Myasthenia Gravis and Related Disorders

C H A P T E R

53 Myasthenia Gravis and Related Disorders Valentina Damato1,2, Stuart Viegas3 and Angela Vincent1,4 1

Nuffield Department of Clinical Neurosciences, Oxford University, Oxford, United Kingdom 2Department of Neuroscience, Institute of Neurology, Catholic University, Rome, Italy 3Department of Neurology, Charing Cross Hospital, Imperial College NHS Trust, London, United Kingdom 4Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom

O U T L I N E Introduction The Neuromuscular Junction Neuromuscular Transmission Acetylcholine Receptor and Muscle-Specific Kinase, the Main Antigenic Targets

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Myasthenia Gravis Epidemiology Etiology of Myasthenia General Clinical Aspects

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Clinical Heterogeneity of Myasthenia Different Forms Related to Antibodies and Thymic Pathology Early-Onset Acetylcholine Receptor-Antibody Positive Myasthenia Gravis Late-Onset Acetylcholine Receptor-Antibody Myasthenia Gravis Thymoma Associated Myasthenia Gravis Muscle-Specific Kinase Antibody Positive Myasthenia Gravis Neonatal Myasthenia Gravis

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Antibodies in Myasthenia Evidence for Pathogenicity of Acetylcholine Receptor and Muscle-Specific Kinase Antibodies Acetylcholine Receptor Antibodies Characteristics and Mechanisms Muscle-Specific Kinase Antibodies

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The Autoimmune Diseases, 6th. DOI: https://doi.org/10.1016/B978-0-12-812102-3.00053-1

The Role of Muscle-Specific Kinase in Neuromuscular Junction Development and Maintenance LRP4 Antibodies Novel Targets

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The Thymus and Cellular Immunity in Myasthenia Gravis 1021 Role of T Lymphocytes in Myasthenia Gravis 1021 Advances in the Cellular Immunology of Acetylcholine Receptor Myasthenia Gravis 1022 The Thymus in Myasthenia Gravis 1023 Thymoma 1024 Treatments in Myasthenia Gravis General Approach Biologics

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Lambert Eaton Myasthenic Syndrome Introduction Epidemiology and Etiology Clinical Features Investigation and Treatment Pathophysiology

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Conclusions and Future Prospects

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References

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Copyright © 2020 Elsevier Inc. All rights reserved.

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53. MYASTHENIA GRAVIS AND RELATED DISORDERS

INTRODUCTION In recent years, the recognition and ability to detect antibodies directed against receptors, ion channels, and relevant proteins within both the peripheral and central nervous system has continued to evolve. Nevertheless, myasthenia gravis (MG) and related autoimmune disorders of the neuromuscular junction remain the paradigm diseases, serving to highlight those features which help define an antibody mediated disorder. The history of the discoveries in this archetypal autoimmune disease is summarized in Table 53.1.

The Neuromuscular Junction The neuromuscular junction (NMJ) consists of the presynaptic motor nerve terminal and the postsynaptic motor “endplate.” At the NMJ, the distal motor axon loses its myelin sheath and expands to form the boutons of the presynaptic nerve terminal. These contain mitochondria and the synaptic vesicles that store the acetylcholine (ACh). The vesicles are organized within specialized active zones alongside voltage-gated calcium channels TABLE 53.1

A History of Myasthenia Gravis Research

Date

Key observation

Reference

1672

Thomas Willis publishes what is arguably the first clinical description of MG

Willis

1895

Jolly shows that defect is at the neuromuscular junction

Jolly

1913

Thymectomy appears to produce clinical improvement in patients with thymoma or nonthymomatous MG

Sauerbruch

1934

Mary Walker demonstrates the effectiveness of cholinesterase inhibitors as treatment

Walker (1934)

1960

Simpson proposes that MG is caused by antibodies to an “endplate” protein

Simpson(1960)

1962

The snake toxin, α-Bungarotoxin, can be used as a label for AChRs at the neuromuscular junction

Chang and Lee (1963)

1964

Elmqvist and colleagues show that the miniature endplate potentials are reduced in MG

Elmqvist et al. (1964)

1971

Several groups begin to purify AChRs from electric organs of electric rays using affinity chromatography on neurotoxin columns

1973

Immunization against purified electric ray AChR leads to an EAMG in rabbits

Patrick and Lindstrom (1973)

1973

AChRs are reduced in number at neuromuscular junctions, as determined by 125I-α-Bungarotoxin binding

Fambrough et al. (1973)

1975

MG can be passively transferred to mice by injection of patients’ IgG

Toyka et al. (1975)

1939

125

I-

Blalock

1976

MG patients have AChR antibodies as shown by radioimmunoprecipitation of α-Bungarotoxin-tagged AChRs

Lindstrom et al. (1976)

1976 78

Plasma exchange produces striking clinical improvement in MG, which correlates inversely with AChR antibody levels

Newsom-Davis et al. (1978)

1977

IgG and complement are present at the neuromuscular junctions in MG patients and in mice with EAMG

Engel et al. (1977)

1980

MG can present with different HLA, thymic pathology, age at onset, and muscle antibodies

Compston et al. (1980)

1981

The MG thymus contains plasma cells making AChR antibody

Scadding et al. (1981)

1977 Present

Experimental autoimmune model used to determine pathogenic and immunological mechanisms

on-going

1984 Present

Study of T cells from MG patients and their responses to the AChR epitopes

on-going

1995 Present

Study of immune mechanisms in the MG thymus

on-going

2001 Present

Discovery of MuSK antibodies and their distinctive mechanisms

on-going

2010 present

Development of new biologics and benefits of Rituximab and Eculizumab demonstrated in MG

on-going

MG, Myasthenia gravis; AChR, acetylcholine receptor; MuSK, muscle-specific kinase; EAMG, experimental autoimmune myasthenia gravis.

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FIGURE 53.1 Ion channel targets for autoantibodies at the neuromuscular junction. Neuromuscular transmission depends on the calcium-dependent release of vesicles of ACh. ACh binds to the AChRs on the postsynaptic membrane resulting in a depolarization which, if it reaches a critical threshold, initiates an action potential in the muscle leading to contraction. ACh is immediately destroyed by acetylcholinesterase. AChRs, VGCCs, and VGKCs are all targets for the antibody mediated neurological diseases. The receptor tyrosine kinase MuSK and the protein that activates it, LRP4, have been found to be targets for antibodies in a proportion of patients with MG without AChR antibodies.

(VGCCs). In addition, voltage-gated potassium channels (VGKCs) are also present on the presynaptic nerve terminal (Fig. 53.1). The postsynaptic membrane is deeply infolded to create “junctional” folds. The crests of these, lying in close alignment to the presynaptic active zones, contain the highest density of acetylcholine receptors (AChR). At the depths of these folds, there are relatively few AChRs, but an abundance of voltage-gated sodium channels (VGSC). The synaptic cleft between the presynaptic and postsynaptic membranes contains the basal lamina composed of collagen IV, heparan sulfate and laminin, and many other proteins. The enzyme acetylcholinesterase is anchored to the basal lamina through its collagen-like tail (ColQ). The clustering of the AChRs is critical for efficient neurotransmission. As will be discussed below, the development and maintenance of the NMJ structure are dependent on a number of key proteins: agrin, low-density lipoprotein receptor related protein 4 (LRP4), muscle-specific kinase (MuSK), docking protein 7 (DOK7), and rapsyn (Singhal and Martin, 2011) (see Fig. 53.1) in addition to other intracellular proteins that are less well characterized.

Neuromuscular Transmission Neuromuscular transmission in mature muscle begins with propagation of the action potential into the motor nerve terminal. This depolarization causes opening of the VGCCs and the resulting calcium influx results in the fusion of the ACh containing vesicles and release of ACh. This is terminated by the closing of VGCCs and the opening of the VGKCs with subsequent repolarization of the nerve terminal. ACh binding to the AChR results in the opening of the AChR central ion pore and a localized depolarization of the motor endplate. If sufficient, this will cause the opening of the VGSCs and propagation of the action potential along the muscle fiber to initiate contraction. The amount of ACh released by a single vesicle is termed a quantum. The spontaneous release of a single quantum is responsible for the generation of a local depolarization, termed a miniature endplate potential (MEPP); a nerve impulse releases multiple vesicles (around 25 30 in humans) leading to a greater depolarization, termed the endplate potential (EPP). It is possible to calculate the number of quanta released, the quantal content, from these parameters (Wood and Slater, 2001). An inherent safety factor exists in normal muscles, whereby more ACh is released than is required to reach the activation threshold for the opening of the VGSCs.

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FIGURE 53.2 The AChR (A,B). The AChR is a transmembrane protein with (α)2, β, γ, and δ subunits in the fetal form and (α)2, β, δ, and ε subunits in the adult form. A high proportion of antibodies in MG bind to the main immunogenic regions that are on both the α subunits. In addition, many patients’ antibodies bind to the fetal-specific γ subunit. In some cases, antibodies that inhibit the function of the fetal form, selectively, cross the placenta causing fetal muscle paralysis with severe and often fatal deformities. MG, Myasthenia gravis; AChR, acetylcholine receptor; MuSK, muscle-specific kinase; VGCC, voltage-gated calcium channel; VGKC, voltage-gated potassium channel; Ach, acetylcholine.

At the neuromuscular junction, as described above, the AChRs are clustered by the agrin/LRP4/Musk/ DOK7 pathway but this clustering process is balanced by binding of ACh that stimulates AChR dispersal. Any defect in the clustering process, as occurs in patients with MuSK antibodies, will be followed by dispersal of the AChRs.

Acetylcholine Receptor and Muscle-Specific Kinase, the Main Antigenic Targets The nicotinic AChR remains the major antigenic target in MG. The AChR is a pentameric ligand-gated ion channel that exists in adult and fetal isoforms. The adult form consists of two α-subunits and one each of the β-, δ-, and ε-subunits, with each subunit being composed of a large extracellular domain, glycosylation sites, and four transmembrane domains. During development, the fetal-specific γ subunit is replaced by the ε-subunit to form the adult isoform. If the muscle is denervated by nerve injury, the fetal isoform expressing the γ subunit is expressed throughout the muscle until reinnervation occurs. The AChR subunits are organized around a central ion channel (Fig. 53.2). The two ACh binding sites are between the α- and ε- or γ-subunits and between the α- and the δ-subunits, respectively. Both sites need to be occupied for the ion channel to be in the open state. The main immunogenic region is a conformation dependent region on the extracellular component of each of the α-subunits (Lindstrom, 2000). MuSK is a receptor tyrosine kinase. The extracellular portion consists of three immunoglobulin-like domains and a cysteine-rich domain. The intracellular portion consists of a juxtamembrane domain, followed by a tyrosine kinase catalytic domain. MuSK is critical both for the development (DeChiara et al., 1996) and ongoing maintenance (Kong et al., 2004) of the NMJ. The role of MuSK and its coreceptor LRP4 is described in detail below (see MuSK antibodies).

MYASTHENIA GRAVIS Epidemiology Myasthenia affects all races and can occur at any age from the first year of life to the age of ninety. In the Western countries, MG with AChR antibodies (AChR-MG) typically shows two age peaks, in the third decade in

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females and in the sixth and seventh decades in males. While the frequency of the early-onset disease has remained unchanged, an increased incidence in the elderly has been consistently reported in the last two decades. The explanation could be the increased longevity of the population and the improvement in diagnosis but these factors only do not appear to be responsible. A metaanalysis of 55 published studies calculated a pooled incidence rate of 5.3 per million person-years (CI 4.4 6.1) and a pooled prevalence rate of 77.7 per million (CI 64 93) (Carr et al., 2010), but a prevalence approaching 150 per million was suggested in a recent study (Andersen et al., 2014). Interestingly, in Asian populations, particularly the Chinese, there is a high prevalence of limited forms of the disease with childhood onset (Zhang et al., 2007). The incidence and prevalence is difficult to evaluate because of different healthcare systems but it is possible that there is a specific genetic predisposition to childhood MG in these populations or that there is an environmental contribution. The incidence of MuSK and LRP4 antibody forms of MG is much lower and difficult to assess. For MuSK-MG, it appears to vary with latitude with the highest rates in Italy, Spain, and Turkey—all Mediterranean countries— compared with much lower rates in northern Europe. It is not uncommon in Japan but relatively infrequent in the Chinese (Yeh et al., 2004).

Etiology of Myasthenia In the majority of cases, no single cause is identifiable. There is genetic predisposition, which most likely reflects the contribution of polymorphic MHC class I and II loci. Other possible genetic susceptibility markers include the AChR alpha subunit (Garchon et al., 1994; Giraud et al., 2007), Immunoglobulin G (IgG) heavy and light chains (Dondi et al., 1994), Fc gamma receptor IIa (Amdahl et al., 2007), TAP (Hjelmstrom et al., 1997), CTLA4 (Wang et al., 2008), and PTPN22 (Provenzano et al., 2012). Molecular mimicry between AChR subunits and microbial proteins has been proposed as a possible initiating process with autosensitization against muscle AChR occurring as a result of determinant spreading, but there is little robust evidence. Certain drugs, notably penicillamine, may also trigger the development of a reversible form of MG in genetically susceptible individuals (Drosos et al., 1993) but changes in clinical practice means that these cases are seldom seen now.

General Clinical Aspects MG is an autoimmune disorder characterized by fatigable muscle weakness. It often involves the extraocular muscles at onset causing diplopia and/or ptosis. If it remains confined to these muscles, it is termed ocular MG. If other muscle groups are involved, often facial, axial, limb, bulbar, and respiratory muscles, it is termed generalized MG. Bulbar and respiratory involvement can be life-threatening. The diagnosis rests on a compatible clinical presentation supported by serological confirmation (see below) and/or electromyographic evidence (with repetitive nerve stimulation and/or single fiber electromyography) of a defect in neurotransmission. MG is commonly associated with thymic abnormalities, notably thymic hyperplasia and thymoma, and appropriate imaging of the thymus is therefore recommended at presentation. Symptomatic treatment includes the use of cholinesterase inhibitors but the majority of cases will also require immunosuppressive agents including the corticosteroids and steroid sparing agents (azathioprine, mycophenolate mofetil, methotrexate, and cyclosporine). Thymectomy is a therapeutic option in younger patients with detectable AChR antibodies and a recent study has shown its beneficial effect (Wolfe et al., 2016). Intravenous immunoglobulin and plasma exchange may be employed in severe, life-threatening, or refractory disease. Newer biological agents, such as the monoclonal anti-CD20 agent, Rituximab (RTX) have shown promise in refractory cases (Maddison et al., 2011). Most of these aspects are discussed in more detail below. The treatment modalities available for MG are more fully reviewed elsewhere (Sanders and Evoli, 2010).

CLINICAL HETEROGENEITY OF MYASTHENIA Different Forms Related to Antibodies and Thymic Pathology MG is not a single disease entity but can be classified into different groups. Defined serologically, it is possible to delineate five main subgroups of MG (see Table 53.2) that differ also by means of age of onset, HLA association, thymic pathology (Compston et al., 1980), and presence of antibodies directed against non-AChR

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53. MYASTHENIA GRAVIS AND RELATED DISORDERS

MG (Myasthenia Gravis) Patients Divided on the Basis of Antibody Status, Age at Onset, Thymic Pathology and HLA

Subtype of MG

Age at onset

Sex M:F

Typical thymic pathology

HLA association

Associated autoantibodies

Early-onset

,51 years

1:3

Thymitis or hyperplasia

B8, DR3

AChR. May have other tissue antibodies, e.g., thyroid

Thymoma associated

Mainly 40 60 years

1:1

Epithelial tumor containing many lymphocytes

No clear association

AChR. Titin and ryanodine receptor antibodies very common. Also cytokine antibodies

Late-onset

.50 years

1.5:1

Normal or atrophied

B7, DR2 in males

AChR. Titin and ryanodine receptor antibodies common, particularly after age 60 years

AChR antibody negative MuSK antibody positive

2 70 years

1:3

Normal or atrophied in most

Not known

MuSK. Other antibodies very uncommon

AChR antibody negative MuSK antibody negative

1 80 years

2:3

Mild thymitis/ hyperplasia in some

DR14.DQ5

Cell-based assays can demonstrate antibodies to clustered AChR, MuSK, and/or LRP4 in a proportion

These subgroups are not appropriate in patients with purely ocular MG and in other ethnic populations. AChR, Acetylcholine receptor; MuSK, muscle-specific kinase.

proteins. The latter group includes MuSK, LRP4 and the striational muscle proteins, titin and ryanodine receptor (RyR).

Early-Onset Acetylcholine Receptor-Antibody Positive Myasthenia Gravis These patients are usually defined as presenting before the age of the 50 years. There is a marked female predominance and an incidence that has remained relatively stable for many years. There is an association with HLA A1, B8, DR3, DR2, and DR52 amongst Northern Europeans (Compston et al., 1980; Janer et al., 1999; Hill et al., 1999) and HLA DPB1, DQB1, and DR9 amongst the Japanese (Horiki et al., 1994). Childhood onset AChRAb MG is relatively rare in North Europeans but more prevalent amongst Oriental populations as mentioned above (Vincent et al., 2001). Clinically, the MG often involves extraocular muscles at onset before generalizing, although a proportion will remain purely ocular. The early response to cholinesterase inhibitors is usually good but the majority of patients will still require some form of immunosuppressive therapy. The thymus is typically hyperplastic and thymectomy is a therapeutic option in early-onset generalized AChR-Ab MG, with current available evidence suggestive of benefit in over half of cases (see below). Antibodies (Abs) against titin and RyR are rarely found in the earlyonset cases and should raise concerns regarding a thymic tumor.

Late-Onset Acetylcholine Receptor-Antibody Myasthenia Gravis By conventional definition, these patients present after 50 years of age and males exceed females by 3:2 ratio. Employing a registry to identify all individuals with positive AChR antibody levels, it has been found that the age specific incidence rises between 45 and 75 years before rapidly falling (Vincent et al., 2003). There is a weak association with HLA B7, DR2 (Compston et al., 1980) and DR4, DQw8 (Carlsson et al., 1990). Clinically, these patients have a similar phenotype to the early-onset form, although ocular MG may be more common (Zivkovic et al., 2012). The overall response to immunosuppressive treatment is similar to early-onset disease but a greater proportion of patients will encounter side effects, presumably due to comorbid disease (Sanders and Evoli, 2010). However, the thymus is typically atrophic unlike the hyperplastic early-onset thymus. The response to thymectomy is poorer and it is not routinely offered to patients over 60 years of age. Over half of these late-onset cases have detectable Abs against titin and RyR (Buckley et al., 2001) whilst 25% have Abs against the cytokines, interferon-α, or interleukin-12 (Meager et al., 2003). Titin and RyR Abs are more prevalent in thymoma cases and some authors have speculated that their presence in late-onset MG may represent an immune response against occult thymomas that are subsequently destroyed (Marx et al., 2010).

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Thymoma Associated Myasthenia Gravis Thymomas are tumors derived from thymic epithelial cells, thereby distinguishing them from lymphoma, neuroendocrine, and germ cell tumors. These are conventionally classified by means of the WHO classification (A, AB, B, and C). A coexisting thymoma is identified in 10% of the MG patients. It can occur at any age but is most common amongst the 40 60 age group. There is no gender difference or consistent HLA association. Clinically, MG is generalized with detectable AChR antibodies, although ocular and seronegative cases have been reported (Maggi et al., 2008). Myasthenia may be more refractory to treatment than other forms of MG (Sanders and Evoli, 2010). Following thymectomy, AChR Ab levels do not necessarily fall without additional treatments but in contrast to early-onset disease, myasthenia rarely improves without further immunotherapies (Somnier, 1994). Serum Abs against striated muscle were recognized first in the 1960s. Their major targets are two intracellular proteins, titin and the RyR, both of which are expressed in thymoma (Skeie et al., 1997; Mygland et al., 1995). These antibodies are typically observed in .90% of the thymoma associated MG cases but there are no convincing data supporting their pathogenic role and Abs against the AChR are invariably identified. Neutralizing Abs against interferon-α and interleukin-12 are observed in approximately 70% and 50% of the cases, respectively (Buckley et al., 2001; Meager et al., 2003). These are useful markers for identification of recurrence which occurs in about 10% of the thymomas.

Muscle-Specific Kinase Antibody Positive Myasthenia Gravis These patients can present at any age, peaking in the 30s with a female predominance. There is significant worldwide variation with a correlation with geographical latitude, suggesting potential environmental influences (Vincent et al., 2008). On the other hand, despite the small numbers in individual studies, a significant association with HLA DR14 and DQ5 in a Dutch cohort (Niks et al., 2006) and DRB16 and DQB5 in an Italian cohort (Bartoccioni et al., 2009) have been identified suggesting genetic susceptibility. It is not yet clear whether environmental or genetic factors or both predispose to this form of MG. Clinically, the phenotype is often different from AChR-MG with prominent ocular, bulbar, neck, and respiratory weakness (Evoli et al., 2003; Sanders et al., 2003). Muscle wasting and atrophy of the tongue and facial muscles may be evident both clinically and radiologically (Farrugia et al., 2006). The response to treatment can also differ with a comparatively poorer response (and frequent intolerance) to cholinesterase inhibitors (Evoli et al., 2003; Pasnoor et al., 2010). A proportion can be refractory to conventional immunosuppressive treatment (Evoli et al., 2003). In such cases, plasma exchange may be more effective than intravenous immunoglobulin (Pasnoor et al., 2010) but interestingly, rituximab (RTX) may be more effective in MuSK patients than in those with AChR antibodies (Maddison et al., 2011; Diaz-Manera et al., 2012). The thymus is typically normal or atrophic (Evoli et al., 2008; Leite et al., 2005) in direct comparison to that seen in AChR-Ab MG, and most centers do not perform thymectomy. Thymoma is very rare in MuSK-MG (Saka et al., 2005).

Neonatal Myasthenia Gravis This is caused by passive transfer of maternal antibodies across the placenta. It may occur in up to 10% of the female patients with AChR antibodies (Vincent et al., 2001). The affected newborn babies exhibit transient symptomatic weakness, requiring the use of cholinesterase inhibitors for a few weeks. Rarely, it can occur in women who are symptom free but have AChR antibodies. Arthrogryposis multiplex congenita is a condition where the newborn have multiple joint contractures as a consequence of absent fetal movement in the uterus. It can occur if there are high levels of maternal antibodies directed against the fetal isoform (see Fig. 53.2) of the AChR (Barnes et al., 1995). These antibodies can block the ion channel function of the fetal isoform leading to paralysis during development. The adult isoform takes over during the third trimester and thus, although the condition is not usually reversible, it does not progress after birth. The pathogenic mechanisms were examined in a mouse model of maternal-to-fetal transfer (Jacobson et al., 1999b). There are also rare case reports of neonatal MG occurring in MuSK MG with both transient (Niks et al., 2008) and more persistent disease (Behin et al., 2008) described.

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ANTIBODIES IN MYASTHENIA Evidence for Pathogenicity of Acetylcholine Receptor and Muscle-Specific Kinase Antibodies Both AChR and MuSK antibodies are pathogenic, satisfying the strict criteria required for establishing causation in autoimmune disease (Rose and Bona, 1993). Both are directed against autoantigens that are highly relevant to a disorder of neurotransmission and are highly specific for MG. The main criteria in these diseases are the passive transfer of MG from man to animal (usually mice or rats) and the response to treatments that reduce Ab levels. Passive transfer models involve injection of IgG from MG patients into animals and lead either to objective weakness or at least to neurophysiological evidence of impaired neurotransmission. Plasma exchange dramatically reduces antibody levels within a few days and leads to striking clinical improvement even in patients with long-standing disease. In addition, maternal fetal transfer of the disease has been reported in both serological forms of MG. Further in vivo evidence comes from replication of the human disease in animals that have been immunized with the relevant antigen, termed experimental autoimmune MG (EAMG). This active immunization model has been used extensively to study the immune biology of MG, as will be mentioned below.

Acetylcholine Receptor Antibodies AChR Abs were first detected by means of a radioimmunoprecipitation assay (RIA) employing 125I α-bungarotoxin that binds strongly to AChRs and labels AChRs in detergent extracts of human muscle (Lindstrom et al., 1976). Modern RIAs employ AChR extracted from muscle cell lines expressing mixtures of fetal and adult AChRs (Beeson et al., 1996). Directly radiolabeled recombinant MuSK are used for detection of MuSK antibodies (Matthews et al., 2004). Enzyme linked immunosorbent assays are not found to be as sensitive or as specific in our hands. AChR antibodies belong to variable IgG subclasses, although IgG1 and IgG3 predominate (Rodgaard et al., 1987; Vincent et al., 1987). A significant proportion of Abs are directed against the MIR on α-subunits, although other sites (Whiting et al., 1986) and other AChR subunits (Jacobson et al., 1999a) can also be targets. These Abs have a high affinity (around 100 pM) and are highly specific for the intact receptor with limited binding to recombinant polypeptides or denatured AChR subunits; the Abs bind predominantly to the extracellular portion of the receptor. The diagnostic sensitivity of the RIA is high in adult-onset generalized MG (80% 85%) but quite low in ocular MG (around 50%) (Wong et al., 2014) and in prepubertal-onset disease (50% 70%) (Finnis and Jayawant, 2011). Between patients, there is variation in the Ab specificity, isoelectric heterogeneity, and avidity for the AChR and no clear correlation with disease activity is observed. Although some reports indicate a poor correlation between Ab levels and clinical severity in MG, the Ab titer can be useful in monitoring disease activity in individual patients (Vincent and Newsom Davis, 1980). This association has been reported after thymectomy, plasma exchange, or immunotherapies (Heldal et al., 2014). Moreover, in a recent study including 223 patients with ocular presentation, AChR antibodies were detected in 71% of the cases and an increased antibody titer predicted the risk for MG generalization (Peeler et al., 2015). In generalized MG, 85% have AChR Abs and 0% 10% have MuSK Abs; a rather variable but usually low percentage may have LRP4 Abs, although there are few systematic studies at present and assays are not well standardized. There are very rare case reports of patients with both AChR and MuSK Abs (Saulat et al., 2007; Rajakulendran et al., 2012) and LRP4 Abs may coexist with much higher levels of MuSK Abs (Higuchi et al., 2011) raising questions about their importance. Cell-based assays (CBAs) have now been developed that use human embryonic kidney (HEK) cells transfected with DNA for the antigen of interest that is then expressed on the cell surface. Indirect immunofluorescence can be used to detect the binding of patients’ Abs. This method is sensitive and, importantly, measures potentially pathogenic Abs that only bind to extracellular determinants of the antigen (Leite et al., 2010). This was demonstrated in previously seronegative cases using cells transfected with AChR subunits which were clustered with the scaffold protein, rapsyn (Leite et al., 2008). CBAs have also been developed to detect MuSK or LRP4 Abs (Higuchi et al., 2011) as mentioned below. The “clustered AChR” Abs were found in 38% of the RIA-seronegative MG patients and were useful in confirming the MG diagnosis especially in childhood, ocular, and mild disease forms (Rodriguez Cruz et al., 2015). More recently, a French study reported the presence of clustered AChR Abs in 16% of the adult patients with generalized MG with a phenotype resembling those positive on standard RIA but with a milder disease course

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as shown previously. Interestingly, thymectomy was performed in a proportion of patients with evidence of thymoma in one case (Devic et al., 2014). In future, these results will need confirmation by larger, multicenter studies. Of relevance, experimental studies of passive transfer with purified patients’ IgG into mice resulted in the reduction of MEPP amplitudes indicating the loss of postsynaptic AChR that mirrors that found in typical MG patients (Jacob et al., 2012) confirming the pathogenicity of the clustered AChR Abs (Vincent and Newsom Davis, 1980).

Characteristics and Mechanisms AChR Abs cause loss of AChR through three principal mechanisms. These include complement mediated destruction, crosslinking and accelerated degradation, and functional blockade. AChR Abs belong predominantly to the complement fixing IgG1 and IgG3 subclasses (Rodgaard et al., 1987) that are divalent for the AChR and can therefore cause neuromuscular transmission failure by internalizing the AChRs or activating complement (Rodgaard et al., 1987; Toyka et al., 1975). Internalization or antigenic modulation is an increase in the normal rate of turnover of the AChRs that is observed in both MG patients (Drachman et al., 1978) and EAMG (Lindstrom and Einarson, 1979). It is most prominent when Abs are directed against the MIR (Tzartos et al., 1991) and lead to reduced AChR expression on the postsynaptic membrane without membrane damage. The activation of complement results in the generation of the membrane activation complex that is responsible for lysis and destruction of the postsynaptic membrane, loss of postsynaptic folding, and ultimately loss of AChR and related proteins. IgG colocalizing with activated complement was shown both in EAMG (Sahashi et al., 1978) and MG (Engel et al., 1977) muscle biopsies. The importance of complement in the pathogenesis of this form of MG came from several lines of evidence. Depletion (Lennon et al., 1978), inhibition (Biesecker and Gomez, 1989), or blockade (Piddlesden et al., 1996) of complement all resulted in resistance to developing the disease in EAMG. Further evidence was provided by the study of transgenic mice lacking components of the classical complement cascade (Tuzun et al., 2003) and complement (Morgan et al., 2006) and recently anticomplement therapy has been found effective in severe cases (see Biologics below). By contrast, direct block of ACh binding to the AChR or of the ion channel itself appears to be uncommon in majority of the patients. Whatever the mechanisms, failure of neuromuscular transmission results from the loss of AChRs, which leads to reduced MEPP and EPP amplitudes. The reduced EPP amplitude falls below the required threshold to initiate an action potential, leading to blocking of neurotransmission. There is partial compensation for this through an increase in AChR synthesis by the muscle and in the number of quanta of ACh released, which appears to be a compensatory mechanism in both MG and EAMG (Plomp et al., 1995).

Muscle-Specific Kinase Antibodies MuSK is a receptor tyrosine kinase critical for the formation and maintenance of the neuromuscular junction. The extracellular portion consists of three immunoglobulin-like domains and a cysteine-rich domain. MuSK-Abs are detected by RIA in 40% of the AChR-negative patients with variable frequency across populations. CBA techniques can be applied to the detection of MuSK-Abs and may improve the diagnosis in RIA-negative patients. A multicenter study investigating seronegative sera from 13 countries searching for MuSK-Abs by CBA achieved positive results in 13% of the samples (Tsonis et al., 2015) but many of the Abs detected were of the IgM type and their specificity is still uncertain. Recently, a study from the Oxford group found that 8% of the RIA-negative MG samples bound MuSK antigen expressed on HEK cells. Their pathogenic role on AChR clustering was confirmed in vitro in C2C12 myotubes (Huda et al., 2017). Interestingly, in this study, the patients with MuSK-Abs only on CBA had a milder phenotype than those positive on standard RIA.

The Role of Muscle-Specific Kinase in Neuromuscular Junction Development and Maintenance To understand the effects of antibodies in MuSK-MG, it is necessary to appreciate the complex mechanism of neuromuscular junction formation and maintenance, for which MuSK has a critical role (Kong et al., 2004). During development, a large soluble protein N-agrin is released from the motor nerve and binds to the MuSK coreceptor, LRP4; this binds to and activates MuSK phosphorylation. Phosphorylated MuSK recruits the intracellular protein DOK7 that is also phosphorylated and together they initiate the complex pathway that results in the clustering of rapsyn and AChRs on top of the postsynaptic folds under the nerve terminals. This process can be

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studied to a large extent in cultured mouse C2C12 myotubes. The C2C12 myoblasts can be induced by serum deprivation to fuse and become myotubes with high AChR expression throughout the membrane. When agrin is added to the myotubes, the LRP4/MuSK/DOK7 pathway is activated and AChRs form high density clusters ( . 3 μm) on the myotube surface. These clusters can be observed and quantified using fluorescently labeled alpha-bungarotoxin, the snake toxin that is used extensively to measure muscle AChRs and for the RIA. If DOK7 is virally transduced into the C2C12 cells, the AChR clusters occur without the requirement of agrin, LRP4, or MuSK, showing that DOK7 is the ultimate initiator of the clustering process. These phenomena can be used to study the effects of MuSK and LRP4 Abs. MuSK Abs belong predominantly to the noncomplement fixing IgG4 subclass (McConville et al., 2004). These Abs are able to undergo Fab arm exchange to produce bispecific Abs that do not cross-link identical antigens (van der Zee et al., 1986), and which therefore function monovalently (Schuurman et al., 1999). This suggests that the effector mechanisms are unlikely to be the same as those in AChR mediated disease. MuSK Abs have been shown to be monovalent for MuSK but they can interfere with protein protein interactions (Koneczny et al., 2017). MuSK-Abs bind to an epitope within the first Ig-like MuSK domain, preventing its interaction with LRP4, which results in the impairment of the AChR clustering pathway (Huijbers et al., 2013; Koneczny et al., 2013). A recent study showed that the level of IgG4 directed against the first Ig-like MuSK domain correlates better with disease severity than the total MuSK Ab titer (Huijbers et al., 2016) while no correlation was found with Abs directed to the other MuSK epitopes (e.g., Frizzled-like domain). Some studies have also found that MuSK interacts with ColQ, a collagen-like protein that anchors AChE to the basal lamina in the synaptic cleft. MuSK Abs inhibit the binding of LRP4 to MuSK and thus interfere with the agrin induced clustering pathway. The divalent IgG1-3 Abs do not block LRP4 binding to MuSK. Nevertheless, both IgG4 and IgG1-3 MuSK Abs inhibit agrin induced AChR clustering in the C2C12 myotubes, suggesting that the IgG1-3 antibodies alter MuSK function in an LRP4-independent manner (Koneczny et al., 2013). Since the IgG1-3 antibodies are less frequent, their functional roles, which could include complement mediated damage, have not been explored adequately in animal models. There are very little data from human muscle, and the only published pathological study has demonstrated normal motor endplates, normal AChR numbers, and little evidence of complement deposition (Shiraishi et al., 2005). By contrast, both active immunization against MuSK (Shigemoto et al., 2006; Viegas et al., 2012) and passive transfer of human MuSK Ab IgG (Cole et al., 2008) models have demonstrated AChR loss in clinically affected animals, although complement deposition has not been observed and complement deficient mice remain susceptible to the disease (Mori et al., 2012). Of particular interest, combined pre- and postsynaptic morphological changes have been observed in animal models (Cole et al., 2008; Richman et al., 2012) and may explain the severe phenotype that is observed in the mice and in patients. There is also electrophysiological evidence of both pre- and postsynaptic defects (Viegas et al., 2012; Klooster et al., 2012) with the failure of the presynaptic compensatory mechanism (Plomp et al., 1995) further impacting on underlying neurotransmission.

LRP4 Antibodies The proportion of LRP4-positive patients among the AChR/MuSK seronegative cases varies widely from 0% to 50% in different reports, probably due to the different techniques applied, such as luciferase reporter immunoprecipitation (Higuchi et al., 2011), ELISA (Zhang et al., 2012), and CBA (Pevzner et al., 2012; Marino et al., 2015). In a recent European multicenter study, LRP4 Abs were detected in 18.7% of the 635 seronegative patients, with a difference in the frequency among different countries, suggesting a possible role of ethnicity in disease susceptibility (Zisimopoulou et al., 2014). A recent Chinese study reported a frequency of LRP4 antibodies of only 4%, associated with AChR and MuSK-Abs in a proportion of cases (Li Y, Muscle Nerve, 2017). LRP4 Abs were predominantly of the IgG1 subclass and their potential pathogenicity has been demonstrated in active immunization models by disrupting agrin LRP4 binding to MuSK and by complement activation (Shen et al., 2013). However, there have been no reports on passive transfer models from human LRP4 Abs. Moreover, somewhat worryingly, LRP4 Abs have been reported in a significant proportion of other neurological diseases including neuromyelitis optica (NMO) (Zhang et al., 2012) and amyotrophic lateral sclerosis (Tzartos et al., 2014). Future studies are needed in order to standardize the assays and clarify the pathogenicity potential of LRP4. Most of the positive patients have a mild generalized disease. In some of the patients, a double association with MuSK (predominantly) or AChR Abs were considered responsible for a more severe disease course

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(Higuchi et al., 2011; Zhang et al., 2012; Pevzner et al., 2012; Marino et al., 2015). There is a female and earlyonset preponderance (only 16% were .50 years). Thymic hyperplasia can occur but there are no reports of thymoma.

Novel Targets While the search for novel antigens in MG is keenly pursued, there are still some patients without detectable Abs. The frequency of the so-called triple-negative cases is variable. Abs to agrin, ColQ, and cortactin have recently been described in MG patients (Zhang et al., 2014; Zoltowska Katarzyna et al., 2015; Gallardo et al., 2014; Cortes-Vicente et al., 2016) but their role in the disease pathogenesis has not been proved and they are not currently used in routine diagnostic assays. Antibodies against rapsyn have also been described (Agius et al., 1998), but as the antigen is intracellular, the Abs are unlikely to be pathogenic. One of the problems with many studies on triple negative cases is that the sera are seldom taken at the onset of symptoms and most patients have been treated with immunotherapies. In addition, it is possible that some of the antibodies that are found are secondary to the disease process and not the primary pathogenic entity.

THE THYMUS AND CELLULAR IMMUNITY IN MYASTHENIA GRAVIS Role of T Lymphocytes in Myasthenia Gravis High affinity AChR Abs are thought to be dependent on CD4 1 T lymphocytes but as these are rarely observed in myasthenic muscle and almost never found at the neuromuscular junction; they are not thought to be effector cells in MG. Nevertheless, their critical role in the autoimmune pathogenesis is demonstrated through several lines of evidence in both MG and EAMG. EAMG was first described following the immunization of rabbits with AChR purified from the electric organ of Torpedo californica (Patrick and Lindstrom, 1973) and later reproduced in a number of other species. In the murine model, there are both disease sensitive and resistant strains, related to their different H-2 alleles (Berman and Patrick, 1980). AChR-specific CD4 1 T lymphocytes occur in both the peripheral blood and thymuses of MG patients. They may also be observed in the healthy controls but the clinical improvement observed following their removal with anti-CD4 monoclonal Abs (Ahlberg et al., 1994) and in HIV (Nath et al., 1990) supports a pathogenic role for these lymphocytes. These isolated CD4 1 T lymphocytes may respond to stimulation with the intact AChR, recombinant subunits or AChR peptides (Conti-Fine et al., 1998; Wang et al., 1998), and T cell lines or clones propagated from MG patients will respond more vigorously to stimulation in vitro than those derived from the healthy controls. The epitopes are most commonly found on the α-subunit of the AChR (e.g., Ong et al., 1991). In EAMG, a dominant epitope within the α146-162 activates MHC class II restricted CD4 1 T lymphocytes, leading to pathogenic antibody production (Christadoss et al., 2000). No clearly immunodominant epitope has been identified reproducibly in a high proportion of MG patients, although an epsilon subunit epitope was identified in some (Hill et al., 1999). The TCR Vβ families show preferential expansion of the Vβ 4 and 6 amongst MG patients (Navaneetham et al., 1998) whilst mice lacking Vβ 6 respond poorly to immunization with AChR (Krco et al., 1991; Ahlberg et al., 1994). Mice genetically deficient in functioning CD4 1 T lymphocytes don’t develop EAMG (Kaul et al., 1994) whilst severe combined immunodeficient mice will only produce AChR Abs and develop myasthenic symptoms if the human grafted cells contain CD4 1 T lymphocytes (Wang et al., 1999). CD4 1 T lymphocytes and the cytokines they secrete will influence the type of autoimmune response generated in both MG and EAMG. Analysis of blood from MG patients has confirmed the presence of T helper 1 (Th1), Th2, Th17, and T regulatory (Treg; Foxp1) cells (Li et al., 2008) but the role of these T lymphocyte subsets in the development of the disease is best examined using transgenic mice. Interleukin-12 (IL-12) is essential for promoting development of Th1 cells and IL-12 2 / 2 mice are resistant to the development of EAMG despite a significant Ab response (Karachunski et al., 2000). The role of IFN-γ remains unclear with conflicting reports of EAMG susceptibility (Balasa et al., 1997; Wang et al., 2007). IL-4 appears to be either neutral (Balasa et al., 1998) or confer a protective effect (Karachunski et al., 1999). There is an apparent increase in Th17 and decrease in Tregs during development of EAMG in rats (Mu et al., 2009) and administration of Tregs to myasthenic rats inhibited progression of EAMG (Aricha et al., 2008). Initial studies identified no change in Treg numbers in MG subjects compared with the healthy controls (Huang et al., 2004), although a specific functional impairment in those Foxp3 1 Tregs (Balandina et al., 2005) was subsequently identified.

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T cell activation requires the interaction of TCR/MHC peptide in addition to the interaction between CD28/ CTLA4 on the T lymphocytes and CD80 (B7) on antigen presenting cells (the CD28 CD80 interaction). It also requires the crosslinking of the CD40 ligand (CD40 CD40L interaction). Using transgenic mice, it has been demonstrated that both interactions are essential for the primary immune response (Shi et al., 1998), although their contribution to the secondary response is thought to be less prominent. Here, the CD278 (inducible T-cell costimulator) is thought to be important for the secondary response (Scott et al., 2004).

Advances in the Cellular Immunology of Acetylcholine Receptor Myasthenia Gravis The breakdown of central and peripheral tolerance checkpoints is an important aspect involved in the pathogenesis of MG. A recent study showed that the frequency of autoreactive B cell receptors was higher in patients with AChR and MuSK MG than in the healthy controls (Lee et al., 2016). In addition, studies on regulatory B and T cells provide new insights into the involvement of defective immune regulatory pathways in the immunopathogenic mechanisms likely responsible for MG. The understanding of the mechanisms that account for B-cell shift from a proinflammatory to a regulatory phenotype and defective B cell tolerance has important therapeutic implications in terms of developing durable B and T cell targeted therapies for MG. The B-cell population is functionally heterogeneous and B cells can be divided into different subsets according to the cytokine that they produce. One functional B cell subset, regulatory B cells (Bregs), has recently been shown to downregulate the immune response through the production of IL-10, IL-35, and TGF-β (Fillatreau et al., 2008; Shen et al., 2014). In particular, IL-10 appears to inhibit dendritic cell production of IL-12 and to suppress Th1 and Th17 cell responses in several models of autoimmune diseases (Li et al., 2012; Quan et al., 2013). The most accepted cell markers to identify Bregs are considered CD1d 1 CD5 1 and CD24 1 CD38 1 B cells, which are phenotypically indistinguishable from transitional immature B cells and which have been shown to produce the highest percentage of IL-10. The involvement of CD19 1 CD24 1 CD38 1 immature Bregs in autoimmunity was first described in studies on RTX-treated Systemic lupus erythematosus patients, in which the disease remission was associated with a reconstituted B cell population predominantly characterized by immature Bregs (Blair et al., 2010). This led to the hypothesis that after the B cell depletion therapy, the newly repopulated B cells are constituted by competent Bregs that can efficiently suppress the immune response and restore the immune balance in favor of tolerance (Palanichamy et al., 2009). Both frequency and function of Bregs were reduced in MG patients compared with the healthy controls and were associated with disease activity (Sheng et al., 2016; Karim et al., 2017; Yi et al., 2017) and response to RTX (Sun et al., 2014). These findings were confirmed in a recent study showing a lower percentage of Bregs in patients with MuSK MG (Guptill et al., 2015). From these findings, Bregs may serve as a marker for disease activity in MG patients and as a promising therapeutic target. Early studies searching for AChR-specific T cells were technically demanding and have not proven to be particularly helpful in understanding the disease etiology or providing new targets for therapies. More recently the emphasis has been on the regulatory T cells. Thymic T CD4 1 lymphocytes include Tregs involved in self-tolerance mechanisms and follicular T helper cells (ThF) that play a critical role in B lymphocyte differentiation and affinity maturation. Treg lymphocytes play important immunosuppressive functions (Sakaguchi et al., 2010; Campbell and Koch, 2011) and both quantitative and functional alterations of CD4 1 CD25 1 FoxP3 1 Tregs have been described in several autoimmune pathologies (Long and Buckner, 2011; Noack and Miossec, 2014) but with contradictory results. In MG, where it is well known that thymus pathology is implicated in the pathogenesis, Treg should be even more relevant. Reduced Tregs were found in a recent study on AChR-MG patients, in which different subpopulations of Treg were significantly reduced after the thymectomy and immunosuppression and were, in general, lower than in healthy subjects (Kohler et al., 2017). Therapies focused on ameliorating Treg function through enhancing (Long and Buckner, 2011) their suppressive activity or their migration appeared to be promising in preclinical models of MG (Sheng et al., 2006) because of the adoptive transfer of ex vivo generated Treg (Aricha et al., 2008). A subpopulation of Treg, called follicular regulatory T (Tfr) cells, are thought to control the function of ThF that promote B cell maturation and high affinity Ab production in germinal centers. An abnormal production of thymus derived ThF might be involved in the development of several autoimmune diseases including MG. A recent study showed an increased frequency of ThF and a decreased rate of Tfr in the peripheral blood of MG patients compared to the healthy control that inversely correlated with the disease severity (Wen et al., 2016). These results were confirmed in the patients with generalized MG in a study which showed a correlation between ThF, plasma cell frequency, and AChR-Ab titer (Zhang et al., 2016).

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The Thymus in Myasthenia Gravis Thymus is involved in the maintenance of self-tolerance. While MuSK-MG is rarely associated with thymus alterations, in AChR-MG thymus changes are common, especially in the early-onset subtype. In case of thymoma, thymectomy is always mandatory while the rationale for thymectomy in nonthymomatous patients is related to the role of the thymus follicular hyperplasia (TFH) in the pathogenesis of the disease. Several studies showed that the TFH includes infiltrates of lymphocytes- and AChR-specific germinal centers that can be targeted by complement factors (Leite et al., 2007). The thymus is an epithelial organ that can be morphologically divided into a distinct cortex, medulla and corticomedullary zone. The cortex contains densely packed immature lymphocytes alongside a sparse population of epithelial cells and bone marrow derived macrophages. The medulla is less cellular containing more mature T lymphocytes, more prominent epithelial cells, dendritic cells, B lymphocytes, and rare myoid cells (Pearse, 2006). The thymus has a critical role in self-tolerance with a fine balance between the generation of protective T lymphocytes and deletion of autoreactive T lymphocytes required. Relevant autoantigens including the α-subunit of the AChR are expressed on medullary thymic epithelial cells (mTECs) under the control of the autoimmune regulator gene (AIRE) (Giraud et al., 2007). Central T cell tolerance relies on the close interaction between these mTECs and the nearby dendritic cells and their effect on the T lymphocyte development and subsequent differentiation. The thymus is thought to have a critical role in the development of early-onset AChR antibody-positive MG. The cortex is typically normal but the medulla contains lymphocytic infiltrates and germinal centers with distinct areas of B lymphocyte proliferation, differentiation, somatic hyper-mutation, and immunoglobulin class switching. A typical pathological section of an MG thymus is shown in Fig. 53.3. These B lymphocytes, when cultured in vitro, are capable of secreting AChR Abs spontaneously (Vincent et al., 1978; Scadding et al., 1981). It is, therefore, to be expected that antibody levels fall post thymectomy (Vincent et al., 1983) but they seldom disappear. Whether early-onset AChR antibody-positive MG begins in the thymus, or whether these changes are a reflection of a systemic process, remains unresolved. Individual AChR subunits are expressed on mTECs (Salmon et al., 1998), presumably as part of a self-tolerance mechanism, and these are targeted by both autoantibodies (Safar et al., 1991) and complement (Leite et al., 2007). Native AChR is also expressed by the muscle-like myoid cells, which whilst comparatively rare, are more abundant in the hyperplastic thymus (Kirchner et al., 1986). The germinal centers appear to be focused around these myoid cells. Given that they lack MHC class II or costimulatory molecules, they rely on the antigen presenting dendritic cells to prime the CD4 1 T lymphocytes. One proposed multistep hypothesis is that the mTECs first present epitopes from isolated AChR subunits to CD4 1 T lymphocytes, evoking the production of early antibodies capable of attacking the thymic myoid cells

FIGURE 53.3 Hyperplastic thymus with the presence of GC in an early-onset MG patient with AChR antibodies.Double CD79a (red)/ CK19 (brown) immunostaining. Intense CD79a 1 staining marks the periphery of germinal centers, CK19 is specific for the thymic epithelial tissue. Original magnifications, 3 250. MG, Myasthenia gravis; AChR, acetylcholine receptor; GC, germinal centers. Source: Courtesy of Professor L. Lauriola, Institute of Pathology, Catholic University, Roma.

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that express the intact AChR. As the immune response continues to proliferate, germinal centers are formed that allow these antibodies to diversify allowing recognition of native AChRs (Willcox et al., 2008). The thymus is typically involuted and atrophic in older patients. The aging thymus is gradually replaced with fat, although residual foci of mTECs may persist and myoid cells are only rarely encountered. The histological analysis of the thymus tissue from the late-onset MG cases previously suggested no differences from normal controls (Myking et al., 1998). Nevertheless, a more recent study looking at young and late-onset MG cases identified residual lymphocyte accumulation amongst the older cohort and no qualitative differences between the two groups (Ishii et al., 2007).

Thymoma Thymomas are heterogenous neoplasms of thymic epithelial cells (TEC) with mixed cortical and medullary markers. They may develop from either early TEC progenitors or from more mature cortical or medullary TECs (Hasserjian et al., 2005). They are responsible for the generation of numerous maturing polyclonal T lymphocytes (thymocytes) capable of maturing into CD4 1 or CD8 1 T lymphocytes. The degree of thymopoiesis is known to vary according to the thymoma subtype (Nenninger et al., 1998). The B2 subtype is the most common type associated with MG (accounting for B50% of the cases) and contains an abundance of mature CD4 1 T lymphocytes ready for export (Strobel et al., 2004). It should be noted that corticosteroids can deplete the immature T lymphocytes and thereby modify the histological subtype. There are certain features in thymomas that are likely to promote inefficient self-tolerance including defective AIRE and HLA class II expression, an absence of myoid cells, failure to generate Foxp3 1 Treg cells as well as defective T lymphocyte signaling (Marx et al., 2010). Other antigenic targets are also recognized which is unsurprising given the wide range of systemic, hematological, endocrine, cutaneous, gastrointestinal, and renal disorders associated with thymoma (Marx et al., 2010). A lower production of Treg was observed in thymoma and confirmed by immunohistochemistry (Scarpino et al., 2007). Genetic aberrations and polymorphisms may also be identified in thymoma. These include HLA genes (notably loci at 6p21) which may affect MHC class II expression and non-HLA genes (including CTLA4 and PTPN22) which influence T-cell receptor signaling.

TREATMENTS IN MYASTHENIA GRAVIS General Approach The current treatment for MG consists of symptomatic therapy with acetylcholinesterase inhibitors and generalized immunosuppression with corticosteroids, azathioprine, mycophenolate, and immunomodulation with intravenous immunoglobulin and plasmapheresis. Thymectomy is common in early-onset MG. In response to the need for more effective or more rapid treatments in some patients, biologics have been applied to MG management as described below. The efficacy of thymectomy in early AChR-MG was suggested in several small case series but results from the thymectomy trial (MGTX) have been recently published (Wolfe et al., 2016). This multicenter (40 centers included) trial was conducted between 2006 and 2012 and recruited 126 nonthymomatous AChR-MG patients comparing prednisone alone versus prednisone with transsternal thymectomy. Patients who underwent thymectomy had a lower time-weighted average Quantitative myasthenia gravis (QMG) score over a 3-year period and required lower average alternate-day prednisone than those who received prednisone alone. Moreover, immunosuppressive treatment, need for hospitalization, and treatment side effects were less in the thymectomy group. The MGTX trial represents a milestone in the management of MG patients even if it leaves some open questions, such as the efficacy of thymectomy in late-onset AChR-MG, where the thymus is normally atrophic or in other MG types and the benefit from less invasive surgical approaches (Keijzers et al., 2015).

Biologics Several recent studies have demonstrated the benefits of Rituximab (RTX)-mediated B cell depletion in refractory MG, especially in MuSK MG (Iorio et al., 2015). RTX, a monoclonal antibody targeting CD20 on B cells, led

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to a sustained clinical improvement in parallel to a reduction or discontinuation of corticosteroid and plasma exchange treatments. The expression of the CD20 antigen is restricted to the late pre-B-cell stage and is maintained until their differentiation to plasma cells when expression is usually lost. Interestingly RTX targeting the precursor of plasma cells depletes the niche of short-lived plasma cells while it has no effect on the long-lived plasma cell subset (Winter et al., 2012). It has been hypothesized that MuSK IgG4 Abs are produced almost exclusively by short-lived plasma cells (Diaz-Manera et al., 2012), which may explain why RTX is particularly effective in MuSK-MG and why MuSK antibody titer markedly decreases after RTX treatment. On the other hand, this finding might be related to a modification of the B-cell repertoire induced by RTX with the disappearance of the initially expanded populations and the reconstitution of a diverse B-cell repertoire that might account for its long-lasting effect. RTX appeared to be safe and effective even in patients with refractory AChR-MG, who were observed to have a long-lasting response after treatment and a significant decrease of the Ab titers that correlated with the clinical improvement (Robeson et al., 2017). The ongoing trial in AChR-MG will provide more definite conclusions on the efficacy and safety of this treatment but one of the main concerns regards the number of reinfusions needed to prevent a relapse. Based on evidences from NMO studies, the reemerging of CD27 1 memory B cells to 0.05% of the peripheral blood cells has been recommended as target value to repeat RTX in MG (Lebrun et al., 2016). Eculizumab, a humanized monoclonal antibody, inhibits the formation of terminal complement complex by preventing the enzymatic cleavage of complement 5 (C5). It was tried for the first time in MG in a small trial involving 14 refractory AChR-MG patients with promising results (Howard et al., 2013). Currently, a phase III study is assessing the efficacy of eculizumab in refractory AChR-MG. Belimumab, a human monoclonal antibody that prevents B-cell activating factor (BAFF) from binding to B cells provoking the inhibition of B cells proliferation and survival, has not been shown to be effective in a phase II trial in patients with AChR-MG and MuSK-MG (clinicaltrialgov.com: NCT01480596).

LAMBERT EATON MYASTHENIC SYNDROME Introduction The Lambert Eaton Myasthenic Syndrome (Wirtz et al.) is clinically and electrophysiologically distinct from MG. Approximately 50% of the LEMS cases are paraneoplastic, typically associated with small cell lung carcinoma (SCLC). There are certain features that make it possible to distinguish paraneoplastic and nonparaneoplastic forms.

Epidemiology and Etiology LEMS is less common than MG with an annual incidence of 0.48 per million in a Dutch study (Wirtz et al., 2003). The median age of onset is 60 years in both the paraneoplastic and nonparaneoplastic forms (O’Neill et al., 1988) although there is another smaller peak at 35 years in the nonparaneoplastic forms (Titulaer et al., 2011a). There is a male predominance in the paraneoplastic form and a slight female predominance in the nonparaneoplastic form. In the latter, similar to early-onset MG, there is an association with HLA B8 and DR3.

Clinical Features The cardinal clinical features of LEMS include muscle weakness, autonomic dysfunction, and areflexia. Typical findings include proximal muscle weakness that is more marked in the lower limbs. Ocular and bulbar symptoms may occur later in the disease course. The speed of progression is often more rapid in the paraneoplastic form (Titulaer et al., 2008). Autonomic involvement is seen in over 80% of the cases. Commonly encountered symptoms include dry mouth, erectile dysfunction, and constipation. Micturition difficulties and orthostatic syncope are less common. In contrast to MG, the muscle strength in LEMS will improve after a period of maximal voluntary contraction. A recent update on the clinical features (Titulaer et al., 2011a) and a clinical method predicting the presence of a small cell cancer can be found elsewhere (Titulaer et al., 2011b).

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Investigation and Treatment Electromyography will confirm a disorder of neurotransmission although a significant increase in the compound muscle action potential following a period of exercise or high frequency stimulation allows electrophysiological differentiation from MG. Serological confirmation involves detection of antibodies against the P/Q type voltage-gated calcium channels which are found in approximately 90% of the LEMS cases and are invariably present in paraneoplastic SCLC cases (see below). To detect the antibodies, the P/Q type VGCCs are extracted from mammalian brain, labeled with 125I ω-conotoxin which binds specifically to these VGCCs and used in an RIA. In addition, antibodies against N type (30% 40%) and L type (25%) VGCCs may be present (Motomura et al., 1997; Johnston et al., 1994). Other antibodies which have been identified include those against Synaptotagmin (Takamori et al., 1995) and more recently against SOX-1 in 65% of the paraneoplastic cases and around 5% of the nonparaneoplastic cases (Sabater et al., 2008; Titulaer et al., 2009). Symptomatic treatment requires 3,4-diaminopyridine (3-4-DAP). If additional treatment is required, the corticosteroids and other immunosuppressive agents are used. Intravenous immunoglobulin, plasma exchange, and RTX may be used in severe and refractory cases. Any underlying SCLC should be treated in appropriate manner. Treatment options are reviewed in more detail (Titulaer et al., 2011a).

Pathophysiology VGCC contains several subunits but the α1-subunit is primarily responsible for the biochemical and electrophysiological functions of the protein. Similar to the MIR on the α-subunit of the AChR, there may be particularly immunogenic sequences; 50% of the LEMS patients have antibodies against linker domains on the α1subunit (Takamori et al., 1997). Freeze fracture electron microscope studies of the presynaptic motor nerve terminal have demonstrated an ordered array of intramembranous particles. These are located close to the site of transmitter exocytosis. There is some evidence that VGCC constitutes at least some of the intramembranous particles in the active zone (Robitaille et al., 1990). LEMS patients have both a reduction in the total number of active zone particles and the number of particles per active zone (Nagel et al., 1988). The evidence for the pathogenic nature of VGCC comes from both clinical and experimental studies. Patients respond to plasma exchange (Newsom-Davis et al., 1982) and cases of maternal-to-fetal transfer of the disease have also been reported (Lecky, 2006). Passive transfer with LEMS plasma or IgG produces the same neurophysiological abnormalities in mice (Lang et al., 1983; Fukunaga et al., 1983), although no weakness was observed. Further studies confirmed the localization of IgG close to the presynaptic active zones (Fukuoka et al., 1987). In addition, active immunization of rats with peptides from the α1 subunit led to mild weakness and compatible neurophysiological changes (Komai et al., 1999). Finally, mice with mutations in the P/Q type VGCC (CACNA1a) share some of the electrophysiological characteristics of LEMS (Kaja et al., 2007).

CONCLUSIONS AND FUTURE PROSPECTS Historically, the classification of MG has been based on AChR Ab status, age of onset, and thymic pathology. MuSK antibodies were first recognized 17 years ago and whilst there was some initial skepticism about their relevance, it is now well established that they are pathogenic, satisfying all the necessary criteria for causation in autoimmune diseases. Defined serologically, this particular form of MG is typically more severe and more refractory to conventional immunosuppressive therapy. From an immunological perspective, the predominance of noncomplement fixing IgG4 Abs in a subclass of MG patients contrasts with the predominantly IgG1 AChR Abs in the classical form and raises interesting questions regarding the mechanisms involved in initiation of these two, otherwise similar, forms of autoimmune disease, and the different treatments that might be optimal. Over recent years further advances in serological assays for MG have been made. Alongside established RIA for detecting AChR and MuSK Abs, cell-based assays have been developed that helped to identify clustered AChR Abs and then more recently LRP4 Abs. Given that LRP4 Abs are predominantly of the IgG1 subclass, it is likely that the role of complement may be more akin to that observed in AChR Ab mediated MG, but LRP4 Abs are usually low titer and most patients have relatively mild disease.

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There have been numerous studies on the thymus histology and immunology that is so clearly different from the thymus in healthy individuals. However, it is still unclear to what extent the observed differences are primary to the disease or secondary features associated with the lymphocytic infiltrations and germinal centers that are hall marks of the early-onset disease. Moreover, very few studies have been done on the late-onset MG patients who now represent the greatest and increasing number of patients. The underlying immunological mechanisms may be similar but they don’t appear to be present in the thymus. Conventional treatment for MG relies on symptomatic treatment, oral immunosuppressive agents, and immunomodulatory treatment with intravenous immunoglobulin and plasma exchange. Newer biological agents, such as RTX have shown promise and may benefit those with refractory disease, particularly in patients with MuSKMG. Thymectomy has been shown to be enhance the beneficial effects of steroids and recent trials inhibiting complement inhibition (Eculizumab) have shown promise for severe cases. In the future, our greater understanding of the immunology of MG may allow the development of other targeted immunotherapies. Potential drugs could include those directed against B lymphocyte proliferation, relevant cytokines and their receptors, lymphocyte adhesion, and migration pathways.

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