The thymus in myasthenia gravis: Site of “innate autoimmunity”?

Myasthenia gravis (MG) is an autoimmune disorder caused, in most cases, by autoantibodies against components of the neuromuscular junction, frequently the acetylcholine receptor (AChR), and less often the muscle-specific kinase receptor. The thymus plays a major role in the pathogenesis of MG with anti-AChR antibodies: it shows marked pathologic alterations (hyperplastic or tumoral) in most AChR-positive patients and contains the elements required to initiate and sustain an autoimmune reaction (AChR autoantigen, AChR-specific T cells, and autoantibody-secreting plasma cells). In this study we review early and more recent findings implicating the thymus as site of AChR autosensitization in MG and briefly discuss the therapeutic role of thymectomy. We also summarize data showing that the MG thymus is in a state of chronic inflammation, and we review emerging evidence of a viral contribution to the onset and maintenance of the thymic autoimmune response.     

Anti-LRP4 autoantibodies in AChR- and MuSK-antibody-negative myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disorder characterized by a defect in synaptic transmission at the neuromuscular junction causing fluctuating muscle weakness with a decremental response to repetitive nerve stimulation or altered jitter in single-fiber electromyography (EMG). Approximately 80% of all myasthenia gravis patients have autoantibodies against the nicotinic acetylcholine receptor in their serum. Autoantibodies against the tyrosine kinase muscle-specific kinase (MuSK) are responsible for 5–10% of all myasthenia gravis cases. The autoimmune target in the remaining cases is unknown. Recently, low-density lipoprotein receptor-related protein 4 (LRP4) has been identified as the agrin receptor. LRP4 interacts with agrin, and the binding of agrin activates MuSK, which leads to the formation of most if not all postsynaptic specializations, including aggregates containing acetylcholine receptors (AChRs) in the junctional plasma membrane. In the present study we tested if autoantibodies against LRP4 are detectable in patients with myasthenia gravis. To this end we analyzed 13 sera from patients with generalized myasthenia gravis but without antibodies against AChR or MuSK. The results showed that 12 out of 13 antisera from double-seronegative MG patients bound to proteins concentrated at the neuromuscular junction of adult mouse skeletal muscle and that approximately 50% of the tested sera specifically bound to HEK293 cells transfected with human LRP4. Moreover, 4 out of these 13 sera inhibited agrin-induced aggregation of AChRs in cultured myotubes by more than 50%, suggesting a pathogenic role regarding the dysfunction of the neuromuscular endplate. These results indicate that LRP4 is a novel target for autoantibodies and is a diagnostic marker in seronegative MG patients.     

Autoantibodies to low-density lipoprotein receptor-related protein 4 in myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction, where acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein (LDL) receptor-related protein 4 (Lrp4) are essential. About 80% and 0% to 10% of patients with generalized MG have autoantibodies to AChR and MuSK, respectively, but pathogenic factors are elusive in others. Here we show that a proportion of AChR antibody-negative patients have autoantibodies to Lrp4. These antibodies inhibit binding of Lrp4 to its ligand and predominantly belong to the immunoglobulin G1 (IgG1) subclass, a complement activator. These findings together indicate the involvement of Lrp4 antibodies in the pathogenesis of AChR antibody-negative MG.     

Pathophysiology of myasthenia gravis

Myasthenia gravis (MG) is arguably the best understood autoimmune disease, and its study has also led to fundamental appreciation of mechanisms of neuromuscular transmission. MG is caused by antibodies against the acetylcholine receptor (AChR), which produce a compromise in the end-plate potential, reducing the safety factor for effective synaptic transmission. It is clear that AChR antibody destruction of the postsynaptic surface is dependent on complement activation. A muscle-specific kinase has been recently found to be an antigenic target in MG patients without antibodies against the AChR. Autoantibody production in MG is a T-cell-dependent process, but how a breakdown in tolerance occurs is not known. In MG there is an interesting differential involvement of muscle groups, in particular, the extraocular muscles. This article reviews normal neuromuscular transmission, mechanisms of the autoimmune process of MG, and differential susceptibility of eye muscles to MG.     

B cells in the pathophysiology of myasthenia gravis

Myasthenia gravis (MG) is an archetypal autoimmune disease. The pathology is characterized by autoantibodies to the acetylcholine receptor (AChR) in most patients or to muscle‐specific tyrosine kinase (MuSK) in others and to a growing number of other postsynaptic proteins in smaller subsets. A decrease in the number of functional AChRs or functional interruption of the AChR within the muscle end plate of the neuromuscular junction is caused by pathogenic autoantibodies. Although the molecular immunology underpinning the pathology is well understood, much remains to be learned about the cellular immunology contributing to the production of autoantibodies. This Review documents research concerning the immunopathology of MG, bringing together evidence principally from human studies with an emphasis on the role of adaptive immunity and B cells in particular. Proposed mechanisms for autoimmunity, which take into account that different types of MG may incorporate divergent immunopathology, are offered. Muscle Nerve 57 : 172–184, 2018     

Prevention of autoimmune attack by targeting specific T-cell receptors in a severe combined immunodeficiency mouse model of myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor. We have previously demonstrated a selection bias of CD4+ T cells expressing the Vbeta5.1 T-cell receptor gene in the thymus of HLA-DR3 patients with MG. To evaluate the pathogenicity of these cells, severe combined immunodeficiency mice engrafted with MG thymic lymphocytes were treated with anti-Vbeta5.1 antibody. Signs of pathogenicity (eg, acetylcholine receptor loss and complement deposits at the muscle end plates of chimeric mice) were prevented in anti-Vbeta5.1-treated severe combined immunodeficiency chimeras. Pathogenicity was mediated by autoantibodies against acetylcholine receptor. Thymic cells depleted of Vbeta5.1-positive cells in vitro before cell transfer were nonpathogenic, indicating that Vbeta5.1-positive cells are involved in the production of pathogenic autoantibodies. Acetylcholine receptor loss was prevented by Vbeta5.1 targeting in HLA-DR3 patients only, demonstrating specificity for HLA-DR3-peptide complexes. The action of the anti-Vbeta5.1 antibody involved both the in vivo depletion of Vbeta5.1-expressing cells and an increase in the interferon-gamma/interleukin-4 ratio, pointing to an immune deviation-based mechanism. This demonstration that a selective and specific T-helper cell population is involved in controlling pathogenic autoantibodies in MG holds promise for the treatment of MG.     

Myasthenia gravis and risks for comorbidity

Myasthenia gravis (MG) is an autoimmune disorder leading to skeletal muscle weakness and fatigability. MG subgroups are defined according to pathogenetic autoantibody (against acetylcholine receptor, muscle‐specific tyrosine kinase or lipoprotein receptor‐related protein 4), thymus pathology and clinical manifestations. MG patients have an increased risk for concordant autoimmune disease, in particular with early onset MG. Most common comorbidities are thyroid disease, systemic lupus erythematosus and rheumatoid arthritis. Cardiomyositis and subclinical heart dysfunction have been described in patients with thymoma MG and late onset MG but represent no major threat. A thymic lymphoepithelioma implies an increased risk for another cancer. Autoimmune MG represents no distinct cancer risk factor, although lymphomas and a few other cancer types have been reported with slightly increased frequency. Severe MG‐related muscle weakness means a risk for respiratory failure and respiratory tract infection. Drug MG treatment can lead to side‐effects. Thymectomy is regarded as a safe procedure both short and long term. Non‐MG‐related comorbidity represents a diagnostic and therapeutic challenge, especially in elderly patients. Diagnostic accuracy and optimal follow‐up is necessary to identify and treat all types of coexisting disease in MG.     

Pathogenic mechanisms of myasthenia gravis induced by antibodies against muscle-specific kinase]

Antibodies to acetylcholine receptor (AChR) are major cause of the human autoimmune disease, myasthenia gravis (MG). Additionally, autoantibodies against Muscle-specific kinase (MuSK) were found in a proportion of patients with generalized MG. After the identification of MuSK antibodies in MG patients, laboratory test for measuring antibodies to MuSK is now required to confirm the diagnosis of MG and the clinical treatment as well as AChR antibodies. MuSK is critical to the clustering of AChR and plays multiple roles at neuromuscular junctions (NMJ). However, it has been dispute concerning the pathogenicity of MuSK antibodies in muscle weakness of MG, as the experimental autoimmune MG caused by MuSK antibodies was absent. Here we describe the recent progress to understand the pathogenic roles of MuSK antibodies in muscle weakness of experimental animals induced by MuSK protein.     

The thymus in autoimmune Myasthenia Gravis: Paradigm for a tertiary lymphoid organ

In autoimmune Myasthenia Gravis (MG), a neuromuscular disease generally mediated by autoantibodies against the acetylcholine receptor (AChR), the muscle is the target organ of the autoimmune attack, while the thymus seems to be the primary production site of the autoantibodies. In the majority of patients with anti-AChR antibodies, it is characterized by the presence of germinal centers, which contain B cells that produce anti-AChR antibodies. In this review, we summarize recent results regarding neoangiogenic processes, cell infiltration and modified chemokine expression in the MG thymus, which are typical features of secondary lymphoid organs. The structural and functional changes in the MG thymus therefore allow us to declare it to be an archetype for tertiary lymphoid neogenesis providing optimal settings for the interaction between lymphocytes and antigen presenting cells in order to elicit an immune response. We further discuss factors that may have a key role in the transformation of the MG thymus into a tertiary lymphoid organ, such as IFN type I and dsRNA signaling. These factors could also be of importance in other autoimmune diseases, especially those characterized by tertiary lymphoid neogenesis.     

Clinical evaluation and management of myasthenia gravis

Myasthenia gravis (MG) is a syndrome of fluctuating skeletal muscle weakness that worsens with use and improves with rest. Eye, facial, oropharyngeal, axial, and limb muscles may be involved in varying combinations and degrees of severity. Its etiology is heterogeneous, divided initially between those rare congenital myasthenic syndromes, which are genetic, and the bulk of MG, which is acquired and autoimmune. The autoimmune conditions are divided in turn between those that possess measurable serum acetylcholine receptor (AChR) antibodies and a smaller group that does not. The latter group includes those MG patients who have serum antibodies to muscle-specific tyrosine kinase (MuSK). Therapeutic considerations differ for early-onset MG, late-onset MG, and MG associated with the presence of a thymoma. Most MG patients can be treated effectively, but there is still a need for more specific immunological approaches.     

AchR-positive myasthenia gravis with MRI evidence of early muscle atrophy

Muscle atrophy, when it occurs in myasthenia gravis (MG), is usually associated with long-standing disease or with chronic corticosteroid treatment. Early muscle atrophy in a steroid-naive patient has been reported previously only in muscle-specific tyrosine kinase (MuSK)-MG. We report a 63-year-old male patient with acetylcholine receptor (AchR)-positive MG with a short duration of disease, no steroid treatment and MRI evidence of muscle atrophy.     

Seronegative myasthenia gravis: clinical features, response to therapy and synthesis of acetylcholine receptor antibodies in vitro

Circulating autoantibodies against the acetylcholine receptor (AChR-Ab) are an important diagnostic tool in myasthenia gravis (MG). Lack of antibodies may cast doubt upon the diagnosis, the immune-mediated mechanism and the nature of the antigen. We examined clinical and laboratory features, response to immunotherapy and production of AChR-Ab in vitro, in 12 seronegative MG patients who were followed up for 2-30 years. It was possible to divide those patients into 2 groups: 7 patients with systemic muscle weakness, with a severe disease and with response to immunosuppressive therapies. The other group of 5 patients was characterized by oculobulbar symptomatology, a relatively benign course and immunotherapy was ineffective in 3 treated patients. Five patients underwent thymectomy and gland histology was normal in all of them. In none of 9 patients examined, were AChR-Ab synthesized in vitro (compared to 65% of seropositive myasthenic patients). Thus seronegative generalized MG is probably an autoimmune disease though the autoantigen is presently unknown and is responsive to immunosuppressive treatment. Seronegative oculobulbar MG might represent a separate disease entity in which immunological mechanisms play no significant role.     

Monozygous twins with neuromuscular transmission defects at opposite sides of the motor endplate

Disorders affecting the postsynaptic side of the neuromuscular junction include autoimmune myasthenia gravis (MG) as well as some of the congenital myasthenic syndromes (CMS). Lambert-Eaton myasthenic syndrome (LEMS) is an acquired autoimmune neuromuscular disorder in which autoantibodies are directed against the presynaptic calcium channels. Here we describe two monozygous twin brothers: case 1 was diagnosed with an indeterminate form of acquired postsynaptic neuromuscular junction defect at age 32 and case 2 with LEMS at age 47. Case 1 presented clinically with mild generalized myasthenic weakness, neurophysiological examination revealed disturbed neuromuscular transmission along with probable myositis and serum analysis regarding antibodies against the acetylcholine receptor and muscle-specific tyrosine kinase was negative. Case 2 presented with proximal muscle fatigue accompanied by areflexia at rest and antibodies against the P/Q-type voltage-gated calcium channels were present. Neurophysiologically, case 2 had reduced baseline compound motor action potential amplitudes on neurography, decrement on low-frequency repetitive nerve stimulation (RNS) and pathological increment on high frequency RNS. To our knowledge this is the first case report of its kind and adds an intriguing contrast to the more common diagnosis of CMS in monozygous twins.     

Frequency of seronegativity in adult-acquired generalized myasthenia gravis

We determined the prevalence of muscle acetylcholine receptor (AChR) antibodies in patients with adult-acquired generalized myasthenia gravis (MG), the seroconversion rate at 12 months, and the prevalence of muscle-specific tyrosine kinase (MuSK) antibody among persistently seronegative patients. We identified 562 consecutive Mayo Clinic patients with MG based on clinical and electrophysiological criteria. At presentation, 508 patients (90.4%) tested positive for AChR binding or AChR modulating antibodies. After 12 months, 15.2% of initially seronegative patients had become seropositive, yielding a seronegativity rate of 8.2% (95% confidence interval: 6.2-9.6%). Among seronegative patients not receiving immunosuppressants, 38% were MuSK antibody-positive and 43% were seropositive for nonmuscle autoantibodies. Classification as seronegative MG should be reserved for nonimmunosuppressed patients with generalized MG who lack muscle AChR binding, AChR modulating, or MuSK antibodies at presentation and at follow-up of at least 12 months.     

AChR抗体阳性重症肌无力合并舌肌萎缩2例报道并文献分析

目的 探讨重症肌无力(MG)患者合并舌肌萎缩的发病特点及机制。方法 分析武汉同济医院神经内科住院部2例乙酰胆碱受体(AChR)抗体阳性MG患者合并舌肌萎缩的临床资料,检索MG合并舌肌萎缩的文献报道,总结此类疾病的特点、萎缩机制、治疗方法及预后。结果 在临床上MG合并舌肌萎缩并不多见,大多数患者伴有血清肌肉特异性酪氨酸肌酶(MuSK)抗体阳性,伴AChR抗体阳性者较为罕见,肌肉萎缩的发病机制具体不详; 该类患者除发病时间较长外,通常提示更严重的病情、更差的治疗反应、更不良的预后。结论 MG舌肌萎缩临床少见,严重影响着患者的言语、咀嚼和吞咽等功能,除病程较长外,其肌肉萎缩机制暂不清楚,有待临床进一步探索,以改善此类患者预后和生活质量。     

Seronegative generalised myasthenia gravis: clinical features,antibodies, and their targets

Myasthenia gravis (MG) is a well-recognised disorder of neuromuscular transmission that can be diagnosed by the presence of antibodies to the acetylcholine receptor (AChR). However, some patients (about 15%) with generalised MG do not have detectable AChR antibodies. There is some evidence, however, that this "seronegative" MG is an antibody-mediated disorder. Plasma from patients with the disorder seems to contain various distinct humoral factors: IgG antibodies that reversibly inhibit AChR function; a non-IgG (possibly IgM) factor that indirectly inhibits AChR function; and an IgG antibody against the muscle-specific kinase (MuSK). The presence of antibodies against MuSK appears to define a subgroup of patients with seronegative MG who have predominantly localised, in many cases bulbar, muscle weaknesses (face, tongue, pharynx, etc) and reduced response to conventional immunosuppressive treatments. Moreover, muscle wasting may be present, which prevents complete response to these therapies.     

Editorial introduction: from the structure and functions of the neuromuscular junction to the diseases

The neuromuscular junction has been recognized as a site for autoimmune and genetic disorders. Myasthenia gravis (MG) is mainly caused by postsynaptic nicotinic acetylcholine receptor (AChR) IgG1 antibodies that are directed against α-subunit 67-76 and 125-147 and activate complement. Thymic abnormalities are present in the autoimmune background. A proportion of MG patients without conformation-dependent AChR antibodies assayed by the cell-based method have muscle-specific tyrosine kinase (MuSK) antibodies which are largely IgG4 and partially IgG1. MuSK is activated by Dok-7 and Lrp4 (agrin receptor) and contributes to AChR clustering at the postsynaptic membrane via various kinase cascades in collaboration with Wnt-MuSK/Frizzled-Dishevelled signaling. Rapsyn interacts with MuSK-linked chaperones to stabilize postsynaptic architecture and also contributes to AChR phosphorylation. MG-associated thymomas express antigens that trigger antibody responses which play a part in disease generation and modification. Among these, ryanodine receptor-1 (RyR1; acts on sarcoplasmic Ca2+ release) antibodies cause muscle contractile weakness. Transient receptor potential canonical-3 (TRPC3) antibodies are also detected in thymoma-associated MG patients; they may participate in muscle contractile weakness because TRPC3 acts on RyR1, and may also impair the refill of sarcoplasmic Ca2+ stores since TRPCs contribute to the receptor-operated Ca2+ influx via the phospholipase C (PLC)-diacylglycerol (DAG) pathway in cooperation with the store-operated, STIM1/Orai1-mediated Ca2+ influx and TRPCs-Homerl-IP3R interaction. Lambert-Eaton myasthenic syndrome (LEMS) is caused by reduced ACh quantal release that occurs mainly because of presynaptic P/Q-type voltage-gated Ca2+ channel (VGCC) antibodies. Physicians should be vigilant for LEMS because it may predict an underlying malignancy, particularly small-cell lung carcinoma; SOX-1 antibodies are usually present in these patients and are absent in those who do not have caucer. Some patients with LEMS have antibodies against synaptotagmin-1, which associates with SNARE complex and functions as Ca2+ sensor for exocytosis. The stimulation of the M1-type presynaptic muscarinic AChR (mAChR)(G-proterin-coupled receptor) can compensate for the deficiency of Ca2+-mediated ACh quantal release via the PLC/DAG-mediated mechanism; This acts in a manner similar to the BDNF/NT4-TrkB interaction. The detection of M1 mAChR antibodies in LEMS suggests an impaired compensatory mechanism and corresponds, at least in part, to autonomic symptoms. Congenital myasthenic syndromes are classified into presynaptic, synaptic basal lamina and postsynaptic defects.     

Myasthenia, thymoma, presynaptic antibodies, and a continuum of neuromuscular hyperexcitability.

BACKGROUND: Autoantibodies specific for the nicotinic acetylcholine receptor (AChR) of skeletal muscle impair neuromuscular transmission in myasthenia gravis (MG). Autoantibodies specific for alpha3 neuronal AChRs or voltage-gated potassium channels have been reported in patients with Isaacs syndrome, an acquired disorder of continuous muscle fiber activity characterized by neuromyotonia. OBJECTIVE: To report the neuromuscular autoantibody profiles of three patients with a syndrome of MG and neuromuscular hyperexcitability. RESULTS: All three patients reported here had clinical and electrophysiologic evidence of MG and neuromuscular hyperexcitability. None had neuromyotonia. Thymoma was proven in two patients and suspected in the third. One had MG and thymoma and subsequently developed cramp-fasciculation syndrome; MG and rippling muscle syndrome appeared simultaneously in the other two. All patients had muscle and neuronal AChR binding antibodies and striational antibodies. Only one had antibodies reactive with alpha-dendrotoxin-complexed potassium channels. CONCLUSIONS: The coexistence of cramp-fasciculation syndrome and acquired rippling muscle syndrome with MG, thymoma, and neuronal AChR autoantibodies suggests that there is a continuum of autoimmune neuromuscular hyperexcitability disorders related pathogenically to Isaacs syndrome. Manifestations of neuromuscular hyperexcitability may be altered and less apparent in the context of MG because of the coexisting defect of neuromuscular transmission.     

The severity of myasthenia gravis correlates with the serum concentration of titin and ryanodine receptor antibodies

BACKGROUND: Myasthenia gravis (MG) is caused by autoantibodies to the acetylcholine receptor (AChR). Non-AChR muscle autoantibodies are present in many MG serum samples, mainly from patients with thymoma or late-onset MG. The exact relationship between MG severity and several non-AChR muscle antibodies is unknown. OBJECTIVE: To study the correlation between the severity of MG and the concentration of antibodies against striated muscle tissue sections, titin, citric acid antigen, ryanodine receptor, and AChR. SETTING: The severity of MG was graded in 146 consecutive patients with MG, and their serum samples were tested for the presence of autoantibodies. Ten patients who were titin antibody positive were observed in longitudinal follow-up. RESULTS: No significant difference was found in MG severity between late-onset and thymoma MG. Titin, citric acid antigen, and ryanodine receptor antibodies occurred significantly more often among patients with severe MG than among patients with less severe disease. Changes in MG severity correlated with changes in titin antibody titer in the individual patient. Titin antibodies showed a better longitudinal correlation with disease severity than the AChR antibodies. CONCLUSIONS: Non-AChR muscle autoantibodies occurred more frequently in severe MG regardless of MG subgroup. Thymoma per se does not generate a more severe MG. It may well be the presence of a humoral immune response to non-AChR muscle antigens such as titin, citric acid antigen, and ryanodine receptor that leads to a severe disease, not the presence of thymoma or a late age of onset. These antibodies can serve as important prognostic markers in MG regardless of the presence of thymoma.     

Extracellular domains of the beta, gamma and epsilon subunits of the human acetylcholine receptor as immunoadsorbents for myasthenic autoantibodies: a combination of immunoadsorbents results in increased efficiency

Myasthenia gravis (MG) is usually caused by autoantibodies against the human muscle acetylcholine receptor (AChR). Plasmapheresis offers a therapeutic option, but, as well as removing the pathogenic anti-AChR autoantibodies, it non-specifically removes indispensable immunoglobulins. An attractive alternative to plasmapheresis would be the extracorporeal specific removal of the autoantibodies using AChR-based immunoadsorbents. Previously, we used the N-terminal extracellular domain (ECD) of the AChR alpha subunit to immunoadsorb anti-alpha subunit autoantibodies from MG sera. In this study, we immobilised the beta -, gamma- and epsilon-AChR ECDs on Sepharose and tested them as immunoadsorbents on 50 MG sera. A given ECD removed a different percentage of autoantibodies from different sera and different ECDs removed different percentages from the same serum; on average, the beta-, gamma- and epsilon-ECDs removed 22%, 20% and 15.5% of the autoantibodies, respectively. Immunoadsorption was completed in 3 min, 1 mug of ECD removed approximately 2 pmol of autoantibodies, and the immunoadsorbent could be recycled approximately 4 times. The combined use of two (alpha+gamma) or four (alpha+beta+gamma+epsilon) ECDs in a single immunoadsorbent resulted in much higher (often additive) immunoadsorption. These results show that MG sera have autoantibodies against several AChR subunits, and suggest that the combined use of all AChR ECDs could provide the basis for a novel, antigen-specific therapy for MG.