Acquired myasthenia gravis: what we have learned from experimental and spontaneous animal models

Veterinary Immunology and Immunopathology 69 (1999) 239±249

Acquired myasthenia gravis: what we have learned from experimental and spontaneous animal models G. Diane Shelton* Department of Pathology, University of California San Diego, La Jolla, CA 92093-0612, USA

Abstract Acquired myasthenia gravis (MG) is a disorder of neuromuscular transmission in which muscle weakness results from an autoantibody mediated depletion of acetylcholine receptors (AChR) at the neuromuscular junction. Experimental autoimmune myasthenia gravis, described in rodents and rabbits, has provided a good model of the effects of the autoimmune response against AChR and has shown that the specificities of the immune response in MG are those that would be obtained by immunization with native AChR. It has provided little information, however, about what initiates and sustains the immune response in MG. Acquired MG occurs spontaneously in dogs and may be the most common neuromuscular disorder that can be diagnosed in this species. As in human MG, an autoimmune response against AChR has been demonstrated and AChR autoantibodies have been implicated in the pathogenesis. The variability in clinical presentation, methods of diagnosis, and occurrence with other autoimmune diseases and neoplasia are identical to that of humans. Future studies of spontaneous canine autoimmune MG may provide clues to the determination of what factors initiate and sustain the autoimmune response to AChR, and in the study of specific suppression of the autoimmune response against AChR. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Canine; Autoimmune; Muscle; Neuromuscular junction; Myasthenia gravis

1. Introduction Acquired myasthenia gravis (MG) is the most completely characterized autoimmune disease affecting the neuromuscular system; and, arguably, of autoimmune diseases in general (Lindstrom et al., 1988). Unlike most other autoimmune diseases, the inciting * Tel.: +619-534-1537; fax: +619-534-0458 E-mail address: [email protected] (G.D. Shelton) 0165-2427/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 9 9 ) 0 0 0 5 8 - 6

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autoantigen is known, there are specific and sensitive diagnostic tests available, and there are relatively specific therapies. As a result of autoantibody-mediated destruction of nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction, there is clinically apparent muscular weakness and excessive fatigability that may affect ocular, facial, oropharyngeal, esophageal and limb muscles. Experimental models of acquired MG have been described in mice, rats, guinea pigs and rabbits (experimental autoimmune myasthenia gravis, EAMG) that reproduce the essential clinical and morphologic correlates of human MG. There are limitations to these models in that the etiology of the autoimmune response cannot be studied and the immune response is not self-perpetuating. Acquired MG occurs spontaneously in dogs and cats. Canine MG has been most extensively studied and it is clear that it is analogous to the human disease. Other animal species that have played an important role in knowledge of MG include krait snakes which contain the specific neuromuscular toxin, a-bungarotoxin (a-Bgt), which has been important in the characterization of nicotinic AChR structure and function, and by the eels Electrophorus electricus and Torpedo californica which contain a rich reservoir of AChR in their electric organs. The a-Bgt, a competitive antagonist of AChR, has proven an especially useful probe in the study of AChRs and in the diagnostic assay for MG. 2. Experimental autoimmune myasthenia gravis The autoimmune nature of MG was discovered following the observation of muscular weakness, reversible by anticholinesterase drugs, in rabbits following repeated immunization with AChR purified from electric eels (Patrick and Lindstrom, 1973). This finding provided support for a previously proposed autoimmune mechanism of antibody mediated blockade of AChR at motor end plates (Simpson, 1960; Lennon and Carnegie, 1971). Additional animal models of EAMG were then established in rats (Lennon et al., 1975; Lindstrom et al., 1976a, b, c), guinea pigs (Lennon et al., 1975), and later mice (Christadoss et al., 1979, 1981). These models clearly showed that sensitization to AChR produces pathogenic autoantibodies resulting in AChR deficiency at the neuromuscular function. The pathological effects of AChR antibodies were extensively studied in Lewis rats with EAMG (Lindstrom et al., 1976a, b). Approximately 30 days after a single immunization with AChR serum antibody titers were maximal and clinical weakness was evident. Serum autoantibody concentrations were in large excess over the amount of muscle AChR (Lindstrom et al., 1976d). Compared to control animals, the amount of muscle AChR was reduced by 50±70% and much of the remaining muscle was bound by antibodies (Lindstrom et al., 1976a). The primary mechanisms by which the antibodies act to reduce AChR content are complement-mediated focal lysis (Engel et al., 1974, 1977a, b; Sahashi et al., 1978) and antigenic modulation (Kao and Drachman, 1977; Heinemann et al., 1977; Drachman et al., 1978; Merlie et al., 1979). Ultrastructurally, endplates in chronic EAMG closely resembled those of MG patients. The postsynaptic membrane was simplified (Engel et al., 1974) and there was evidence of complement fixation (Sahashi et al., 1978). The importance of the lytic components in the

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pathogenesis of AChR deficiency was indicated by the fact that C5-deficient mice immunized with AChR produce AChR antibodies but show no clinical signs of EAMG (Christadoss, 1988). IgG from rats with chronic EAMG also induces acute passivetransfer EAMG in recipient rats (Lindstrom et al., 1976b). The antigenic specificities of AChR antibodies in EAMG sera were determined by the ability of monoclonal antibodies against defined determinants on the AChR molecule to inhibit binding of the serum antibodies to receptor. A main immunogenic region (MIR), a conformationally dependent epitope, was defined on receptors from fish electric organs to which 60±70% of the antibodies were directed (Tzartos and Lindstrom, 1980; Tzartos et al., 1981). The MIR is located on the extracellular surface of  subunits and is distinct from the acetylcholine binding sites (Gullick et al., 1981). Monoclonal antibodies to this region can cause antigenic modulation of AChR (Conti-Tronci et al., 1981) and passively transfer EAMG (Tzartos et al., 1987). Using a similar panel of monoclonal antibodies, the pattern of specificities in the sera of human (Tzartos et al., 1982) and canine (Shelton et al., 1988) patients with MG were tested and it was found that MG patients produced fundamentally the same pattern of specificities as that produced by animals immunized with receptor purified from fish electric organs or mammalian muscle. If MG were caused by cross-reaction with a viral antigen, due to a defect in the immune system or alterations in the endogenous immunogen that caused other parts of the molecule to appear more immunogenic, MG sera should differ from EAMG sera. The finding that rats, human and canine MG patients made most of their AChR antibodies to the MIR supports the inference that the immunogen in MG is native receptor (Lindstrom et al., 1978). The cellular immunology of autoantibody production has been studied extensively in the EAMG model and several lines of evidence suggest that antibody production against AChR is T cell dependent. Neonatally thymectomized rats or lethally irradiated rats that are reconstituted with B cells do not respond to AChR (Lennon et al., 1974), and lymph node cells from AChR-immunized rats do not produce antibodies against AChR in vitro after treatment with anti-rat brain serum plus complement (DeBaets et al., 1982). Antibody production could be reconstituted by adding back T cells (Krolick and Urso, 1987). In EAMG, T cells from AChR-immunized rats proliferate only in the presence of syngeneic antigen-presenting cells (Hohlfeld et al., 1981). Unlike antibody recognition that depends on the native conformation of the AChR and is lost by denaturation (Tzartos and Lindstrom, 1980; Tzartos et al., 1982), T cell receptors recognize denatured AChR subunits (DeBaets et al., 1982; Krolick and Urso, 1987) or small synthetic peptides (Lennon et al., 1985; Atassi et al., 1987) only in association with a major histocompatibility (MHC) encoded molecule of their own type on the surface of an antigen presenting cell (Hohlfeld et al., 1981; Schwartz, 1985). Using synthetic peptides made from the  subunit sequence of AChR or bacterially expressed peptide fragments, some of the T cell epitopes recognized by mice (Atassi et al., 1987), rats (Fujii and Lindstrom, 1988; Zhang et al., 1988), or humans (Hohlfeld et al., 1987) have been identified. Strains differed markedly in the peptides to which they responded and there did not appear to be a consistent restricted pattern. Susceptibility to EAMG has been mapped to the I±A subregion using mice congenic in their MHC region (Christadoss et al., 1979). A monoclonal antibody to an allele of I±A

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was reported to suppress anti-AChR antibody production (Waldor et al., 1983). In human MG, genetic restriction of the response to AChR has been suggested by the association of HLA B8 and DR3 with young female patients (Naeim et al., 1978; Compston et al., 1980). While EAMG has provided a good model of the effects of the autoimmune response against AChR, it has provided little information about what initiates the immune response in MG. EAMG does show that the specificities of the immune response in MG are those that would be obtained by immunization with native AChR, and that the immune response to AChR is not self-sustaining once initiated, implying that MG must have both an initiating and a sustaining immune response to persist. Studies of naturally occurring canine autoimmune MG may be valuable in these areas as factors that initiate and sustain the autoimmune response to AChRs are probably similar in humans and dogs. 3. Spontaneous autoimmune canine myasthenia gravis Myasthenia gravis was initially described in dogs as an infrequent disorder presenting clinically as reduced tolerance to exercise with development of weakness which was alleviated by rest and administration of anticholinesterase drugs (Ormrod, 1961; Palmer and Barker, 1974). A decremental response of the muscle action potential during repetitive stimulation of the motor nerve was described. An association with cranial mediastinal tumors was noted in some cases. The autoimmune nature of canine acquired MG was described in 1978 by Lennon et al. (1978) with the documentation of autoantibodies against nicotinic AChRs, a reduction in muscle AChR content, and demonstration of remaining AChRs complexed with antibody. Since these original descriptions, much has been learned about the diverse clinical presentations of acquired canine MG (Shelton et al., 1990; Dewey et al., 1995; King and Vite, 1998), breed predispositions (Shelton et al., 1997), and clinical course of the disorder (Dau et al., 1979; Shelton, unpublished observations). While recent textbooks of veterinary internal medicine still describe acquired MG as an infrequent disorder, acquired MG may actually be the most common neuromuscular disorder we can diagnose and treat in the dog (Shelton, 1998a). 4. Clinical presentation Due to the propensity of canine MG to mimic other myopathic and neuropathic disorders, acquired MG should be high on the list of differential diagnoses in any dog with focal or generalized neuromuscular weakness. Presenting clinical signs may be focal in nature and limited to regurgitation (as a result of a megaesophagus), dysphagia (due to pharyngeal dysfunction), voice change (as a result of laryngeal paresis), or multiple cranial nerve abnormalities in the absence of generalized muscle weakness. In a recent study (Shelton et al., 1997), 43% of the dogs with a confirmed diagnosis of MG by the demonstration of a positive acetylcholine receptor antibody titer did not have clinically detectable limb muscle weakness. Generalized weakness was present in the remaining

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57% with 13% of these dogs having generalized weakness in the absence of esophageal or pharyngeal dysfunction. Recently a severe form of MG, acute fulminating MG, has been described (Dewey et al., 1997; King and Vite, 1998). These cases were associated with a sudden onset of esophageal dilatation, rapid progression to quadraparesis, respiratory failure, and high mortality. Several of these dogs had concurrent thymoma. In humans, the acute fulminating form of MG is associated with high mortality, myasthenic crisis and with the highest percentage of thymomas (Osserman and Genkins, 1971). 5. Diagnosis of canine MG While a dramatic improvement in muscle strength following the short acting anticholinesterase drug edrophonium chloride provides a presumptive diagnosis of MG in dogs, a subjective improvement in muscle strength may be found in other myopathic or neuropathic disorders. Conversely, a lack of improvement in muscle strength does not eliminate a diagnosis of MG. A decrement in the amplitude of the compound muscle action potential in response to repetitive nerve stimulation also provides a presumptive diagnosis of MG (Sims and McLean, 1990), however, there is a similar lack of sensitivity and specificity and the requirement for general anesthesia in a possibly critical patient. Single-fiber electromyography is a sensitive method for detecting delayed or failed neuromuscular transmission and a methodology has been established for the dog (Hopkins et al., 1993). Specificity, however, is lacking, with positive findings in other disorders of the nerve, muscle and neuromuscular junction (Oh et al., 1992). The `gold standard' for the diagnosis of MG in humans (Lennon, 1997) and animals (Shelton, 1998b) remains the demonstration of serum autoantibodies against native AChR by immunoprecipitation radioimmunoassay. This assay involves precipitation of serum IgG and IgM antibodies that bind to solubilized AChR complexed with a high-affinity peptide antagonist, 125I-labeled a-bgt. The precipitate's g-emission reflects the amount of AChR bound to immunoglobulin. This assay is specific, sensitive and documents an autoimmune response against muscle AChRs. Although there is some cross-reactivity in AChR recognition of antibodies between species, the assay is relatively species specific and a canine specific assay system should be used. Antibody titers in dogs are in general lower than in humans, and low-titer positives may be missed if human AChR is used as antigen. The AChR binding antibody assay is the first choice for confirming a diagnosis of acquired MG (Lennon, 1997). It is not, however, predictive of the degree of weakness. In the assay conditions of the authors laboratory, sera of healthy dogs bind <0.6 nmol/l of AChR per liter. During the time-period 1991±1995, 1154 dogs were diagnosed with acquired MG by this assay system. During the same time period, 20 dogs with clinical signs consistent with acquired generalized MG and a positive Tensilon test were `seronegative' by this test method. Of these 20 dogs, five were shown to have a low muscle AChR content (<0.2 pmol/gm tissue) and antibody bound to remaining muscle AChR (>1%) by biochemical quantification (Lindstrom et al., 1976a). This would suggest that the AChR binding assay detects 98% of the dogs with generalized acquired MG. Antibody titers may also be negative early in the disease and retesting would be suggested

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if clinical signs were recent in onset. Immunosuppressive therapy for longer than 7±10 days will lower antibody titers, so a pretreatment blood sample is advisable. In humans, positive results for AChR binding have been described in patients with autoimmune liver disorders, in 13% of patients with Lambert Eaton Syndrome, and in 3% of patients with primary lung cancer uncomplicated by neurologic autoimmunity (Lennon, 1997). To date, we have not detected AChR antibodies in dogs with any disorder other than acquired MG or paraneoplastic MG. If the AChR binding assay is negative and there is a suspicion of acquired MG, variations on the standard radioimmunoassay may be performed. The AChR blocking antibody assay detects antibodies that bind near the a-bungarotoxin binding sites. While blocking antibodies may be found in 50% of myasthenic dogs with binding antibodies, we have detected this specificity alone in only two dogs (Shelton, 1998b). In humans, only 1% of MG patients have AChR blocking antibody without detectable AChR binding antibodies (Lennon, 1997). The AChR modulating antibody assay is a measure of AChR blockage and degradation in a tissue culture system. If AChR binding and blocking assays are negative, a positive result in the AChR modulating antibody assay should not be considered valid unless repeatable with a fresh serum sample (Lennon, 1997). False positive results can occur and may be attributable to hemolysis, muscle relaxant drugs, or microbial growth. 6. Seronegative MG Seronegative MG has been described in humans (Mossman et al., 1986; Soliven et al., 1988; Sanders et al., 1997) and numbers vary between 6% and 34% of patients. Seronegative and seropositive patients manifest similar clinical features. Abnormal neuromuscular transmission can be transferred to animals by injecting immunoglobulin from patients with seronegative MG (Burges et al., 1994). Criteria used to classify dogs as having seronegative myasthenia gravis (Shelton, 1998) include consistent clinical signs, consistent pharmacologic (positive edrophonium response) and electrophysiologic (decrement) findings, normalization of limb muscle weakness following anticholinesterase therapy, and at least one negative serum acetylcholine receptor antibody titer by radioimmunoassay. In 20 dogs that fit this criteria (Shelton, 1998), five were shown to have antibody bound to AChR and a low AChR content by biochemical quantification of an external intercostal muscle sample. This would support the presence of AChR antibody titers in a range undetectable by standard serum assay. Since seroconversion may occur, serum antibody titers should be repeated 1±2 months following a negative AChR antibody titer. Possible explanations for seronegative MG include: (1) Antibodies are directed against non-AChR end-plate determinants (Newsom-Davis et al., 1986); (2) Antibodies are directed against the toxin binding site, so patients with antibodies to this site would appear to be seronegative; (3) Antibodies are bound to the end-plates without detectable circulating serum antibodies (antigen excess); and (4) Antibodies are directed against antigenic determinants that may be lost during the AChR extraction procedure.

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7. Other serologic tests In humans with MG, autoantibodies against striated muscle proteins have been described including antibodies against myosin, actin, and a-actinin (Williams and Lennon, 1986), titan (Gautel et al., 1993), and the sarcoplasmic reticulum. These have been collectively referred to as striational antibodies (StrAb). Since these antigens are located in the muscle's cytoplasm and are not normally accessible to circulating antibodies, they are believed to be non-pathogenic. Positive assays for StrAb in humans may support a clinical diagnosis of acquired MG when tests for AChR antibodies are negative (Lennon, 1997). StrAb are detected in 80% of human MG patients with thymoma, in 24% of patients having thymoma without clinical signs of MG, and in 30% of adult MG patients without thymoma. While the StrAb have been reported in canine MG (Garlepp et al., 1984) and the author has observed StrAb in canine patients with thymoma, the actual incidence has not yet been determined. Studies are currently in progress to address this issue. Recently, antibodies against the ryanodine receptor (RyR) have been described in humans associated with thymoma (Mygland et al., 1992) and in late-onset MG (Skie et al., 1995). The presence of autoantibodies against RyR was associated with a severe form of thymoma related MG and a higher mortality rate. The RyR is a calcium release channel in striated muscle involved in muscle contraction and in the mechanism of excitation±contraction coupling. In vitro studies have shown that RyR antibodies from MG patients have high affinity for the RyR and affect RyR function by inhibition of ryanodine binding (Skeie et al., 1998). The binding of antibodies likely affects calcium release from SR by locking the RyR ion channel in a closed position (Skeie et al., 1998). While the in vivo effect of MG antibodies on RyR function has not yet been established, a spontaneous thymoma rat model has recently been described that produces anti-RyR antibodies and shows muscle weakness in the absence of AChR antibodies (Iwasa et al., 1988). Electrophysiological studies in muscles from these animals suggested a defect in excitation±contraction coupling. Autoantibodies against RyR have also been documented in dogs with thymoma and older onset MG (Shelton, manuscript in preparation). Studies are currently in progress in canine MG correlating the presence of RyR antibodies with clinical severity. The presence of RyR antibodies that play a functional role may begin to explain the lack of correlation between AChR antibody titer and severity of muscle weakness. 8. Other associated conditions Acquired MG may occur concurrently with other autoimmune disorders and following the diagnosis of MG, a search for other diseases should be made. MG may be associated with hypothyroidism (Dewey et al., 1995), hypoadrenocorticism, thrombocytopenia, hemolytic anemia (Shelton, 1998) and as a paraneoplastic syndrome associated with thymoma (Aronsohn et al., 1984; Klebanow, 1992), osteogenic sarcoma (Moore et al., 1990), cholangiocellular carcinoma (Krotje et al., 1990), and anal sac adenocarcinoma (Shelton, unpublished results). Third-degree heart block has also been found in some

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dogs with MG, thymoma and polymyositis (Hackett et al., 1995). As in human MG, disease onset or exacerbation may be preceded by infection with bacterial or viral agents. Heat cycles, gestation and whelping may initiate or exacerbate MG in female dogs so ovariohysterectomy is recommended. 9. Feline MG Compared to canine MG, acquired feline MG is uncommon (Joseph et al., 1988). During the time period from 1988 to 1997, 79 cats were diagnosed in the author's laboratory with acquired MG based on demonstration of circulating AChR antibodies. Nine of the cats were of Abyssinian or Somali breeds. While information was not complete in all cases, 15/79 (19%) of the confirmed MG cases were associated with a thymoma. This is in contrast to the canine MG population where 3.4% of the cases were associated with a cranial mediastinal mass (Shelton et al., 1997). 10. Future directions While much is known about the pathogenesis, clinical presentations, and diagnosis of acquired MG in dogs, several important areas remain to be investigated. The role of the thymus in non-thymomatous canine MG has not been explored. It has been established in humans that the thymus plays a central role in the pathogenesis of this disease and thymic hyperplasia and thymoma are common in human MG patients. Thymectomy has been a cornerstone of therapy in human MG patients (Jaretzki III, 1997). While we know the B lymphocyte response results in autoantibody specificities similar to those of human MG and EAMG (Shelton et al., 1988), the T cell response has not been investigated. Spontaneous remissions occur in canine MG from a few weeks following diagnosis (Dau et al., 1979; Garlepp et al., 1984) until up to one year after diagnosis (Shelton, unpublished observations). The author has followed several dogs in which the AChR antibody titer has remained positive for over one year, and while the information is not yet complete, neoplasias including thymoma have been found to develop months following the diagnosis of MG. Remissions can occur in the absence of immunosuppressive therapy. Evaluation of T-lymphocyte activation during the active phase of the immune response to AChR and during the remission phase may provide clues to antigen specific immunotherapy of MG for both humans and dogs. Although rarely the case in humans, spontaneous remission in canine MG is associated with AChR antibody titers that return to the normal range with resolution of clinical signs without the necessity of anticholinesterase or immunosuppressive therapy (Shelton, unpublished observation). Unequivocal evidence indicates that susceptibility to autoimmune disease is largely genetically determined, and the most clearly established genetic association with autoimmune disease predisposition is related to the major histocompatibility complex class II genes (Compston et al., 1980). In human MG, significant associations with particular DRB and DBQ alleles are seen in class II genes in early-onset, non-thymoma-

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associated MG (Vieira et al., 1993). A significant role for MHC class II genes has been shown in several animal models of EAMG with elimination of MHC class II molecules or binding of inhibitory peptide analogues of MHC class II preventing onset of EAMG (Kaul et al., 1994). In a recent study (Shelton et al., 1997), the relative risk of acquired MG for 61 different breeds of dogs was determined and the Akita was found to be at highest relative risk. The Akita, in general, has a higher than expected level of immunemediated disease compared to a cross-bred population. Newfoundland siblings and closely related dogs with acquired MG have also recently been identified (Lipsitz et al., in press). There are two important aspects of MG that still remain a mystery: what initiates and sustains the autoimmune response to AChR and how does one specifically suppress the autoimmune response? Dogs may play an important role in the study of both of these aspects, because the disorder develops spontaneously in dogs, indicating that triggers may be similar to those initiating the disease in humans. Dogs also have spontaneous remissions, indicating that their immune system is able to correct the imbalance that results in clinical disease. References Aronsohn, M.G., Schunk, K.L., Carpenter, J.L., King, N.W., 1984. J. Am. Vet. Med. Assn. 184, 1355. Atassi, M.Z., Mulac-Jericevic, B., Yokoi, T., Manshouri, T. 1987. Fed. Proc. 46, pp. 2538. Burges, J., Vincent, A., Molenaar, P.C., Newsom-Davis, J., Peers, C., Wray, D., 1994. Muscle Nerve 17, 1393. Christadoss, P., Lennon, V.A., David, C., 1979. J. Immunol. 123, 2540. Christadoss, P., Krco, C.J., Lennon, V.A., David, C.S., 1981. J. Immunol. 126, 1646. Christadoss, P., 1988. J. Immunol. 140, 2589. Conti-Tronci, B., Tzartos, S., Lindstrom, J., 1981. Biochemistry 20, 2181. Compston, D.A.S., Vincent, A., Newson-Davis, J., Batchelor, J.R., 1980. Brain 103, 579. Dau, P.C., Yano, C.S., Ettinger, S.J., 1979. Neurology 29, 1065. DeBaets, M.H., Einarson, B., Lindstrom, J.M., Weigle, W.O., 1982. J. Immunol. 128, 2228. Dewey, C.W., Shelton, G.D., Bailey, C.S., Willard, M.D., Podell, M., Collins, R.L., 1995. Prog. Vet. Neurol. 6, 117. Dewey, C.W., Bailey, C.S., Shelton, G.D., Kass, P.H., Cardinet III, G.H., 1997. J. Vet. Intern. Med. 11, 50. Drachman, D.B., Angus, C.W., Adams, R.N., Kao, I., 1978. Proc. Natl. Acad. Sci. USA 75, pp. 3422. Engel, A.G., Tsujihasta, M., Lambert, E.H., Lindstrom, J.M., Lennon, V.A., 1974. J. Neuropathol. Exp. Neurol. 35, 569. Engel, A.G., Lindstrom, J.M., Lambert, E.H., Lennon, V.A., 1977a. Neurology 27, 307. Engel, A.G., Lambert, E.H., Howard, F.M., 1977b. Mayo Clin. Proc. 52, 267. Fujii, Y., Lindstrom, J., 1988. J. Immunol. 140, 1830. Garlepp, M.J., Kay, P.H., Farrow, B.R., Dawkins, R.L., 1984. Clin. Immunol. Immunopath 31, 301. Gautel, M., Lakey, A., Barlow, D.P., Holmes, B.A., Scales, S., Leonard, K., Labeit, S., Mygland, A., Gilhus, N.E., Aarli, J.A., 1993. Neurology 43, 1581. Gullick, W., Tzartos, S., Lindstrom, J.M., 1981. Biochemistry 20, 2173. Hackett, T.B., Vanpelt, D.R., Willard, M.D., Martin, L.G., Shelton, G.D., Wingfield, W.E., 1995. J. Am. Vet. Med. Assn. 206, 1173. Heinemann, S., Bevan, S., Kullberg, R., Lindstrom, J., Rice, J., 1977. Proc. Natl. Acad. Sci. USA 74, pp. 3090. Hohlfeld, R., Kalies, I., Heinz, F., Kalden, J.R., Wekerle, H., 1981. J. Immunol. 126, 1355±1359. Hohlfeld, R., Toyka, K., Tzartos, S., Carson, W., Conti-Tronconi, B., 1987. Proc. Natl. Acad. Sci. USA 84, pp. 5379. Hopkins, A.L., Howard, J.F., Wheeler, S.J., Kornegay, J.N., 1993. J. Small Anim. Pract. 34, 271.

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