Life S iences, Vol . 18, pp . 1031-1038, 1976 Printe is the U..S .A .
Pergamon Preas
MINIREVIEW RECENT ADVANCES IN MYASTHENIA GRAVIS Stanton B . Elias and Stanley H . Appel Division of Neurology, Duke University Medical Center Durham, North Carolina 27710
h~yasthenia gravis is a remitting and relapsing neuromuscular disease of man characterized by muscle fatiguability which increases with exertion and improves with rest . Although the etiology and pathogenesis of myasthenia gravis are incompletely understood, extensive studies of the acetylcholine receptor and the immune system in the last three years have clarified many features of this disorder . Prior to this time there was no apparent way to relate the involvement of the immunological system with the documented alterations of the neuromuscular junction . The evidence supporting the participation of the immunological system was largely circumstantial . It consisted of a high incidence of thymic hyperplasia in myasthenic patients (1), the demonstration of antibodies directed against muscle structural proteins (2), the presence of lymphocytes toxic to muscle culture (3), and the beneficial effects of repeated lymphocytec drainage (4) . However, none of these studies demonstrated myasthenic antibody or lymphocyte reactivity directed against the neuromuscular junction . Physiological studies of skeletal muscle had localized the functional defect to the neuromuscular junction and more specifically to the presynaptic terminal (5) . This localization was based upon the detailed investigation of myasthenic intercostal muscles in which MEPP amplitude was found to be decreased and postsynaptic sensitivity to bath-applied carbamyl choline or décamethonium was found to be normal . Thus, as of several years ago, myasthenia gravis was considered to be a presynaptic disorder with immunological alterations possibly unrelated to the physiological defects . Our present view of the myasthenia gravis has changed considerably . We now localize the functional defect to the postsynaptic surface of the neuromuscular junction and suspect that the alterations in the morphology of the neuromuscular junction and in the density of the acetylcholine receptor are brought about by the humoral and possibly cell-mediated immune factors which result in the impairment of neuromuscular transmission . This change in the formulation has come largely from the availability of neurotoxins which bind specifically to the nicotinic acetylcholine receptor and which made possible the isolation and characterization of this receptor . Fo11ow ing this basic advance, an experimental model of myasthenia gravis was developed in animals by inoculation of electric eel acetylcholine receptor (6) ; an apparent reduction in an acetylcholine receptor content was demonstrated in muscle biopsies of myasthenic patients (7) ; and antibodies against the acetylcholine receptor were detected in the sera of myasthenic patients (8) . All of these findings have helped to clarify the functional disturbances in myasthenia gravis and have renewed interest in basic approaches to the etiology and pathogenesis of this disorder . 1031
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Presyna~ti c Al terations Attention was drawn to the presynaptic terminals as the site of the defect in myasthenia gravis by the early morphological studies of r~yasthenic muscle biopsies . Lü th the intravital stainin technique, multiple elongated endplates (9), terminal axonal sprouting ~10), abnormal branching of distal nerves, and swelling and beading of axons were demonstrated (11) . Later studies employed electron microscopy to reveal "growth cones" on apparently regenerating axon tips, and shrunken nerve terminals conveyed by and often replaced by Schwann cell processes (12-14) . However, only when quantitative morphological techniques were employed did it become apparent that the area of nerve terminals at the neuromuscular junction was actually decreased and that this decrease was in direct proportion to a decrease in the surface area of the postsynaptic membrane (14) . The number, size, and configuration of synaptic vesicles were found to be nornial . Such alterations in presynaptic morphology were not specific to rt~yasthenia gravis but were observed in other disorders and could be produced by chronic administration of anticholinesterase compounds to rats (15) . Thus, the morphological studies could not by themselves designate the presynaptic terminals as the site of primary pathology . The pharmacological and physiological studies were of no great help in clarifying the role of the presynaptic terminal . Hemicholinium was initially thought to simulate the neuromuscular block of rt~yasthenia gravis by reducing choline uptake in acetylcholine synthesis in axon terminals (16) . However, the resulting post-activation exhaustion of neuromuscular transmission differed from ir~yasthenia gravis in that quantum size was not reduced with fatigue, did not return to normal with rest, and did not recover with choline administration in myasthenia gravis (17) . The major physiological defect in myasthenic muscle was demonstrated to be a reduced amplitude of miniature end-plate potentials (0 .98 ± 0 .3 MV in normals vs 0 .20 ± 0 .11 MV in myasthenics), but a normal MEPP frequency and quantal con tent . The decreased MEPP amplitude was felt to be due to reduced acetylcholine content of each vesicle because bath-applied carbamyl choline and iontophoretically applied acetylcholine resulted in normal end-plate depolarization (5) . This demonstration of normal end-plate sensitivity in myasthenic muscle was a most crucial experiment because it focused a decade of myasthenic research on the presynaptic terminal . Atl attempts to substantiate a presynaptic theory have proven fruitless and no direct evidence supports an impairment of acetylcholine synthesis, packaging, or release . Postsynaptic Localization Despite the circumstantial evidence implicating a presynaptic site for the physiological defect in ~rasthenia gravis, several findings actually supported a postsynaptic localization . Grob (18) had demonstrated that more intra-arterial acetylcholine was required to increase the spontaneous potentials recorded from Furthermore, the end-plate zone of opponens pollicis of ~yasthenic patients . the duration of the increased activity after acetylcholine was much shorter in myasthenics than in normal individuals . These findings may be related to the dramatically altered morphology of the end-plate in myasthenia gravis . Poorly developed synaptic folds, sparse secondary synaptic clefts, and a widened distance between the nerve terminal and muscle receptor surface are all prominent By histometric features of the rt~yasthenic neuromuscular junction (10,14) . analysis, the mean area of the postsynaptic region per nerve terminal is decreased (normal, 10 .58 t 0 .79 u 2 ; ir~yasthenics, 6 .55 t 0 .36 u Z ) as is the concentration of membrane profiles (normal, 5 .83 ± 0 .25 u/u 2 ~ myasthenic, 3 .95 t 0 .21 u/u 2 ) (14) .
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The availability of purified a-bungarotoxin which could be radiolabeled with ixsl and could bind to the acetylcholine receptor permitted Fambrough et al to demonstrate a decreased number of bungarotoxin binding sites in myasthenic intercostal muscle biopsies with autoradiography (11-30% of normal) (7) . Such data presumably reflect a decreased acetylcholine receptor population, but final judgement must await a direct biochemical demonstration that such binding is 1) specifically inhibited by d-tubocurarine, decamethonium, or cholinergic ligands, and 2) is not the result of a decreased affinity of the myasthenic acetylcholine receptor for a-bungarotoxin . Additional evidence for a postsynaptic localization of the defect is the ability of cobra neurotoxin, which binds reversibly to the nicotinic acetylcholine receptor, to simulate the neuromuscular transmission defect of myasthenia gravis (19) . ImnunoloQiçal Studies Early evidence for the involvement of the immune system in myasthenia gravis derived primarily from thymic involvement in a large percentage of myasthenic patients and the beneficial effects of early thymectomy . Approximately 5 to 15% of patients with myasthenia gravis have thymoma and another 70 to 80% have "thymic hyperplasia" . Germinal centers are extremely prominent in patients with thymic hyperplasia and the number of such germinal centers may correlate with the likelihood of remission following thymectomy (20,21) . The shorter the duration of disease, the smaller the number of germinal centers and the earlier the remission . Immunosuppresant medication and corticosteroids produced beneficial effects possibly related to their interference with irtmune processes, to their anti-inflammatory effects, or to a direct action at the neuromuscular junction (22) . The population of lymphocytes derived from the thymus of patients with myasthenia gravis appears to be altered (23) . There is an increased percentage of IgM-bearing lymphocytes and an increased number of Ig-bearing cells . ~ Poke weed mitogen can induce a greater proliferative response in cultured thymocytes from myasthenic patients than thymic lymphocytes from control patients . In addition, thymic lymphocytes fram . patients with myasthenia gravis appear to be cytotoxic for fetal muscle when stimulated by phytohemagglutinin (3) . Cell mediated immunity to acetylcholine receptor is also increased in patients with asthenia gravis, especially those with long-standing disease or with thymoma (24~ . Such studies documented the importance of both altered T-cell and B-cell function in the pathogenesis of myasthenia gravis . The beneficial effects of thoracic duct drainage of lymphocytes upon the symptomatology of myasthenia gravis (4), the association of myasthenia gravis with other autoimmune diseases (25), and the presence of antibodies against muscle structural protein also provide circumstantial support for the role of the immune system . The structural protein antibodies are present in 30% of patients with myasthenia gravis especially older males, patients with longstanding disease, and patients with thymoma . However, 90% of patients with thymoma were found to have such antibodies, whether or not symptoms of myasthenia were present, thereby suggesting that such antibodies were secondary to rather than causely related to functional defects of the neuromuscular junction (2) . Acetylçholine Receptor Antibodies Although antibodies directed at constituents of the neuromuscular junction had been actively sought, the lack of a sensitive assay prevented their discovery until the last several years . Such an assay was developed which depended upon the inhibition of a-bungarotoxin binding to acetylcholine receptor extracted
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from denervated rat muscle . With this assay the presence of serum IgG antibodies directed against the acetylcholine receptor was documented in 5 of 15 patients with myasthenia gravis (8) . An additional 6 of 15 patients had borderlinè positive tests, while the remaining four patients were negative . No control patients demonstrated any inhibition of a-bungarotoxin binding . The maximal inhibition of bungarotoxin binding was 50X regardless how much antibody was added . Furthermore, the blockade appeared to be non-competitive, and the antibody appeared to be bound to a site on the acetylcholine receptor other than the a-bungarotoxin binding site . A more sensitive radioimmunoassay was then devised (26) which permitted detection of antibodies in 70 to 80X of myasthenic patients (27) . Normal rat acetylcholine receptor was a very poor antigen for such studies while denervated rat acetylcholine receptor (26) or normal human acetylcholine receptor (28,29) proved to be excellent antigens . Lindstrom et al have reported positive results in 91 of 93 sera from 59 patients including positive tests for antibody in twins born to a rgyasthenic mother (30) . The acetylcholine receptor antibody can also be detected in the majority of patients with immunohistochemical (28), or micro-complement fixation tests (31) . It is important to note that the antigen specificity of the acetylcholine receptor antibody is not identical to the muscle structural protein antibodies previously reported since a number of patients positive for receptor antibodies are negative for the structural muscle protein antibodies (32) . The role of such antibodies in the pathologic alterations of the neuromuscular junctions are unclear . The possibility of simple direct blockade of the acetylcholine site by antibody giving rise to the altered physiology appears unlikely since globulin added to rat or human neuromuscular junctions in vitro does not alter either the end-plate potential or miniature end-plate potentials (33) . It is possible that interaction of antibody with the acetylcholine receptor might accelerate receptor desensitization or might accelerate the turnover of postsynaptic membrane and its receptor in a manner analogous to the acceleration of the lymphocyte capping by membrane surface antibodies . The net result of either process would be a decreased density of postsynaptic receptors available to interact with acetylcholine released from the presynaptic terminal . The length of time necessary to accelerate receptor turnover would preclude the demonstration of short-term effects (less than 5 hours) of antibody upon the neuromuscular junction in vitro . Such an explanation may provide an explanation of the prolonged time necessary to demonstrate physiologic effects when myasthenic antibodies are injected into mice (34) . Several reports have suggested that myasthenic populations can be segregated into two subpopulations : young females with a high rate of HL-A8 and older males with a predominence of HL-A2 (35,36) . The clinical syndrome in each of the groups appears slightly different with respect to susceptibility to drugs, However, likely benefit by thymectomy, and clinical progression of the disorder . both males and females possess high titers of circulating antibodies directed against the acetylcholine receptor (27) . Experimental Myasthenia The most recent and most cogent evidence for myasthenia gravis as an autoimmune disease is the experimental production of rt~yasthenia gravis in animals following immunization with electric eel acetylcholine receptor (6) . Clinically such animals show weakness, easy fatiguability, and improvement with neostigmine . A decremental response to IOHz stimulation is noted by EMG and can be reversed with neostigmine (37) . A decreased amplitude of MEPP is present in those animals showing the full spectrum of disease as well as in animals with very little weakness (38 ) . The rats with experimental disease have antibodies against the electric eel acetylcholine receptor in titers which are 10,000 times greater
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than their response to homologous rat acetylcholine receptor . EAMG could be prevented by early thymectomy or treatment with anti-T-cell serum but late treatment had no effect (39) . The first pathologic changes in EAMG demonstrate destruction of the postsynaptic region of the muscle fiber by an inflammatory infiltrate . Axon termiBy nals are intact but separated from muscle by debris and inflammatory cells . 12 to 20 days, inflammatory cells have left the end-plate region and new neuromuscular junctions are formed with less secondary folds and membrane profiles in a pattern similar to that noted in human myasthenic junctions (40) . The similarity of physiologic and morphologic findings make the extrapolation from the animal model to the human disease an easy one . However, the initiating insult in the human disorder is presently unknown, and the dramatic inflam matory response seen in the acute stage in the animal has not been described in man . Çoncludi~ Remarks The presence of experimental models for myasthenia gravis induced by the administration of acetylcholine receptor, the presence of acetylcholine receptor antibodies in the patients with myasthenia gravis, and the altered postsynaptic morphology of the neuromuscular junction all place an entirely new perspective on the human rt~yasthenic disorder . For the first time in the investigation of this disorder, it is now possible to propose a scheme which relates the immunological alterations to the functional neuromuscular defect . The initiating factor might be a break in immunologic tolerance possibly secondary to a viral infection of the susceptible host . Such dysfunction might alter the normal Tcell control of B-cell function and the recognition of self and non-self antigens . T-cell proliferation may itself become uncontrolled and give rise to thyB-cell proliferation might occur in the thymus and alter the response to momas . antigens previously recognized as self, such as structural proteins and acetylcholine receptors both of which have been described in thymus tissue . These antibodies as well as specific cell mediated immunity would then have reactivity against peripheral skeletal muscle constituents . In the periphery, inflammatory responses at the neuromuscular junction might alter the geometry and widen the synaptic cleft . The lack of dramatic cellular infiltrate in man might suggest that circulating antibodies are a more important factor in this process than cell mediated cytotoxicity . The altered morphology would give rise to a decreased safety factor of neuromuscular transmission, a reduction of miniature end-plate potential amplitude, and the likelihood of fatiguability associated with the exertion . The interaction of antibody with acetylcholine receptor might induce a state of desensitization of the receptor and possibly accelerate the turnover of both postsynaptic membranes and acetylcholine receptors leading to their relative depletion . Such a scheme would predict a decreased sensitivity of the postsynaptic junction to iontophoretically applied acetylcholine . Although Elmgvist et al (5) could not demonstrate this with their studies in the early 1960's, the availabil ity of Nomarski Optics has recently permitted Albuquerque (33,41) to document the loss of postsynaptic sensitivity to acetylcholine . The significant difference between Albuquerque's experiments and previous ones was his ability to localize the recording micro-electrodes closer to the end-plate region than had been previously possible and to achieve a high sensitivity of applied acetylcholine (3000 mV/nc) . The present scheme would suggest that the beneficial effect of steroids may be related to their ability to improve the altered geometry of the neuromuscular
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junction as well as to their anti-inflammatory and anti-immune properties . In addition the beneficial effects of withdrawal from anticholinesterase medication after the onset of clinical refractoriness might be related to the removal of the morphological and desensitizing effects of anticholinesterase medication superimposed on similar effects produced by the acetylcholine receptor antibody . Furthermore, the sensitivity to curare and resistance to decamethonium of myasthenic patients might similarly be explained by the altered geometry of the neuromuscular junction and the altered postsynaptic structures . Such a model changes the emphasis from presynaptic to postsynaptic structures and suggests that both experimental and therapeutic efforts should be directed more intensively to the altered geometry of the myasthenic junction, to the postsynaptic acetylcholine receptors and membrane components, and to the immunologic factors which may be inducing such alterations . Acknowledgements This work was supported in part by grants NS12213 and NS07872 from the National Institute of Neurological and Communicative Disorders and Stroke and grant 558-D-5 from the National Multiple Sclerosis Society . We are grateful to Mildred Washington and Winfred Clingenpeel for technical assistance in several of the studies from our laboratory reported in this review and to Eleanor Chapman for secretarial assistance . References l. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
B . CASTLEMAN, Ann . N .Y . Aced . Sci . 135 496-503 (1966) . A . J . STRAUSS, B . C . SEGAL, K . C . HSII, P . M . BURKHOLDER, W . L . NASTUR, and K . E . OSSERMAN, Proc . Soc . Exp . Biol . Med . 105 184-191 (1960) . A . M . ARMSTRONG, R . M . NOWAK, and R . E . FALK,Neurology 23 1078-1083 (1973) . K. BERGSTROM, C . FRANKSSON, G . MATELL, and f . VON REIS, Éur . Neurol . _9 157 (1973) . D . ELMQVIST, W . W . HOFFMAN, J . KUGELBERD, and D . M . J . QUASTEL, J . Physiol . 174 417-434 (1964) . .PATRICK, J and J . M . LINDSTROM, Science 180 871-872 (1973) . D . M . FAMBROUGH, D . B . DRACHMAN, and S . S TYAMURTI, Science _182 293-295 (1973) . R . R . ALMON, C . G . ANDREW, and S . H . APPEL, Science 186 55-57 (1974) . A . L . WOOLF, Ann . N .Y . Aced . Sci . 135 35-56 (1966) . E . R . BICKERSTAFF, and A . L . WOOLF, Brain _83 10-23 (1960) . V . MACDERMOT, Brain 83 24-36 (1960) . A . G . JOHNSON, and A.L . WOOLF, Acta Neuropathologica 4 436-441 (1965) . R . A . BERGMAN, R . J . JOHNS, and A . K . AFIFI, Ann . N .Y . Âcad . Sci . 183 88122 (1971) . A . G . ENGEL, and T . SANTA, Ann . N .Y . Aced . Sci . 18 3 46-64 (1971) . A . G . ENGEL, E . H . LAMBERT, and T . SANTA, Neurology 23 1273-1281 (1973) . J . E . DESMEDT, Ann . N .Y . Aced . Sci . 135 209-246 196 W . W . HOFFMAN, Ann . N .Y . Aced . Sci . 1~ 276-286 ~1966~. D . GROB, R . J . JOHNS, and A . M . HARVEY, Bull . Johns Hopkins Hosp . 99 153181 (1956) . S . SATYAMURTI, D . B . DRACHMAN, and F . SLONE, Science 187 955-957 (1975) . V . P . PERLO, B . ARNASON, D . POSKANZER, B . CASTLEMAN, ~S . SCHWAB, K . E . OSSERMAN, C . ALPERT, and A . KARK, Ann . N .Y . Aced . Sci . 18 3 308-315 (1971) . G . GENKINS, A . E . PAPATESTIS, S . H . HOROWITZ, and P . KO N~IELD, Am . J . Med . 58 517-523 (1975) . G . GENKINS, P . KORNFIELD, K . OSSERMAN, T . NAMBA, D . GROB, and N . G . BRUNNER, Ann . N .Y . Aced . Sci . 183 369-374 (1971) .
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23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 . 33 . 34 . 35 . 36 . 37 . 38 . 39 . 40 . 41 .
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N . J . ABDOU, R . P . LISAK, B . Z . ZWEIMAN, I . ABRAHAMSON, and A . S . PENN, New Eng . J . Med . 291 1271-1275 (1974) . 0 . ABRAMSKY, .AHARONOV, A D . TEITELBAUM, and S . FUCHS, Arch . Neurol . _32 684687 (1975) . J . A . SIMPSON, Ann . N .Y . Acad . Sci . 13 5 506-516 (1966) . R . R . ALMON, and S . H . APPEL, Biochim Biophys . . Acta 393 66-77 (1975) . S . H . APPEL, R . R . ALMON, and N . LEVY, New Eng . J . Me~293 760-761 (1975) . A . N . BENDER, S . P . RINGEL, and W . R . ENGEL, Lancet 1 60708 (1975) . R . R . ALMON, and S . H . APPEL, Ann . N .Y . Acad . Sci . (in press) . J . LINDSTROM, and V . LENNON, Ann . N .Y . Acad . Sci . (in press) . A . AHARONOV, 0 . ABRAMSKY, R . TARRAB-NAZDAI, and S . FUCHS, Lancet _1 340-342 (1975) . A . N . BENDER, S . P . RINGEL, W . K . ENGEL, Z . VOGEL, and M . P . DANIELS, Ann . N .Y . Acad . Sci . (in press) . E . X . ALBUQUERQUE, R . R . ALMON, S . H . APPEL, F . C . KAUFFMAN, F . J . LEBEDA, R . F . MAYER, T . NARAHASHI, and J . Z . YEH, Ann . N .Y . Acad . Sci . (in press) . K . V . TOYKA, D . B . DRACHMAN, A . PESTRONK, and I . KAO, Science _190 397-399 (1975) . D . FRITZE, C . HERMAN, JR ., F . NAEIM, G . SMITH, and R . WALFORD, Lancet _1 240242 (1974) . T . E . W . FELTKAMP, P . M . VAN DEN HERG-LOONEN, L . E . NIZENHUIS, G . P . ENGEL FRIET, A . L . VAN ROSSUM, J . J . VAN LONHON, and N . J . G . N . OOSTERHUIS, Brit . Med . J . 1 131-133 (1974) . V . A . LENNON, J . M . LINDSTROM, and M . E . SEYBOLD, J . Exp . Med . _141 1365-1375 (1975) . E . H . LAMBERT, J . M . LINDSTROM, and V . A . LENNON, Ann . N .Y . Acad . Sci . (in press) . V . A . LENNON, and J . M . LINDSTROM, Ann . N .Y . Acad . Sci . (in press) . A . G . ENGEL, M . TSUJIHATA, J . LINDSTROM, and V . A . LENNON, Ann . N .Y . Acad . Sci . (in press) . E . ALBUQUERQUE, (personal communication) .
Pergamon Preas
MINIREVIEW RECENT ADVANCES IN MYASTHENIA GRAVIS Stanton B . Elias and Stanley H . Appel Division of Neurology, Duke University Medical Center Durham, North Carolina 27710
h~yasthenia gravis is a remitting and relapsing neuromuscular disease of man characterized by muscle fatiguability which increases with exertion and improves with rest . Although the etiology and pathogenesis of myasthenia gravis are incompletely understood, extensive studies of the acetylcholine receptor and the immune system in the last three years have clarified many features of this disorder . Prior to this time there was no apparent way to relate the involvement of the immunological system with the documented alterations of the neuromuscular junction . The evidence supporting the participation of the immunological system was largely circumstantial . It consisted of a high incidence of thymic hyperplasia in myasthenic patients (1), the demonstration of antibodies directed against muscle structural proteins (2), the presence of lymphocytes toxic to muscle culture (3), and the beneficial effects of repeated lymphocytec drainage (4) . However, none of these studies demonstrated myasthenic antibody or lymphocyte reactivity directed against the neuromuscular junction . Physiological studies of skeletal muscle had localized the functional defect to the neuromuscular junction and more specifically to the presynaptic terminal (5) . This localization was based upon the detailed investigation of myasthenic intercostal muscles in which MEPP amplitude was found to be decreased and postsynaptic sensitivity to bath-applied carbamyl choline or décamethonium was found to be normal . Thus, as of several years ago, myasthenia gravis was considered to be a presynaptic disorder with immunological alterations possibly unrelated to the physiological defects . Our present view of the myasthenia gravis has changed considerably . We now localize the functional defect to the postsynaptic surface of the neuromuscular junction and suspect that the alterations in the morphology of the neuromuscular junction and in the density of the acetylcholine receptor are brought about by the humoral and possibly cell-mediated immune factors which result in the impairment of neuromuscular transmission . This change in the formulation has come largely from the availability of neurotoxins which bind specifically to the nicotinic acetylcholine receptor and which made possible the isolation and characterization of this receptor . Fo11ow ing this basic advance, an experimental model of myasthenia gravis was developed in animals by inoculation of electric eel acetylcholine receptor (6) ; an apparent reduction in an acetylcholine receptor content was demonstrated in muscle biopsies of myasthenic patients (7) ; and antibodies against the acetylcholine receptor were detected in the sera of myasthenic patients (8) . All of these findings have helped to clarify the functional disturbances in myasthenia gravis and have renewed interest in basic approaches to the etiology and pathogenesis of this disorder . 1031
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Vol. 18, No . 10
Presyna~ti c Al terations Attention was drawn to the presynaptic terminals as the site of the defect in myasthenia gravis by the early morphological studies of r~yasthenic muscle biopsies . Lü th the intravital stainin technique, multiple elongated endplates (9), terminal axonal sprouting ~10), abnormal branching of distal nerves, and swelling and beading of axons were demonstrated (11) . Later studies employed electron microscopy to reveal "growth cones" on apparently regenerating axon tips, and shrunken nerve terminals conveyed by and often replaced by Schwann cell processes (12-14) . However, only when quantitative morphological techniques were employed did it become apparent that the area of nerve terminals at the neuromuscular junction was actually decreased and that this decrease was in direct proportion to a decrease in the surface area of the postsynaptic membrane (14) . The number, size, and configuration of synaptic vesicles were found to be nornial . Such alterations in presynaptic morphology were not specific to rt~yasthenia gravis but were observed in other disorders and could be produced by chronic administration of anticholinesterase compounds to rats (15) . Thus, the morphological studies could not by themselves designate the presynaptic terminals as the site of primary pathology . The pharmacological and physiological studies were of no great help in clarifying the role of the presynaptic terminal . Hemicholinium was initially thought to simulate the neuromuscular block of rt~yasthenia gravis by reducing choline uptake in acetylcholine synthesis in axon terminals (16) . However, the resulting post-activation exhaustion of neuromuscular transmission differed from ir~yasthenia gravis in that quantum size was not reduced with fatigue, did not return to normal with rest, and did not recover with choline administration in myasthenia gravis (17) . The major physiological defect in myasthenic muscle was demonstrated to be a reduced amplitude of miniature end-plate potentials (0 .98 ± 0 .3 MV in normals vs 0 .20 ± 0 .11 MV in myasthenics), but a normal MEPP frequency and quantal con tent . The decreased MEPP amplitude was felt to be due to reduced acetylcholine content of each vesicle because bath-applied carbamyl choline and iontophoretically applied acetylcholine resulted in normal end-plate depolarization (5) . This demonstration of normal end-plate sensitivity in myasthenic muscle was a most crucial experiment because it focused a decade of myasthenic research on the presynaptic terminal . Atl attempts to substantiate a presynaptic theory have proven fruitless and no direct evidence supports an impairment of acetylcholine synthesis, packaging, or release . Postsynaptic Localization Despite the circumstantial evidence implicating a presynaptic site for the physiological defect in ~rasthenia gravis, several findings actually supported a postsynaptic localization . Grob (18) had demonstrated that more intra-arterial acetylcholine was required to increase the spontaneous potentials recorded from Furthermore, the end-plate zone of opponens pollicis of ~yasthenic patients . the duration of the increased activity after acetylcholine was much shorter in myasthenics than in normal individuals . These findings may be related to the dramatically altered morphology of the end-plate in myasthenia gravis . Poorly developed synaptic folds, sparse secondary synaptic clefts, and a widened distance between the nerve terminal and muscle receptor surface are all prominent By histometric features of the rt~yasthenic neuromuscular junction (10,14) . analysis, the mean area of the postsynaptic region per nerve terminal is decreased (normal, 10 .58 t 0 .79 u 2 ; ir~yasthenics, 6 .55 t 0 .36 u Z ) as is the concentration of membrane profiles (normal, 5 .83 ± 0 .25 u/u 2 ~ myasthenic, 3 .95 t 0 .21 u/u 2 ) (14) .
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The availability of purified a-bungarotoxin which could be radiolabeled with ixsl and could bind to the acetylcholine receptor permitted Fambrough et al to demonstrate a decreased number of bungarotoxin binding sites in myasthenic intercostal muscle biopsies with autoradiography (11-30% of normal) (7) . Such data presumably reflect a decreased acetylcholine receptor population, but final judgement must await a direct biochemical demonstration that such binding is 1) specifically inhibited by d-tubocurarine, decamethonium, or cholinergic ligands, and 2) is not the result of a decreased affinity of the myasthenic acetylcholine receptor for a-bungarotoxin . Additional evidence for a postsynaptic localization of the defect is the ability of cobra neurotoxin, which binds reversibly to the nicotinic acetylcholine receptor, to simulate the neuromuscular transmission defect of myasthenia gravis (19) . ImnunoloQiçal Studies Early evidence for the involvement of the immune system in myasthenia gravis derived primarily from thymic involvement in a large percentage of myasthenic patients and the beneficial effects of early thymectomy . Approximately 5 to 15% of patients with myasthenia gravis have thymoma and another 70 to 80% have "thymic hyperplasia" . Germinal centers are extremely prominent in patients with thymic hyperplasia and the number of such germinal centers may correlate with the likelihood of remission following thymectomy (20,21) . The shorter the duration of disease, the smaller the number of germinal centers and the earlier the remission . Immunosuppresant medication and corticosteroids produced beneficial effects possibly related to their interference with irtmune processes, to their anti-inflammatory effects, or to a direct action at the neuromuscular junction (22) . The population of lymphocytes derived from the thymus of patients with myasthenia gravis appears to be altered (23) . There is an increased percentage of IgM-bearing lymphocytes and an increased number of Ig-bearing cells . ~ Poke weed mitogen can induce a greater proliferative response in cultured thymocytes from myasthenic patients than thymic lymphocytes from control patients . In addition, thymic lymphocytes fram . patients with myasthenia gravis appear to be cytotoxic for fetal muscle when stimulated by phytohemagglutinin (3) . Cell mediated immunity to acetylcholine receptor is also increased in patients with asthenia gravis, especially those with long-standing disease or with thymoma (24~ . Such studies documented the importance of both altered T-cell and B-cell function in the pathogenesis of myasthenia gravis . The beneficial effects of thoracic duct drainage of lymphocytes upon the symptomatology of myasthenia gravis (4), the association of myasthenia gravis with other autoimmune diseases (25), and the presence of antibodies against muscle structural protein also provide circumstantial support for the role of the immune system . The structural protein antibodies are present in 30% of patients with myasthenia gravis especially older males, patients with longstanding disease, and patients with thymoma . However, 90% of patients with thymoma were found to have such antibodies, whether or not symptoms of myasthenia were present, thereby suggesting that such antibodies were secondary to rather than causely related to functional defects of the neuromuscular junction (2) . Acetylçholine Receptor Antibodies Although antibodies directed at constituents of the neuromuscular junction had been actively sought, the lack of a sensitive assay prevented their discovery until the last several years . Such an assay was developed which depended upon the inhibition of a-bungarotoxin binding to acetylcholine receptor extracted
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from denervated rat muscle . With this assay the presence of serum IgG antibodies directed against the acetylcholine receptor was documented in 5 of 15 patients with myasthenia gravis (8) . An additional 6 of 15 patients had borderlinè positive tests, while the remaining four patients were negative . No control patients demonstrated any inhibition of a-bungarotoxin binding . The maximal inhibition of bungarotoxin binding was 50X regardless how much antibody was added . Furthermore, the blockade appeared to be non-competitive, and the antibody appeared to be bound to a site on the acetylcholine receptor other than the a-bungarotoxin binding site . A more sensitive radioimmunoassay was then devised (26) which permitted detection of antibodies in 70 to 80X of myasthenic patients (27) . Normal rat acetylcholine receptor was a very poor antigen for such studies while denervated rat acetylcholine receptor (26) or normal human acetylcholine receptor (28,29) proved to be excellent antigens . Lindstrom et al have reported positive results in 91 of 93 sera from 59 patients including positive tests for antibody in twins born to a rgyasthenic mother (30) . The acetylcholine receptor antibody can also be detected in the majority of patients with immunohistochemical (28), or micro-complement fixation tests (31) . It is important to note that the antigen specificity of the acetylcholine receptor antibody is not identical to the muscle structural protein antibodies previously reported since a number of patients positive for receptor antibodies are negative for the structural muscle protein antibodies (32) . The role of such antibodies in the pathologic alterations of the neuromuscular junctions are unclear . The possibility of simple direct blockade of the acetylcholine site by antibody giving rise to the altered physiology appears unlikely since globulin added to rat or human neuromuscular junctions in vitro does not alter either the end-plate potential or miniature end-plate potentials (33) . It is possible that interaction of antibody with the acetylcholine receptor might accelerate receptor desensitization or might accelerate the turnover of postsynaptic membrane and its receptor in a manner analogous to the acceleration of the lymphocyte capping by membrane surface antibodies . The net result of either process would be a decreased density of postsynaptic receptors available to interact with acetylcholine released from the presynaptic terminal . The length of time necessary to accelerate receptor turnover would preclude the demonstration of short-term effects (less than 5 hours) of antibody upon the neuromuscular junction in vitro . Such an explanation may provide an explanation of the prolonged time necessary to demonstrate physiologic effects when myasthenic antibodies are injected into mice (34) . Several reports have suggested that myasthenic populations can be segregated into two subpopulations : young females with a high rate of HL-A8 and older males with a predominence of HL-A2 (35,36) . The clinical syndrome in each of the groups appears slightly different with respect to susceptibility to drugs, However, likely benefit by thymectomy, and clinical progression of the disorder . both males and females possess high titers of circulating antibodies directed against the acetylcholine receptor (27) . Experimental Myasthenia The most recent and most cogent evidence for myasthenia gravis as an autoimmune disease is the experimental production of rt~yasthenia gravis in animals following immunization with electric eel acetylcholine receptor (6) . Clinically such animals show weakness, easy fatiguability, and improvement with neostigmine . A decremental response to IOHz stimulation is noted by EMG and can be reversed with neostigmine (37) . A decreased amplitude of MEPP is present in those animals showing the full spectrum of disease as well as in animals with very little weakness (38 ) . The rats with experimental disease have antibodies against the electric eel acetylcholine receptor in titers which are 10,000 times greater
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than their response to homologous rat acetylcholine receptor . EAMG could be prevented by early thymectomy or treatment with anti-T-cell serum but late treatment had no effect (39) . The first pathologic changes in EAMG demonstrate destruction of the postsynaptic region of the muscle fiber by an inflammatory infiltrate . Axon termiBy nals are intact but separated from muscle by debris and inflammatory cells . 12 to 20 days, inflammatory cells have left the end-plate region and new neuromuscular junctions are formed with less secondary folds and membrane profiles in a pattern similar to that noted in human myasthenic junctions (40) . The similarity of physiologic and morphologic findings make the extrapolation from the animal model to the human disease an easy one . However, the initiating insult in the human disorder is presently unknown, and the dramatic inflam matory response seen in the acute stage in the animal has not been described in man . Çoncludi~ Remarks The presence of experimental models for myasthenia gravis induced by the administration of acetylcholine receptor, the presence of acetylcholine receptor antibodies in the patients with myasthenia gravis, and the altered postsynaptic morphology of the neuromuscular junction all place an entirely new perspective on the human rt~yasthenic disorder . For the first time in the investigation of this disorder, it is now possible to propose a scheme which relates the immunological alterations to the functional neuromuscular defect . The initiating factor might be a break in immunologic tolerance possibly secondary to a viral infection of the susceptible host . Such dysfunction might alter the normal Tcell control of B-cell function and the recognition of self and non-self antigens . T-cell proliferation may itself become uncontrolled and give rise to thyB-cell proliferation might occur in the thymus and alter the response to momas . antigens previously recognized as self, such as structural proteins and acetylcholine receptors both of which have been described in thymus tissue . These antibodies as well as specific cell mediated immunity would then have reactivity against peripheral skeletal muscle constituents . In the periphery, inflammatory responses at the neuromuscular junction might alter the geometry and widen the synaptic cleft . The lack of dramatic cellular infiltrate in man might suggest that circulating antibodies are a more important factor in this process than cell mediated cytotoxicity . The altered morphology would give rise to a decreased safety factor of neuromuscular transmission, a reduction of miniature end-plate potential amplitude, and the likelihood of fatiguability associated with the exertion . The interaction of antibody with acetylcholine receptor might induce a state of desensitization of the receptor and possibly accelerate the turnover of both postsynaptic membranes and acetylcholine receptors leading to their relative depletion . Such a scheme would predict a decreased sensitivity of the postsynaptic junction to iontophoretically applied acetylcholine . Although Elmgvist et al (5) could not demonstrate this with their studies in the early 1960's, the availabil ity of Nomarski Optics has recently permitted Albuquerque (33,41) to document the loss of postsynaptic sensitivity to acetylcholine . The significant difference between Albuquerque's experiments and previous ones was his ability to localize the recording micro-electrodes closer to the end-plate region than had been previously possible and to achieve a high sensitivity of applied acetylcholine (3000 mV/nc) . The present scheme would suggest that the beneficial effect of steroids may be related to their ability to improve the altered geometry of the neuromuscular
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junction as well as to their anti-inflammatory and anti-immune properties . In addition the beneficial effects of withdrawal from anticholinesterase medication after the onset of clinical refractoriness might be related to the removal of the morphological and desensitizing effects of anticholinesterase medication superimposed on similar effects produced by the acetylcholine receptor antibody . Furthermore, the sensitivity to curare and resistance to decamethonium of myasthenic patients might similarly be explained by the altered geometry of the neuromuscular junction and the altered postsynaptic structures . Such a model changes the emphasis from presynaptic to postsynaptic structures and suggests that both experimental and therapeutic efforts should be directed more intensively to the altered geometry of the myasthenic junction, to the postsynaptic acetylcholine receptors and membrane components, and to the immunologic factors which may be inducing such alterations . Acknowledgements This work was supported in part by grants NS12213 and NS07872 from the National Institute of Neurological and Communicative Disorders and Stroke and grant 558-D-5 from the National Multiple Sclerosis Society . We are grateful to Mildred Washington and Winfred Clingenpeel for technical assistance in several of the studies from our laboratory reported in this review and to Eleanor Chapman for secretarial assistance . References l. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
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