Pre- and postsynaptic neuromuscular junction abnormalities in musk myasthenia.

Autoantibodies to muscle-specific kinase (MuSK) can cause myasthenia gravis (MG). The pathophysiological mechanism remains unknown. We report in vitro electrophysiological and histological studies of the neuromuscular junction in a MuSK MG patient. Low levels of presynaptic acetylcholine release and small miniature endplate potentials were found. This combination of pre- and postsynaptic abnormalities was supported by histology, revealing partially denervated postsynaptic areas, and some degeneration of postsynaptic folds. Results suggest that anti-MuSK antibodies reduce the stability of muscle-nerve contact.

Suppression of terminal axonal sprouting at the neuromuscular junction by monoclonal antibodies against a muscle-derived antigen of 56,000 daltons.

After the partial denervation or paralysis of a muscle, the remaining motor axon terminals may sprout fine, neuritic processes (terminal sprouts) which escape the endplate region of the neuromuscular junction. We previously identified a muscle-derived, protein antigen of 56,000 daltons (56 kD) which plays a necessary role in terminal sprouting. A panel of monoclonal antibodies have been produced against the 56-kD antigen, some of which also partially suppress motor axon terminal sprouting. These monoclonal antibodies define at least two different epitopes upon the surface of the antigen, one of which is necessary for it to effect its biological role in vivo.

Ultrastructural localization of the terminal and lytic ninth complement component (C9) at the motor end-plate in myasthenia gravis.

The terminal and lytic complement component (C9) was localized at the motor end-plate in acquired autoimmune myasthenia gravis (MG) by the immunoperoxidase method, with adequate preservation of fine structure and negligible background staining. C9 was localized on short segments of the postsynaptic membrane on degenerated fragments of the junctional folds shed into the synaptic space, and on disintegrating junctional folds. An inverse relationship was noted between the structural integrity of the junctional folds and the abundance of C9 at a given end-plate region. Destruction of junctional folds by complement may induce relocation of the nerve terminal and increased spatial separation of end-plate regions on the muscle fiber. Destruction of junctional folds by the complement membrane attack complex is a cause of the acetylcholine receptor deficiency at the MG end-plate, but antibody-dependent modulation of the receptor may also contribute to deficiency of the receptor. In certain disorders other than autoimmune MG, pathological mechanisms other than complement-mediated lysis may affect the structural integrity of the postsynaptic region.

Ultrastructural localization of immune complexes (IgG and C3) at the end-plate in experimental autoimmune myasthenia gravis.

Rats immunized with purified torpedo acetylcholine receptor (AChR) plus adjuvants developed chronic experimental autoimmune myasthenia gravis (EAMG) after day 28. Forelimb muscles from EAMG rats 29 to 103 days after immunization and from control animals were used for the ultrastructural localization of IgG and C3. IgG was demonstrated with rabbit anti-rat IgG followed by treatment with peroxidase-labeled staphylococcal protein A; and C3 with peroxidase-labeled rabbit anti-rat C3, or with unlabeled rabbit anti-rat C3 followed by peroxidase-labeled protein A. In EAMG rats both IgG and C3 were localized on the terminal expansions of the junctional folds, where AChR is known to be located, and on detached, degenerated parts of the folds in the synaptic space. Background staining was negligible. The findings provide unambiguous evidence for a destructive autoimmune reaction involving the postsynaptic membrane in EAMG, implicate the complement system in this reaction and show that detachment of the tips of the junctional folds is one way by which immune complexes, and AChR, are eliminated from the postsynaptic membrane. The immuno-electron microscopic findings in chronic EAMG closely resemble those described in human myasthenia gravis.

Antibodies to acetylcholine receptor in patients with thymoma but without myasthenia gravis.

Antibodies to acetylcholine receptor were found in 3 of 11 patients with a thymoma removed by operation but without myasthenia gravis. Because masthenia gravis may appear after removal of the thymoma, detection of antiacetylcholine receptor antibodies may have predictive value.

Myasthenia gravis: passive transfer to mice of antibody to human and mouse acetylcholine receptor.

Mice were given intraperitoneal injections of serum from three patients with myasthenia gravis who had different titers of antibody against mouse acetylcholine receptor (AChR). Almost all mice treated with a high titer of serum antigens showed generalized paralysis, reduced amplitudes of miniature endplate potential (MEPP), or reduced numbers of AChRs. The effects were less marked in serum with lower titers of antibody to mouse AChR, when there was no relation to the severity of effects to the titer of antibody to human AChR.

Passively transferred experimental autoimmune myasthenia gravis. Sequential and quantitative study of the motor end-plate fine structure and ultrastructural localization of immune complexes (IgG and C3), and of the acetylcholine receptor.

Experimental autoimmune myasthenia gravis (EAMG) was passively transferred with immunoglobulin from rats with chronic EAMG to normal recipients. IgG and C3 were localized on terminal expansions of junctional folds of end-plates by 6 hours. Segments of folds rich in acetylcholine receptor (AChR) and coated with IgG and C3 were shed into the synaptic space by 24 hours, resulting in AChR deficiency of the postsynaptic membrane. Many sensitized postsynaptic regions were destroyed by macrophages by day 2, but effective nerve-muscle contacts were reestablished by day 5. On day 10, end-plates were still structurally abnormal and showed AChR deficiency, but the animals were clinically recovered. On day 54, postsynaptic regions were still reduced in size, with slight reduction of postsynaptic AChR. Throughout the study, the miniature end-plate potential amplitude tended to vary directly with morphometric estimates of the abundance of the postsynaptic membrane reacting for AChR. Complement-mediated injury to the junctional folds and opsonization of the postsynaptic region can explain the morphologic changes. It is not yet known why phagocytic invasion of the end-plate occurs in acute EAMG and in passively transferred EAMG induced by chronic EAMG immuglobulin, but not in chronic EAMG and only rarely in the human disease.

Experimental autoimmune myasthenia gravis and cholinergic ionophore: an electrophysiologic study.

Rabbits were immunized with purified acetylcholine receptor from Narke electroplax japonica. A defect of neuromuscular transmission, physiologically identical to human myasthenia gravis, developed when antibodies against the receptor were found in serum. To clarify the mode of action of these antibodies, changes in the endplate current of frog muscle fibers were recorded after exposure to immune rabbit sera. The rabbit sera depressed the amplitude of the endplate current, but caused no change in the time course or the dependence of amplitude and half-decay time on membrane potential. Antibodies may affect acetylcholine binding without impairing ionophore function.

Immune complexes (IgG and C3) at the motor end-plate in myasthenia gravis: ultrastructural and light microscopic localization and electrophysiologic correlations.

Although there is strong evidence that myasthenia gravis (MG) is caused by an autoimmune reaction to the nicotinic postsynaptic acetylcholine receptor (AChR) protein, immune complexes have never been directly demonstrated at the end-plate by immunocyto-chemistry or immunoelectron microscopy. Staphylococcal protein A (which binds to the Fc region of human IgG subclasses 1, 2, and 4) and rabbit anti-human C3 conjugated with peroxidase were used for the ultrastructural (5 patients) and light microscopic (12 patients) localization of IgG and C3, respectively, at MG end-plates. Both IgG and C3 were localized on segments of the postsynaptic membrance and fragments of degenerating junctional folds in the synaptic space. In nonmyasthenic control patients no immune complexes were evident at the end-plate. As judged by morphometric analysis of electron micrographs, the immune complexes were more abundant in the less severely affected MG patients than in the more severely affected ones. A linear correlation was demonstrated between the length of the postsynaptic membrance binding immune complexes and the amplitude of the miniature end-plate potential. The less intense reaction for immune complexes in the more severely affected MG patients can be attributed to the smaller quantity of AChR remaining at their end-plates. The findings provide unambiguous evidence for a destructive auto-immune reaction involving the postsynaptic membrance in MG. Immunopharmacologic blockade of AChR and IgG-induced modulation of AChR may also contribute to the AChR deficiency at the MG end-plates.

The immunopathology of acquired myasthenia gravis.

Specific probes (alpha-bungarotoxin for acetylcholine receptor (AChR), staphylococcal protein A for IgG, monospecific antibodies against C3 and C9) labelled with peroxidase were applied to study of the ultrastructure of the MG end plate. In each case of MG there was postsynaptic AChR deficiency, usually greatest at end plates with marked degeneration of junctional folds. Morphometric estimates of postsynaptic AChR correlated linearly with the MEPP amplitude. In each case of MG, IgG was localized on the postsynaptic membrane where AChR is known to be located and on debris in the synaptic space. The abundance of antibody was proportionate to the amount of AChR remaining at the end plate. The localization of C3 was essentially identical with that of IgG. For most cases of MG it can be inferred that binding of IgG and C3 to AChR does not interfere with receptor function. C9, the terminal lytic complement component, was localized on debris in the synaptic space and on remnants of junctional folds. This proves that complement mediated destruction of junctional folds occurs in human MG. Studies in experimental auto-immune MG indicate that antibody-dependent internalization of AChR occurs in subclinical, mild and more severe diseases but increased AChR synthesis can compensate for this in subclinical and mild myasthenia. Complement-mediated injury of the postsynaptic membrane appears to be a requirement for induction of more severe MG.

Differences in acetylcholine receptor-antibody interactions between extraocular and extremity muscle fibers.

There are two types of motor nerve innervation patterns and AChR distributions in human EOM: single and multiple. The latter is further divided into two subgroups that are restricted to EOM and are not found in limb muscles. Epitopes that are unique to EOM end-plates exist. Some OMG patients have antibodies that are specifically targeted to those epitopes. These antibodies are functionally active and can cause AChR loss in EOM end-plates. In addition to AChRs, specific components constructing the microenvironment surrounding them may also be involved in the susceptibility of EOM in myasthenia gravis.

Immunoregulation in experimental autoimmune myasthenia gravis--about T cells, antibodies, and endplates.

Experimental autoimmune myasthenia gravis (EAMG) can be induced in a large number of animal species by active immunization (AI) AChR, by passive transfer (PT) of anti-AChR antibodies, by autologous bone marrow transplantation and cyclosporin (BMT-Cy), or spontaneously. Depending on the model used, different immunological mechanisms are operational. In the AI model, the T cell is pivotal in directing the anti-AChR antibody production towards pathogenic, that is, cross-linking and complement-fixing antibodies. Injection of anti-AChR antibodies alone suffices to induce EAMG, excluding the role of specific cell-mediated immune responses in the effector phase of the disease. Aged animals are resistant to the induction of AI and PT EAMG. This resistance is localized at the postsynaptic membrane containing more AChR-anchoring proteins, including S-laminin and rapsyn in aged animals. In BMT-CyA EAMG, a dysregulation of the immune system in the absence of immunization is capable of inducing myasthenia. The role of these animal models in relation to pathogenesis and immunotherapy is discussed.

Antigenic difference of acetylcholine receptor between single and multiple form endplates of human extraocular muscle.

The acetylcholine receptor (AChR) of human extraocular muscle (EOM) has been studied by the immunological method using anti-AChR antibodies obtained from the sera of patients with myasthenia gravis (MG) of ocular type, whose symptoms have been restricted to EOM. Those antibodies could distinguish the AChR of multiple (en grappe) form endplates from that of single (en plaque) form endplates. This result indicates the antigenic difference of AChR between those two forms of endplates of human EOM.

Immunochemical properties of junctional and extrajunctional acetylcholine receptor.

Immunological assays were performed to compare two distinct forms of the nicotinic acetylcholine receptor (AChR): junctional (JR) and extrajunctional receptor (EJR). Antibodies from myasthenia gravis patients' sera inhibited the binding of [125I]alpha-bungarotoxin (BGT), to EJR more effectively than binding to JR. Immunological differences between JR and EJR were confirmed by other assay methods. In all cases, EJR appeared to have antigenic determinants not found on JR. It was established that enzymatic removal of carbohydrates from EJR caused it to more closely resemble JR. Thus differences between JR and EJR may be due, in part, to carbohydrate residues found on EJR that are absent on JR. The extent of antibody binding to EJR was examined by gel filtration methods. Immunochemical studies of bands from SDS gels showed that antibodies are present in myasthenic serum which react with the 3 subunits (42, 53, 64 kdaltons) of AChR to varying degrees.

Effect of myasthenic immunoglobulin G on motor end-plate morphology.

This study was undertaken to clarify the role of complement in acetylcholine receptor loss and degeneration of the postsynaptic membrane in myasthenia gravis (MG). We examined the end-plate morphology in rats with passively transferred immunoglobulin G (IgG) from myasthenic patients and the effect of complement by treatment of the rats with cobra venom factor. We injected peroxidase-labeled alpha-BuTx (P-BuTx) into the extensor digitorum longus (EDL) muscle to label the motor end-plates. Three hours later, 100 mg of IgG from MG patients or healthy controls was injected into the tail vein. The EDL was removed 48 hours after the injection of IgG. The presence of macrophages and degeneration of the postsynaptic membrane were seen in 4 of 6 IgG samples from MG patients and a decrease in AChRs in the other 2 samples. These changes were reversed completely by treatment with cobra venom factor in all but one case in which the end-plates were severely degenerated. Injection of MG IgG only never induced end-plate morphology changes. The results suggest that complement has a critical role in degeneration of the postsynaptic membrane and AChR loss at the motor end-plates in the passively transferred model and probably in human MG.

Galanin-immunoreactive nerve terminals innervating the striated muscle fibers of the rat esophagus.

Galanin (GAL) immunohistochemistry combined with acetylcholinesterase (AChE) histochemistry was applied to demonstrate the innervation of the rat esophageal muscle coats. GAL immunoreactivity was found in a number of nerve cell bodies in the myenteric ganglia and in numerous varicose and non-varicose nerve fibers in the myenteric plexus and around blood vessels. Many GAL-positive varicose fibers ran in the internodal strands and along the striated muscle fibers. They often ramified and terminated on the muscle fibers to form arborizing structures, which were most abundant in the thoracic portion of the esophagus. Such GAL-positive terminals were localized in most (87.7%) of AChE-reactive motor endplates on the esophageal striated muscles. Left supranodose vagotomy caused a significant decrease of the GAL-arborizing terminals on the striated muscles of the esophagus. This suggests that they are terminals of efferent fibers in the vagus nerve.

T-lymphocytes in experimental autoimmune myasthenia gravis. Isolation of T-helper cell lines.

The role of T-lymphocytes in Experimental Autoimmune Myasthenia Gravis (EAMG) was investigated. We generated highly purified, acetylcholine receptor (AChR)-specific T-cell populations and subsequently characterized these cell lines with respect to their membrane phenotype and their function. Using a series of mouse monoclonal antibodies directed against rat lymphocyte surface differentiation antigens, the vast majority of line cells was shown to express a leucocyte common antigen, a T-common antigen and a T-helper antigen. Small subpopulations were Ia or T suppressor antigen-positive. Adaptive transfer to sublethally irradiated, thymectomized recipients revealed that 1 X 10(6) AChR-specific line cells could cooperate effectively with 10 X 10(6) AChR-primed, complement (C3) receptor-bearing (B-cell enriched) spleen cells in the production of anti-AChR autoantibodies. Recipients of B-cells along with relevant line cells developed an acute myasthenic syndrome 6-7 days after cell transfer. Electron-microscopical examination revealed the typical features of "acute phase" EAMG with heavy mononuclear infiltration. There was, however, no evidence antibody-independent cytotoxic activity exerted by AChR-specific line cells.

Passive transfer of myasthenia gravis by immunoglobulins: lack of correlation between AChR with antibody bound, acetylcholine receptor loss and transmission defect.

The effects of serum Ig from 7 myasthenia gravis patients on neuromuscular transmission was investigated by passive transfer to mice. A protocol of 60 mg/day for 3 days produced mouse serum levels of anti-mouse AChR that were similar to those in the MG patients, and resulted in corresponding levels (2-76%) of mouse muscle AChR with antibody bound in situ. However, AChR loss was only greater than 20% with one MG preparation. Nevertheless, there was a marked neuromuscular defect in mice injected with 3 preparations which did not necessarily correlate with the degree of AChR loss or the amount of AChRs with antibody bound in situ. We conclude that in some MG patients part of the defect in neuromuscular transmission may result from antibodies binding to other components of the neuromuscular junction.

Acquired myasthenia gravis.

Myasthenia gravis, an antibody-mediated disorder of neuromuscular transmission that produces clinical weakness, may be ocular or generalized. Clinical diagnostic evaluation may be supplemented by electrophysiologic studies and antibody testing. Therapeutic options, including anticholinesterase inhibitors, immunosuppressive agents, plasmapheresis and thymectomy, are tailored for the individual patient. This article emphasizes the key aspects of the clinical evaluation, diagnosis, and therapy.

Myasthenia gravis: demonstration of membrane attack complex in muscle end-plates.

The membrane attack complex (MAC) assembles from C5b-9 complement components and has neoantigenic properties. Antihuman-MAC rabbit immunserum was applied in order to localize the MAC in myasthenic muscles. Using the indirect immunoperoxidase method MAC was demonstrated at the motor end-plates in eleven myasthenic patients who underwent thymectomy. This result provides direct evidence of antibody-dependent complement-mediated injury of acetylcholine receptors in myasthenia gravis.