Stuart Viegas

IgG1 antibodies to acetylcholine receptors in ‘seronegative’ myasthenia gravis

Only around 80% of patients with generalized myasthenia gravis (MG) have serum antibodies to acetylcholine receptor [AChR; acetylcholine receptor antibody positive myasthenia gravis (AChR-MG)] by the radioimmunoprecipitation assay used worldwide. Antibodies to muscle specific kinase [MuSK; MuSK antibody positive myasthenia gravis (MuSK-MG)] make up a variable proportion of the remaining 20%. The patients with neither AChR nor MuSK antibodies are often called seronegative (seronegative MG, SNMG). There is accumulating evidence that SNMG patients are similar to AChR-MG in clinical features and thymic pathology. We hypothesized that SNMG patients have low-affinity antibodies to AChR that cannot be detected in solution phase assays, but would be detected by binding to the AChRs on the cell membrane, particularly if they were clustered at the high density that is found at the neuromuscular junction. We expressed recombinant AChR subunits with the clustering protein, rapsyn, in human embryonic kidney cells and tested for binding of antibodies by immunofluorescence. To identify AChRs, we tagged either AChR or rapsyn with enhanced green fluorescence protein, and visualized human antibodies with Alexa Fluor-labelled secondary or tertiary antibodies, or by fluorescence-activated cell sorter (FACS). We correlated the results with the thymic pathology where available. We detected AChR antibodies to rapsyn-clustered AChR in 66% (25/38) of sera previously negative for binding to AChR in solution and confirmed the results with FACS. The antibodies were mainly IgG1 subclass and showed ability to activate complement. In addition, there was a correlation between serum binding to clustered AChR and complement deposition on myoid cells in patients’ thymus tissue. A similar approach was used to demonstrate that MuSK antibodies, although mainly IgG4, were partially IgG1 subclass and capable of activating complement when bound to MuSK on the cell surface. These observations throw new light on different forms of MG paving the way for improved diagnosis and management, and the approaches used have applicability to other antibody-mediated conditions.

Myasthenia Gravis Seronegative for Acetylcholine Receptor Antibodies

Antibodies to muscle‐specific kinase (MuSK) are found in a variable proportion of patients with myasthenia without typical acetylcholine receptor (AChR) antibodies, but their characteristics and pathogenic mechanisms are not fully understood. We discuss the incidence and pathogenicity of MuSK antibodies and how clinical studies, animal models, and cultured cell lines can be used to elucidate their pathogenic mechanisms. Patients without either AChR or MuSK antibodies (seronegative myasthenia) appear to present another disease subtype that is highly similar to that of typical myasthenia gravis. We demonstrate a new method that detects AChR antibodies in these patients and show that these low‐affinity AChR antibodies are predominantly IgG1 and can activate complement C3b deposition. Similarly MuSK antibodies, although mainly IgG4, are partially IgG1 and can activate C3b deposition. Overall, these results suggest that complement‐activation may be an important pathogenic mechanism even in patients without conventional AChR antibodies.

Myasthenia gravis and other neuromuscular junction disorders

Myasthenia gravis is the most common autoimmune disease affecting the neuromuscular junction and is characterised by painless fatigable muscle weakness. It is caused by autoantibodies against neuromuscular junction proteins, either the nicotinic acetylcholine receptor (AChR) or the muscle specific tyrosine kinase (MuSK). Mutations in neuromuscular junction proteins cause congenital myasthenic syndromes. Other antibody mediated conditions affecting the neuromuscular junction include Lambert Eaton myasthenic syndrome and neuromyotonia. Figure 1 The neuromuscular junction and the proteins involved in neuromuscular transmission. Several of the proteins at the neuromuscular junction are targets for autoimmune disorders (AChR and MuSK in myasthenia gravis, VGCC in Lambert–Eaton myasthenic syndrome and VGKC in neuromyotonia). Genetic mutations can affect several of these proteins (AChR, Rapysn, MuSK, Dok-7, etc) causing congenital myasthenic syndromes. ACh, acetylcholine; AChE, acetylcholinesterase; AChR, acetylcholine receptor; MuSK, muscle specific tyrosine kinase; VGCC, voltage gated calcium channel; VGKC, voltage gated potassium channel; VGSC voltage gated sodium channel.

Myasthenia Gravis and Related Disorders

Myasthenia gravis (MG) and the Lambert–Eaton myasthenic syndrome (LEMS) are the prototypical autoimmune channelopathies of the peripheral nervous system. The predominant neuromuscular junction antigenic targets are either postsynaptic (MG; nicotinic acetylcholine receptor (AChR)) or presynaptic (LEMS; voltage-gated calcium channels (VGCCs)). Over the last 35 years the development of active immunization, passive transfer models, and use of transgenic animals has greatly enhanced our understanding of the biology of these disorders. The search for other antibodies in previously “seronegative” MG cases has led to the recognition of antibodies against clustered AChRs, muscle-specific kinase (MuSK) and low density lipoprotein receptor-related protein 4 (Lrp4). The pathogenic mechanisms that underlie these newer serological subclasses of MG are not yet fully understood, but are likely to be different from AChR antibody-mediated disease. While many still would consider MG an antibody-mediated disorder, it is well recognized that AChR-specific T lymphocytes and the thymus are critical to the autoimmune pathogenesis. In LEMS, new antibodies have been found including those against the intracellular protein SOX-1, which may help better predict those patients with a paraneoplastic disorder. This chapter aims to summarize the important clinical, immunological, and therapeutic aspects of both MG and LEMS, as well as highlighting recent developments in the field.

Serological and experimental studies in different forms of myasthenia gravis

Antibodies to the acetylcholine receptor (AChR) have been recognized for over 40 years and have been important in the diagnosis of myasthenia gravis (MG), and its recognition in patients of different ages and thymic pathologies. The 10–20% of patients who do not have AChR antibodies are now known to comprise different subgroups, the most commonly reported of which is patients with antibodies to muscle‐specific kinase (MuSK). The use of cell‐based assays has extended the repertoire of antibody tests to clustered AChRs, low‐density lipoprotein receptor–related protein 4, and agrin. Autoantibodies against intracellular targets, namely cortactin, titin, and ryanodine receptor (the latter two being associated with the presence of thymoma), may also be helpful as biomarkers in some patients. IgG4 MuSK antibodies are clearly pathogenic, but the coexisting IgG1, IgG2, and IgG3 antibodies, collectively, have effects that question the dominance of IgG4 as the sole pathologic factor in MuSK MG. After a brief historical review, we define the different subgroups and summarize the antibody characteristics. Experiments to demonstrate the in vitro and in vivo pathogenic roles of MuSK antibodies are discussed.

Clinical and immunological correlates in myasthenia gravisassociated with musk antibodies [Abstract]

HALL A From basic to clinical neuroimmunology: An introductory course Chairs: G. Martino, H. Willison SESSION Basic Neuroimmunology 09.00–09.45 Microglia at the interface between the immune and the nervous system Serge Rivest 09.45–10.30 Regulatory and effector T cells in the central nervous system Roberto Furlan 10.30–11.00 COFFEE BREAK 11.00–11.45 Ion channels and nerve degeneration Stephen G. Waxman 11.45–12.30 Genetics of neuroinflammation Maja Jagodic 12.30–14.30 LUNCH