisone use, lasting several months or longer, is the primary initial therapy for my... more High-dose prednisone use, lasting several months or longer, is the primary initial therapy for myasthenia gravis (MG). Upwards of a third of patients do not respond to treatment. Currently no biomarkers can predict clinical responsiveness to corticosteroid treatment. We conducted a discovery-based study to identify treatment responsive biomarkers in MG using sera obtained at study entry to the thymectomy clinical trial (MGTX), an NIH-sponsored randomized, controlled study of thymectomy plus prednisone versus prednisone alone.
We recently demonstrated that hepatic stellate cells (HSC) induce myeloid derived suppressor cell... more We recently demonstrated that hepatic stellate cells (HSC) induce myeloid derived suppressor cells (MDSC) differentiation from myeloid progenitors. In this report, we found that adoptive transfer of the MDSC was effective in reversing disease progression in experimental autoimmune myasthenia gravis (EAMG), a T cell-dependent, and B cell-mediated model for myasthenia gravis (MG). In accordance with ameliorated disease severity, the MDSC-treated EAMG mice showed suppressed AchR-specific T cell responses, decreased levels of serum anti-AChR IgGs and reduced complement activation at the neuromuscular junctions. We also discovered that the MDSC inhibited the proliferation of, and antibody production from B cells activated by anti-IgM or anti-CD40 antibodies in vitro, and inhibited the production of antibodies against a T cell-independent antigen in vivo, indicating that in addition to their T cell inhibitory activities, MDSC also directly inhibit B cells. These results demonstrated that ...
A characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic ge... more A characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic germinal centers (GC). However, the mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Dysregulation of small, non-coding microRNAs (miRNAs) and their target genes has been identified in the pathology of several autoimmune diseases. We assessed the miRNA and mRNA profiles of the MG thymus and have investigated their role in GC formation and maintenance. MG thymus samples were assessed by histology and grouped based upon the appearance of GC; GC positive and GC negative. A systems biology approach was used to study the differences between the groups. Our study included miRNA and mRNA profiling, quantitative real-time PCR validation, miRNA target identification, pathway analysis, miRNA-mRNA reciprocal expression pairing and interaction. Thirty-eight mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>...
Biomarkers that assess treatment response for patients with the autoimmune disorder, myasthenia g... more 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 associ...
The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis... more The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis are poorly understood. In this investigation, we evaluate the role of survivin, a member of the inhibitor of apoptosis protein family, in humans and in two animal models. We identified survivin expression in cells with B lymphocyte and plasma cells markers, and in the thymuses of patients with myasthenia gravis. A portion of survivin-expressing cells specifically bound a peptide derived from the alpha subunit of acetylcholine receptor indicating that they recognize the peptide. Thymuses of patients with myasthenia gravis had large numbers of survivin-positive cells with fewer cells in the thymuses of corticosteroid-treated patients. Application of a survivin vaccination strategy in mouse and rat models of myasthenia gravis demonstrated improved motor assessment, a reduction in acetylcholine receptor specific autoantibodies, and a retention of acetylcholine receptor at the neuromuscular ...
Myasthenia gravis demonstrates a distinct predilection for involvement of the extraocular muscles... more Myasthenia gravis demonstrates a distinct predilection for involvement of the extraocular muscles (EOM), and we have hypothesized that this may be due to a unique immunological environment. To assess this hypothesis, we took an unbiased approach to analyze RNA expression profiles in EOM, diaphragm, and extensor digitorum longus (EDL) in rats with experimentally acquired myasthenia gravis (EAMG). Experimentally acquired myasthenia gravis was induced in rats by intraperitoneal injection of antibody directed against the acetylcholine receptor (AChR), whereas control rats received antibody known to bind the AChR but not induce disease. After 48 hours, animals were killed and muscles analyzed by RNA expression profiling. Profiling results were validated using qPCR and immunohistochemical analysis. Sixty-two genes common among all muscle groups were increased in expression. These fell into four major categories: 12.8% stress response, 10.5% immune response, 10.5% metabolism, and 9.0% tran...
The site of pathology in myasthenia gravis (MG) is the neuromuscular junction (NMJ). Our goal was... more 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. 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. 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. We demonstrate a method to effectively target a therapeutic agent to the NMJ.
Although myasthenia gravis (MG) is often considered the best-understood autoimmune disorder and e... more Although myasthenia gravis (MG) is often considered the best-understood autoimmune disorder and effective treatments have controlled life-threatening complications, the pathogenesis of ocular myasthenia (OM) remains enigmatic, and its clinical consequences offer therapeutic challenges. About half of patients with MG present with visual complaints of droopy eyelids or double vision, and many will remain with purely ocular muscle weakness without generalized weakness, defined as OM. OM may be confused with disorders of the brainstem, ocular motor nerves, and eye muscles. Frustrating for the clinician, confirmatory tests such as the edrophonium test, serum acetylcholine receptor antibodies, and standard electrodiagnostic evaluations may fail to positively identify the clinical suspicion of OM. Patients may derive relief from nonpharmacologic interventions and cholinesterase inhibitors, but most will desire better symptom control with corticosteroids or need other immunosuppression. Early corticosteroid therapy may reduce the probability of generalization of the disease. The reasons for ocular muscle involvement by OM include physiologic and cellular properties of the ocular motor system and the unique immunology of OM, which, when better understood, will lead to novel treatments. OM is a challenging disorder for the clinician and scientist, with both learning from the other for the betterment of the patient. The future requires answers to why the ocular muscles are so frequently involved by MG, whether the generalization of the disease may be limited by early corticosteroid treatment, and what treatment options may be developed which will improve symptoms without long-term complications.
Biomarkers are defined as characteristics (e.g., proteins, RNA, single nucleotide polymorphisms, ... more Biomarkers are defined as characteristics (e.g., proteins, RNA, single nucleotide polymorphisms, imaging) that are objectively measured and evaluated as indicators of pathogenic processes or pharmacologic responses to therapeutic intervention. Biomarkers are important in clinical trials where the robust biomarker reflects the underlying disease process in a sensitive and reliable manner. For myasthenia gravis (MG), acetylcholine receptor and muscle‐specific kinase antibodies, as well as single‐fiber electromyography, serve as excellent biomarkers for diagnosis but do not adequately substitute for clinical evaluations to predict treatment response. New technologies are emerging that enable broad biomarker discovery in biological fluids. Biomarker evaluation is ideally done in the context of longitudinal clinical trials. The MGTX trial has collected plasma and serum for RNA and protein analysis and thymus, which will allow robust biomarker discovery. The ultimate goal will be to ident...
To determine the expression of fetal and adult acetylcholine receptor (AChR) isoforms among extra... more To determine the expression of fetal and adult acetylcholine receptor (AChR) isoforms among extraocular muscle (EOM) en plaque and en grappe endplates. Antibodies against peptide fragments of the gamma- and epsilon-subunits of the fetal and adult AChRs and alpha-bungarotoxin were used in immunofluorescence experiments to stain rat neonatal leg, adult diaphragm, and extraocular rectus muscle endplates. Anti-epsilon antibodies intensely stained diaphragm endplates weakly stained rare neonatal endplates. Anti-gamma antibodies stained neonatal, but not diaphragm, endplates. Anti-epsilon antibodies bound to all en plaque and en grappe endplates of extraocular muscle. Anti-gamma antibodies bound to global and orbital en grappe endplates. All en plaque endplates of the orbital region and a subset of endplates in the global region stained with anti-gamma antibodies. All en grappe endplates and certain en plaque endplates of EOM are the only mature endplates that coexpress the adult and feta...
High-dose prednisone use, lasting several months or longer, is the primary initial therapy for my... more High-dose prednisone use, lasting several months or longer, is the primary initial therapy for myasthenia gravis (MG). Upwards of a third of patients do not respond to treatment. Currently no biomarkers can predict clinical responsiveness to corticosteroid treatment. We conducted a discovery-based study to identify treatment responsive biomarkers in MG using sera obtained at study entry to the thymectomy clinical trial (MGTX), an NIH-sponsored randomized, controlled study of thymectomy plus prednisone versus prednisone alone.
We recently demonstrated that hepatic stellate cells (HSC) induce myeloid derived suppressor cell... more We recently demonstrated that hepatic stellate cells (HSC) induce myeloid derived suppressor cells (MDSC) differentiation from myeloid progenitors. In this report, we found that adoptive transfer of the MDSC was effective in reversing disease progression in experimental autoimmune myasthenia gravis (EAMG), a T cell-dependent, and B cell-mediated model for myasthenia gravis (MG). In accordance with ameliorated disease severity, the MDSC-treated EAMG mice showed suppressed AchR-specific T cell responses, decreased levels of serum anti-AChR IgGs and reduced complement activation at the neuromuscular junctions. We also discovered that the MDSC inhibited the proliferation of, and antibody production from B cells activated by anti-IgM or anti-CD40 antibodies in vitro, and inhibited the production of antibodies against a T cell-independent antigen in vivo, indicating that in addition to their T cell inhibitory activities, MDSC also directly inhibit B cells. These results demonstrated that ...
A characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic ge... more A characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic germinal centers (GC). However, the mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Dysregulation of small, non-coding microRNAs (miRNAs) and their target genes has been identified in the pathology of several autoimmune diseases. We assessed the miRNA and mRNA profiles of the MG thymus and have investigated their role in GC formation and maintenance. MG thymus samples were assessed by histology and grouped based upon the appearance of GC; GC positive and GC negative. A systems biology approach was used to study the differences between the groups. Our study included miRNA and mRNA profiling, quantitative real-time PCR validation, miRNA target identification, pathway analysis, miRNA-mRNA reciprocal expression pairing and interaction. Thirty-eight mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>...
Biomarkers that assess treatment response for patients with the autoimmune disorder, myasthenia g... more 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 associ...
The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis... more The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis are poorly understood. In this investigation, we evaluate the role of survivin, a member of the inhibitor of apoptosis protein family, in humans and in two animal models. We identified survivin expression in cells with B lymphocyte and plasma cells markers, and in the thymuses of patients with myasthenia gravis. A portion of survivin-expressing cells specifically bound a peptide derived from the alpha subunit of acetylcholine receptor indicating that they recognize the peptide. Thymuses of patients with myasthenia gravis had large numbers of survivin-positive cells with fewer cells in the thymuses of corticosteroid-treated patients. Application of a survivin vaccination strategy in mouse and rat models of myasthenia gravis demonstrated improved motor assessment, a reduction in acetylcholine receptor specific autoantibodies, and a retention of acetylcholine receptor at the neuromuscular ...
Myasthenia gravis demonstrates a distinct predilection for involvement of the extraocular muscles... more Myasthenia gravis demonstrates a distinct predilection for involvement of the extraocular muscles (EOM), and we have hypothesized that this may be due to a unique immunological environment. To assess this hypothesis, we took an unbiased approach to analyze RNA expression profiles in EOM, diaphragm, and extensor digitorum longus (EDL) in rats with experimentally acquired myasthenia gravis (EAMG). Experimentally acquired myasthenia gravis was induced in rats by intraperitoneal injection of antibody directed against the acetylcholine receptor (AChR), whereas control rats received antibody known to bind the AChR but not induce disease. After 48 hours, animals were killed and muscles analyzed by RNA expression profiling. Profiling results were validated using qPCR and immunohistochemical analysis. Sixty-two genes common among all muscle groups were increased in expression. These fell into four major categories: 12.8% stress response, 10.5% immune response, 10.5% metabolism, and 9.0% tran...
The site of pathology in myasthenia gravis (MG) is the neuromuscular junction (NMJ). Our goal was... more 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. 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. 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. We demonstrate a method to effectively target a therapeutic agent to the NMJ.
Although myasthenia gravis (MG) is often considered the best-understood autoimmune disorder and e... more Although myasthenia gravis (MG) is often considered the best-understood autoimmune disorder and effective treatments have controlled life-threatening complications, the pathogenesis of ocular myasthenia (OM) remains enigmatic, and its clinical consequences offer therapeutic challenges. About half of patients with MG present with visual complaints of droopy eyelids or double vision, and many will remain with purely ocular muscle weakness without generalized weakness, defined as OM. OM may be confused with disorders of the brainstem, ocular motor nerves, and eye muscles. Frustrating for the clinician, confirmatory tests such as the edrophonium test, serum acetylcholine receptor antibodies, and standard electrodiagnostic evaluations may fail to positively identify the clinical suspicion of OM. Patients may derive relief from nonpharmacologic interventions and cholinesterase inhibitors, but most will desire better symptom control with corticosteroids or need other immunosuppression. Early corticosteroid therapy may reduce the probability of generalization of the disease. The reasons for ocular muscle involvement by OM include physiologic and cellular properties of the ocular motor system and the unique immunology of OM, which, when better understood, will lead to novel treatments. OM is a challenging disorder for the clinician and scientist, with both learning from the other for the betterment of the patient. The future requires answers to why the ocular muscles are so frequently involved by MG, whether the generalization of the disease may be limited by early corticosteroid treatment, and what treatment options may be developed which will improve symptoms without long-term complications.
Biomarkers are defined as characteristics (e.g., proteins, RNA, single nucleotide polymorphisms, ... more Biomarkers are defined as characteristics (e.g., proteins, RNA, single nucleotide polymorphisms, imaging) that are objectively measured and evaluated as indicators of pathogenic processes or pharmacologic responses to therapeutic intervention. Biomarkers are important in clinical trials where the robust biomarker reflects the underlying disease process in a sensitive and reliable manner. For myasthenia gravis (MG), acetylcholine receptor and muscle‐specific kinase antibodies, as well as single‐fiber electromyography, serve as excellent biomarkers for diagnosis but do not adequately substitute for clinical evaluations to predict treatment response. New technologies are emerging that enable broad biomarker discovery in biological fluids. Biomarker evaluation is ideally done in the context of longitudinal clinical trials. The MGTX trial has collected plasma and serum for RNA and protein analysis and thymus, which will allow robust biomarker discovery. The ultimate goal will be to ident...
To determine the expression of fetal and adult acetylcholine receptor (AChR) isoforms among extra... more To determine the expression of fetal and adult acetylcholine receptor (AChR) isoforms among extraocular muscle (EOM) en plaque and en grappe endplates. Antibodies against peptide fragments of the gamma- and epsilon-subunits of the fetal and adult AChRs and alpha-bungarotoxin were used in immunofluorescence experiments to stain rat neonatal leg, adult diaphragm, and extraocular rectus muscle endplates. Anti-epsilon antibodies intensely stained diaphragm endplates weakly stained rare neonatal endplates. Anti-gamma antibodies stained neonatal, but not diaphragm, endplates. Anti-epsilon antibodies bound to all en plaque and en grappe endplates of extraocular muscle. Anti-gamma antibodies bound to global and orbital en grappe endplates. All en plaque endplates of the orbital region and a subset of endplates in the global region stained with anti-gamma antibodies. All en grappe endplates and certain en plaque endplates of EOM are the only mature endplates that coexpress the adult and feta...
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