Nature medicine
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| NATURE MEDICINE • VOLUME 7 • NUMBER 3 • MARCH 2001 365
A RT I C L E S Myasthenia gravis (MG) is an antibody-mediated autoimmune disease of the neuromuscular junction. In approximately 80% of patients, auto-antibodies to the muscle nicotinic acetylcholine receptor (AChR) are present 1 . These antibodies cause loss of AChR numbers and function, and lead to failure of neuromuscu- lar transmission with muscle weakness 2 . The pathogenic mecha- nisms acting in the 20% of patients with generalized MG who are seronegative for AChR-antibodies (AChR-Ab) 3 have not been elucidated, but there is evidence that they also have an anti- body-mediated disorder 4,5 , with the antibodies directed towards another, previously unidentified muscle-surface–membrane tar- get 6–8 . Here we show that 70% of AChR-Ab–seronegative MG pa- tients, but not AChR-Ab–seropositive MG patients, have serum auto-antibodies against the muscle-specific receptor tyrosine ki- nase, MuSK. MuSK mediates the agrin-induced clustering of AChRs during synapse formation, and is also expressed at the mature neuromuscular junction 9–12 . The MuSK antibodies were specific for the extracellular domains of MuSK expressed in transfected COS7 cells and strongly inhibited MuSK function in cultured myotubes. Our results indicate the involvement of MuSK antibodies in the pathogenesis of AChR-Ab–seronegative MG, thus defining two immunologically distinct forms of the disease. Measurement of MuSK antibodies will substantially aid diagnosis and clinical management. We first tested coded plasmas from AChR-Ab–seronegative MG patients and healthy individuals using COS7 cells transfected with rat MuSK constructs (Fig. 1a). IgG from all five AChR- Ab–seronegative MG plasmas (Fig. 1b, AChR-Ab–neg/MuSK), but not from the three healthy control plasmas, bound to MuSK ag- gregates on the cell surface at dilutions of up to 1:1000. The pat- tern of immunoreactivity was indistinguishable from that observed with polyclonal antibodies raised against recombinant MuSK in rabbits 13 . Each of the AChR-Ab–seronegative MG plas- mas recognized the extracellular domains of MuSK, as we ob- served no immunoreactivity with COS7 cells expressing the transmembrane and cytoplasmic domains only (Fig. 1b, AChR- Ab–neg/MuSK ∆ Ig1-4).
Immunoprecipitation experiments confirmed that the IgG an- tibodies in AChR-Ab–seronegative MG plasmas recognized the native MuSK protein. As a source of MuSK, we used detergent ex- tracts from either MuSK-expressing COS7 cells or mouse C2C12 myotubes that express functional MuSK 10,11,13
. In each case, after the plasma incubations, we precipitated the IgG antibodies with immobilized G protein and ran the precipitates on an SDS-PAGE. We used a rabbit serum against MuSK as a positive control. Antibodies from the AChR-Ab–seronegative plasmas immuno- precipitated bands of 110 kD that were identified as MuSK by binding of a specific antibody against MuSK (Fig 1c). MuSK was not immunoprecipitated by healthy control plasmas, AChR- Ab–seropositive plasmas or from an AChR-Ab–seronegative plasma that had been pre-absorbed with the extracellular do- main of MuSK (Ig1-4). Currently, AChR-Ab–seronegative MG is thought to represent about 10–20% of all MG patients 3 , but the true prevalence is dif- ficult to assess because of differences in patient ascertainment and referral to specialist centers. Most AChR-Ab–seropositive MG patients present in adult life, often after the age of 50 years 14 , but children with MG are frequently AChR-Ab–seronegative 3 . To establish an assay that could be used for future diagnosis and epi- demiological studies, we tested sera and plasmas in an ELISA using plates coated with fragments of the extracellular domains of MuSK, expressed in secreted form from COS7 cells. We calcu- lated a cut-off (0.08 optical density units [OD]) on the basis of the mean ± 3 s.d. of the values with healthy control plasmas or sera. Raised levels of IgG antibodies to MuSK were found in 17/24 samples from patients with AChR-Ab–seronegative gener- alised MG, including the 2 children who had presented before the age of 10 years. Titers were borderline or in the control range in 19 AChR-Ab–seropositive MG patients without thymoma, and in 20 with thymoma, as well as in 33 sera from patients with other neurological disorders (Fig. 2a). The lack of detectable MuSK antibodies in MG patients with thymoma is significant be- cause these patients frequently have antibodies against a range of muscle proteins 15 . Detection of MuSK antibodies will provide a new and much needed tool for the diagnosis of AChR- Ab–seronegative MG in children and in adults and facilitate epi- demiological studies. It will also strengthen the case for immunosuppressive therapy and provide a means of monitoring its effectiveness. Importantly, the thymus gland usually appears normal in AChR-Ab–seronegative MG (ref. 16), indicating that thymectomy (often used therapeutically in AChR-Ab–seroposi- tive MG) may not be appropriate in those cases with MuSK anti- bodies. Also, the presence of MuSK antibodies should help to exclude a thymoma. We noted a broad correlation between optical density values Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies W ERNER
H OCH
1 , J
OHN M C C ONVILLE
2 , S
IGRUN H ELMS 1 , J
OHN N EWSOM -D AVIS
2 , A RTHUR M ELMS 3 & A
NGELA V INCENT 2 1 Max Planck Institute for Developmental Biology, Tübingen, Germany 2 Neurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK 3 Department of Neurology, University of Tübingen, Tübingen, Germany Correspondence should be addressed to W.H.; email: werner.hoch@tuebingen.mpg.de or A.V.; email: angela.vincent@imm.ox.ac.uk © 2001 Nature Pub lishing Gr oup http://medicine .nature .com © 2001 Nature Publishing Group http://medicine.nature.com 366 NATURE MEDICINE • VOLUME 7 • NUMBER 3 • MARCH 2001 A RT I C L E S for IgG binding to MuSK and IgG binding to the human TE671 cell line measured in an earlier study 8 (r 2 = 0.72; P < 0.001; n = 12), indicating that the previously defined, but unidentified, cell-surface antigen on TE671 cells might be MuSK. The patients who were negative for both MuSK and AChR antibodies were not clinically distinguishable from those who had MuSK antibodies, but 4 of 7 MuSK-negative samples were from patients who had received immunosuppressive treatments, compared with only 2 of 17 of the MuSK-positive samples. This indicates that the cur- rent assay may lack sensitivity for patients who are already im- munosuppressed. Further analysis showed that most of the MuSK antibodies (Fig. 2b) were directed against the amino-ter- minal sequences (construct Ig1-2 in Fig. 1a) rather than the membrane-proximal half (construct Ig3-4 in Fig. 1a). Overall, there is 94% homology between rat MuSK and the published human sequence 9 , and they differ by only two amino acids in the Ig1-2 construct. Nonetheless, use of human MuSK, as well as further refinement of the assay, may increase the sensitivity of the test. In addition, as we did not detect IgM antibodies to MuSK (data not shown), the target for the putative non-IgG anti- bodies in some patients 6 , including two of the MuSK-Ab–nega- tive patients studied here, requires further investigation. These data indicate that the MuSK antibodies are directed against extracellular determinants and could therefore be di- rectly involved in the pathogenesis of the disease. To determine whether the antibodies can interfere with MuSK function, we ex- amined agrin-induced AChR clustering in myotubes derived from the mouse cell line, C2C12. In the absence of agrin (Fig. 3a, upper panels), very few spontaneous clusters of AChRs were found in the presence of healthy control plasma, whereas MuSK- Ab–positive MG plasma induced some AChR aggregates along the surface of the myotubes. Similar results have previously been reported for rabbit antibodies raised against purified MuSK (ref. 13). When agrin was added together with the plasmas (Fig. 3a, lower panels), the marked agrin-induced clustering which oc- curred in the presence of healthy control plasma was not seen in the presence of MuSK-Ab–positive MG plasma, indicating that the MuSK antibodies had inhibited the agrin-induced AChR clus- tering. A quantitative analysis of the data revealed that both the clustering (Fig. 3b), and the inhibitory activity (Fig. 3c), were found with MuSK-Ab–positive plasmas or IgG, but not with the one MuSK-Ab–seronegative preparation or the healthy controls tested. Moreover, both the clustering (Fig. 3b) and inhibitory (Fig. 3c) activity of an AChR-Ab–negative plasma were prevented by absorption with soluble MuSK, demonstrating that the MuSK antibodies were responsible for these effects. By contrast, plas- mas from two patients with AChR-Ab–seropositive MG (Fig. 3c), and the non-IgG fractions of MuSK-Ab–positive MG patients
Antibodies from AChR-Ab–seronegative MG patients bind to MuSK. a, Domain structure of the MuSK molecule and schematic view of constructs used in Fig. 1b and c and Fig. 2. b, AChR-Ab–negative MG IgGs bound to COS7 cells expressing full-length MuSK (AChR-Ab–neg/MuSK), whereas healthy control IgG did not (Healthy control/MuSK). MuSK immunoreactivity appeared as a speckled pattern, similar to that seen previously with rabbit anti-MuSK antibodies 13 . Non-transfected cells in the same field, demonstrated by phase contrast microscopy (arrows), showed non-specific binding only. No specific binding of AChR-Ab–seronegative MG IgG to cells expressing MuSK lacking the extracellular domains (AChR-Ab–neg/MuSK ∆ Ig1-4) was detected. c, IgG from 2 AChR-Ab–seronegative MG plasmas (Neg), but not from control plasmas (HC), precipitated MuSK from detergent extracts of COS7 cells expressing MuSK, and from C2C12 (C2) myotubes. The same band was immunoprecipitated by IgG from a rabbit immunized against MuSK (Rb). MuSK was not precipitated by AChR-Ab–seropositive MG plasmas (Pos) or after absorption of AChR-Ab–seronegative plasma (Abs) with soluble MuSK Ig1-4. MuSK appears as a 110 kD band from COS-cells and as several bands representing different MuSK splice variants in the C2C12 cells 11 .
Neg Pos
Thy ON -1 0 1 2 3 OD (A
492 ) 0.25 0.75 0.00
0.50 1.00
Dilution of plasma (1:6 serial dilutions) 0 1
3 4 5 6 Fig. 2 MuSK antibodies can be detected by ELISA. a, Antibodies to MuSK were found in 17 of 24 AChR-Ab–seronegative MG patients (Neg) com- pared with 13 healthy controls (HC). Negative or borderline values were found in anti-AChR–seropositive MG patients (Pos), including those with thymomas (Thy), and in patients with other neurological disorders (ON). b, Titration of one AChR-Ab–seronegative MG plasma against different do- mains of MuSK. The antibodies bound strongly to MuSK constructs express- ing the N-terminal domains, Ig1-4 ( ˾) and Ig1-2 (̆; see Fig. 1a), but binding to the membrane-proximal Ig3-4 ( b) domains was similar to that of a healthy control ( ć).
a b OD (A
492 )
2001 Nature Pub lishing Gr oup http://medicine .nature .com © 2001 Nature Publishing Group http://medicine.nature.com NATURE MEDICINE • VOLUME 7 • NUMBER 3 • MARCH 2001 367
A RT I C L E S (data not shown) did not affect agrin-induced clustering. We show that AChR-Ab–seronegative IgG preparations con- taining MuSK antibodies interfere with the agrin/MuSK/AChR clustering pathway in myotubes and have the potential to alter MuSK function at the adult neuromuscular junction. As agrin does not appear to bind directly to MuSK, but via a hypothetical agrin-binding component called MASC (refs. 10,12), we specu- late that the antibodies in AChR-Ab–seronegative patients bind to MuSK in such a manner as to prevent its interaction with MASC. This interaction is known to depend on the N-terminal half of the extracellular domains of MuSK (ref. 17) that we find to be the main target for the IgG antibodies in AChR- Ab–seronegative patients (Fig. 2b). How these antibodies cause seronegative MG is not yet clear. AChR-Ab–seronegative MG is mediated by humoral IgG antibodies that lead to a defect in neu- romuscular transmission 4 . A few limited human biopsy stud- ies 18,19
indicate that there is a reduction in AChR function at the neuromuscular junction, although total AChR numbers were variable. We propose that interference with MuSK function at the mature neuromuscular junction is one pathogenic mecha- nism in MuSK-Ab–seropositive MG patients, leading secondarily to reduced numbers and/or altered distribution of AChRs and other postsynaptic proteins. By analogy with AChR-Ab–positive MG (ref. 2), MuSK antibodies may not only inhibit MuSK func- tion directly but also increase the turnover of MuSK, further re- ducing its activity. In addition, there could be complement-mediated damage to the AChR-containing postsy- naptic membrane following IgG binding to MuSK and comple- ment activation. Some evidence for complement deposition at the endplates of muscle from AChR-Ab–negative MG patients has been reported 20 . Elucidating the contribution of different mechanisms to the defect in neuromuscular transmission will re- quire further in vitro and in vivo studies. We have defined a novel specific target antigen, the receptor tyrosine kinase MuSK, for the auto-antibodies in AChR- Ab–seronegative MG. The antibodies have functional effects on agrin-induced AChR distribution in cultured myotubes, indicat- ing that they could interfere with AChR numbers or distribution in mature muscle. The IgG antibodies were found in 70% of AChR-Ab–seronegative MG patients and not in AChR- Ab–seropositive MG patients, clearly distinguishing the two forms of the disease. Moreover, the antibodies can be detected by a simple ELISA that could be used for routine diagnosis and clinical management. Our results add a receptor tyrosine kinase (RTK) to the AChR and voltage-gated calcium and potassium channels that are already defined targets for antibody-mediated disorders at the neuromuscular junction 21 . Auto-antibodies to other RTKs have occasionally been implicated in some forms of endocrine disease 22 . Since RTKs are cell-surface molecules, regu- lating intracellular functions and activated by dimerization, they are attractive candidate antigens and should be considered in other antibody-mediated disorders. Moreover, as members of the RTK family are mutated in inherited diseases 23 , MuSK may be in- volved not only in this acquired autoimmune disorder but also in congenital muscle diseases. Methods Patients. Samples were obtained from 24 patients (18 female, 6 male) with moderate or severe generalized MG in whom the standard radio-immuno- precipitation assay for anti-AChR antibodies 24 was negative on several occa- sions. All had typical fatigable muscle weakness. The diagnosis was confirmed by electromyographic evidence of a defect in neuromuscular transmission (a decrement of more than 10% in the amplitude of the com- pound muscle action potential on repetitive nerve stimulation at 3 Hz and/or an increase in jitter on single fiber studies), or by a positive response to anti- cholinesterase medication (edrophonium or pyridostigmine). The age at onset was 2–68 years (median 24) and the duration of symptoms at sam- pling was between 1 month and 13 years (median 1.0 year). In 18 cases, plasma was obtained during therapeutic plasmapheresis, which improved muscle strength. The remaining 6 samples were sera taken on first examina- tion. Six of the patients had received corticosteroids for up to two months before sampling. Sera or plasmas were also obtained from healthy volun- teers, from patients with AChR-Ab–positive MG, and from patients with other immune-mediated neurological disorders. IgG preparations were made using a ImmunoPure (G) IgG purification kit (Pierce, Rockford, IL)
MuSK (ref. 13) and the soluble fragment s-agrin (4/19) 25 have been de- scribed. MuSK deletion fragments comprising the entire extracellular do- main (Ig1-4; aa 1–490) 9 , or the first half containing two Ig-domains (Ig1-2; aa 1–231), were generated by insertion of artificial stop signals at these po- sitions by a PCR approach. N-terminal fragments of MuSK comprising the membrane-proximal extracellular domains, including Ig-domains 3 and 4 (Ig3-4; aa 203–490), or the transmembrane region and intracellular domain (MuSK–Ig1-4, aa 491–868) were generated. The corresponding cDNA-frag- ments, including a newly introduced SphI site, were linked to a vector con- taining an artificial signal sequence followed by 6 histidines and a 10-aa epitope-tag 25 . All constructs were transiently transfected into COS7 cells 11 . For the production of soluble agrin and MuSK constructs, cells were switched to serum-free medium the second day after transfection. - Ag
+ Ag 1 1 2 2 3 3 4 4 5 5 6 6 HC HC
0 5 10 15 20 25 30 35 40 - Agrin Number of clusters/ field - Ag + Ag
1 1 2 2 3 3 4 4 5 5 6 6 HC HC Abs Pos Pos
0 5 10 15 20 25 30 35 40 + Agrin Number of clusters/ field a b c Fig. 3 MuSK IgG antibodies induce AChR clusters but inhibit agrin-in- duced clustering. a, In the absence of agrin, a moderate number of AChR clusters, visualised by rhodamine- α -bungarotoxin, were induced in the presence of MuSK-Ab–positive MG plasma compared with that in healthy control plasma. Conversely, in the presence of agrin, large numbers of clus- ters were induced in healthy control plasma-treated myotubes, but not in myotubes treated with MuSK-Ab–positive MG plasma. b and c, The AChR clusters without (b) or with (c) added agrin (Ag) in cultures treated with plasma (
í) or IgG ( ). Only the MuSK-Ab–positive plasmas and IgG prepa- rations (1–5) affected AChR clusters; there was no effect of a MuSK-Ab–neg- ative plasma (6), of 2 healthy control plasmas (HC), or of 1 MuSK-Ab–positive plasma after absorption with MuSK (Abs). Two AChR- Ab–seropositive samples (Pos) did not affect agrin-induced clustering. Clusters in control cultures without plasma or IgG ( २).
368 NATURE MEDICINE • VOLUME 7 • NUMBER 3 • MARCH 2001 A RT I C L E S Conditioned media, containing MuSK or agrin fragments, were removed 24 hours later and analyzed by western blotting to confirm expression.
onto chamber slides the day after transfection. Two days later, cells were fixed with 2% paraformaldehyde and stained as described 13 . Plasmas of MG patients and controls were analyzed at various dilutions (between 1:20 and 1:5000). Bound antibodies were visualized with secondary antibodies conju- gated to Cy3 (anti-human IgG, Dianova, Hamburg, Germany). In all experi- ments, expression of transfected MuSK constructs was confirmed by staining parallel slides with rabbit antibodies against MuSK (ref. 13).
MuSK-transfected COS7 cells or from C2C12 myotubes that had been fused for 5 days 11 . The immunoprecipitation was performed as de- scribed 11,13
. AChR-Ab–seronegative MG, AChR-Ab–seropositive MG plasmas and control plasmas were incubated with the extracts at 1:20. Rabbit anti- MuSK serum was used at 1:100. MuSK in the immunoprecipitates was ana- lyzed by western blotting using affinity-purified serum antibodies directed against the MuSK cytoplasmic sequence 13 . One AChR-Ab–seronegative MG plasma was absorbed with soluble MuSK, by addition of an equal volume of conditioned medium of COS7 cells transfected with the soluble MuSK frag- ment Ig1-4, for 2 hours. The mixture was then diluted further before use.
MuSK-transfected COS7 cells or from control cells mock-transfected with salmon sperm DNA, was diluted 1:1 with 100 mM NaHCO3 buffer (pH 9.5) and applied overnight to ELISA plates. Plasmas were first tested at 1:5 in triplicates and subsequently at 1:10 in duplicates. Bound antibodies were detected by horseradish peroxidase-protein A (Amersham, Braunschweig, Germany) followed by o-phenylenediamine and measuring A 492
. For each sample, nonspecific immunoreactivity, determined by incubation of plates coated with conditioned medium from mock-transfected COS7 cells, was subtracted. The efficient immobilization of deletion fragments of MuSK was confirmed by ELISA with an antibody directed against the HA-epitope.
determine functional effects of antibodies. Cells were plated onto chamber slides, fused and treated with or without agrin and/or plasmas or IgGs for 5 hours
13 . After fixation, AChRs were visualised with rhodamine- α -bungaro- toxin and the number of aggregates from more than 20 microscopic fields, and at least 2 independent cultures, were measured as described 25 . Control experiments showed that the MuSK fragment, used for preincubation of some plasmas, did not inhibit AChR aggregation in the final concentration present in the assay. Results are expressed as mean ± s.e.m. Acknowledgments We thank U. Schwarz for his support; C. Hopf for participating in cloning some of the constructs; A. Evoli and S. Robb for two of the plasmas and clinical infor- mation; and C.-M. Becker, C. Schuster and D. Roberts for critical reading of the manuscript. JMcC is supported by a Wellcome Trust Research Training Fellowship. J.N.-D. and A.V. thank the Muscular Dystrophy Campaign/Myasthenia Gravis Association for support. RECEIVED 2 AUGUST 2000; ACCEPTED 22 JANUARY 2001 1. Lindstrom, J., Seybold, M.E., Lennon, V.A., Whittingham, S. & Duane, D.D. Antibody to acetylcholine receptor in myasthenia gravis: prevalence, clinical cor- relates and diagnostic values. Neurology 26, 1054–1059 (1976). 2. Drachman, D.B. Myasthenia gravis. New Engl. J. Med. 330, 1797–1810 (1994). 3. Sanders, D.B., Andrews, I., Howard, J.F. & Massey, J.M. Seronegative myasthenia gravis. Neurology 48, S40–S45 (1997). 4. Mossman, S., Vincent, A. & Newsom-Davis, J. Myasthenia gravis without acetyl- choline-receptor antibody: a distinct disease entity. Lancet 1, 116–119 (1986). 5. Miers, A.K. & Havard, C.W.H. Diaphragmatic myasthenia in mother and child.
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7. Brooks, E.B., Pachner, A.R., Drachman, D.B. & Kantor, F.S. A sensitive rosetting assay for detection of acetylcholine receptor antibodies using BC3H-1 cells: posi- tive results in ‘antibody-negative’ myasthenia gravis. J. Neuroimmunol. 28, 83–93 (1990). 8. Blaes, F., Beeson, D., Plested, P., Lang, B. & Vincent, A. IgG from “seronegative” myasthenia gravis patients binds to a muscle cell line, TE671, but not to human acetylcholine receptor. Ann. Neurol. 47, 504–510 (2000). 9. Valenzuela, D.M. et al. Receptor tyrosine kinase specific for the skeletal muscle lin- eage: expression in embryonic muscle, at the neuromuscular junction, and after injury. Neuron 15, 573–584 (1995). 10. Glass, D.J. et al. Agrin acts via a MuSK receptor complex. Cell 85, 513–523 (1996). 11. Hopf, C. & Hoch, W. Tyrosine phosphorylation of the muscle-specific kinase is ex- clusively induced by acetylcholine receptor-aggregating agrin fragments. Eur. J. Biochem. 253, 382–389 (1998). 12. Sanes, J.R. & Lichtman, J.W. Development of the vertebrate neuromuscular junc- tion. Ann. Rev. Neurosci. 22, 389–442 (1999). 13. Hopf, C. & Hoch, W. Dimerization of the muscle-specific kinase induces tyrosine phosphorylation of acetylcholine receptors and their aggregation on the surface of myotubes. J. Biol. Chem. 273, 6467–6473 (1998). 14. Robertson, N.P., Deans, J. & Compston, D.A. Myasthenia Gravis: a population based epidemiological study in Cambridgeshire, England. J. Neurol. Neurosurg. Psychiatry 65, 492–496 (1998). 15. Aarli, J.A., Skeie, G.O., Mygland, A. & Gilhus, N.E. Muscle striation antibodies in myasthenia gravis. Diagnostic and functional significance. Ann. NY Acad. Sci. 841, 505–515 (1998). 16. Willcox, N., Schluep, M., Ritter, M.A. & Newsom-Davis, J. The thymus in seroneg- ative myasthenia gravis patients. J. Neurol. 238, 256–261 (1991). 17. Zhou, H., Glass, D.J., Yancopoulos, G.D. & Sanes, J.R. Distinct domains of MuSK mediate its ability to induce and to associate with postsynaptic specializations. J. Cell Biol. 146, 1133–1146 (1999). 18. Drachman, D.B., De Silva, S., Ramsay, D. & Pestronk, A. Humoral pathogenesis of myathenia gravis. Ann. NY Acad. Sci. 505, 90–105 (1987). 19. Vincent, A. et al. Seronegative myasthenia gravis. Ann. NY Acad. Sci. 681, 529–538 (1993). 20. Nakano, S. & Engel, A.G. Myasthenia gravis: quantitative immunocytochemical analysis of inflammatory cells and detection of complement membrane attack complex at the end-plate in 30 patients. Neurology 43, 1167–1172 (1993). 21. Newsom-Davis, J. Autoantibody-mediated channelopathies at the neuromuscular junction. The Neuroscientist 3, 337–346 (1997). 22. Taylor, S.I., Barbetti, F., Accili, D., Roth, J. & Gorden, P. Syndromes of autoimmu- nity and hypoglycaemia. Autoantibodies directed against insulin and its receptor. Endocrinol. Metab. Clin. North Am. 18, 123–143 (1989). 23. Robertson, S.C., Tynan, J.A. & Donoghue, D.J. RTK mutations and human syn- dromes: when good receptors turn bad. Trends Genet. 16, 265–271 (2000). 24. Vincent, A. & Newsom-Davis, J. Acetylcholine receptor antibodies as a diagnostic test for myasthenia gravis: results in 153 validated cases and 2967 diagnostic as- says. J. Neurol. Neurosurg. Psychiatry 48, 1246–1252 (1985). 25. Hopf, C. & Hoch, W. Heparin inhibits acetylcholine receptor aggregation at two distinct steps in the agrin-induced pathway. Eur. J. Neurosci. 9, 1170–1177 (1997). © 2001 Nature Pub lishing Gr oup http://medicine .nature .com © 2001 Nature Publishing Group http://medicine.nature.com Yüklə 85,66 Kb. Dostları ilə paylaş:
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