Immunology 1979 37 807
Antibodies to the acetylcholine receptor in myasthenic dogs
M. GARLEPP, B. FARROW*, P. KAY& R. L. DAWKINS Department of Clinical Immunology, Hospital & University Pathology Services, Queen Elizabeth II Medical Centre, Perth, Western Australia and *Department of Veterinary Medicine, University of Sydney
Acceptedfor publication 1 February 1979
Summary. Antibodies to the AChR and anti-striational antibodies have been detected in dogs suffering from myasthenia gravis. The detection of these antibodies adds to the known similarities between the human and canine disease. This animal model will facilitate investigation of agents involved in the induction of spontaneous myasthenia gravis.
regurgitation of food and mega-oesophagus. Administration of anti-cholinesterase leads to rapid but transient recovery (Palmer & Barker, 1974). Although thymomata have been reported in these dogs (Darke et al., 1975) there has been no previous demonstration ofautoantibodies. We report the detection of anti-AChR in three myasthenic dogs and the detection of anti-striational antibody in one of them. We also present some preliminary evidence suggesting that canine myasthenia gravis may provide insights into the development of the anti-AChR.
INTRODUCTION
Myasthenia gravis in man is a disease characterized clinically by excessive muscle fatigue which may be alleviated by administration of anti-cholinesterase. The disease is associated with thymic abnormalities (i.e. thymoma and thymic hyperplasia), anti-striational antibodies (Peers, McDonald & Dawkins, 1977) and antibodies to the acetylcholine receptor (AChR) (Lindstrom, 1977). It is very likely that the anti-AChR interferes with neuromuscular transmission, but little is known concerning the development of the antibody. A similar disease has been reported in a few dogs (Palmer & Barker, 1974; Johnson, Watson, Smith & Cooper, 1975; Darke, McCullagh & Geldart, 1975). Clinical features of the animal disease include muscular weakness after exercise, excessive salivation,
MATERIALS AND METHODS Dogs The clinical features of the dogs diagnosed to be suffering from myasthenia gravis are summarized in Table 1. Dogs 1, 2 and 3 presented with a 2 week history of weakness on exercise, excessive salivation and regurgitation of food. Dog I the most severely affected, showed a poor response to anti-cholinesterase, and died after developing aspiration pneumonia. Dogs 2 and 3 showed a dramatic response to intravenous edrophonium, and underwent spontaneous recovery I month after onset, dog 2 having been maintained on anti-cholinesterase throughout this time. Dog 3 gave a decremental response to repetitive nerve stimulation
Correspondence: Dr R. L. Dawkins, Dept of Clinical Immunology, Hospital & University Pathology Services, Queen Elizabeth II Medical Centre, Perth, Western Austra-
(Fig. 1). Dogs 4-12 were pups from the litter of dog 13. At 12 weeks of age dogs 4-11 developed a neostigmine-responsive weakness, having been vaccinated 3 weeks
lia. 00 19-2805/79/0800-0807$02.00 ©) 1979 Blackwell Scientific Publications
807
808
M. Garlepp et al. Table 1. Clinical features of myasthenic dogs Dog
1 2 3
Age
Diagnosis
21 years Kelpie Airedale I year Kelpie Cross 6 years
Old English Sheepdog 12 Old English Sheepdog 13 Old english Sheepdog 14-24 Various 4-1 1
Sex
12 weeks 12 weeks Unknown Various
M
MG; Clinical F MG; Clinical, Pharmacologic F MG; Clinical, Pharmacologic EMG ?MG; Transient weakness M&F neostigmine responsive M Nil Nil
F
M&F
Normal controls
Triton X-l00 (Rohm-Haas Co., Philadelphia) according to the standard method (Lindstrom, 1977).
Figure 1. Electromyogram from dog 3. The ulnar nerve was stimulated at a rate of 30 s- and recording made from the interosseus muscle.
previously with a combined measles and distemper vaccine. Spontaneous recovery occurred during 2 weeks without treatment. Healthy dogs of various ages and breeds were used to supply normal dog sera. Reagent.s
7-Bungarotoxin (Sigma, St Louis, Missouri) was labelled according to a previously published method (Lindstromn Lennon, Seybold & Whittingham, 1976). Goat anti-dog IgG was prepared in our laboratory by hyperimmunization of a goat with dog IgG in Freund's complete adjuvant (FCA). Benzoquinonium was a gift from Sterling Winthrop Research Institute. i)-Penicillamine was given as 125 mg tablets (DISTA Products Ltd, Speke, Liverpool, England).
Assay of aniti-A ChR Anti-AChR was detected by a modification of the standard assay (Lindstrom, 1977). A mixture of I ml of AChR and I ml of 1 x 10-9 M ot-bungarotoxin were incubated overnight at 4'. Ten microlitres of test serum was then added to tubes in triplicate and after a further overnight incubation sufficient goat anti-dog IgG (usually 100 pl) was added to precipitate the dog IgG in 24 h. The precipitate was washed once with 4 ml of 0 1 M sodium phosphate buffer pH 7 0 and the radioactivity counted in a Packard gamma spectrometer (model 5285). Parallel assays in which the AChR was pre-incubated with 10- M benzoquinonium for 6 h were performed for each serum. The counts precipitated in the presence of benzoquinonium were subtracted from those precipitated in its absence and the anti-AChR activity of each serum was then expressed as the number of moles of o-bungarotoxin binding sites precipitated per litre of serum. .Anlti-.srliationabl anHlthodi'
All sera were tested by indirect immunofluorescence on glycerinated guinea-pig skeletal muscle myofibrils as previously described (Peers et a1., 1977). Rat ACi R Sprague Dawley rat skeletal muscle was extracted as above and purified according to Lindstrom et al. (1976).
Extraction oftA Cli R
Dog AChR was extracted from fresh gastrocnemius muscle of normal dogs using the non-ionic detergent
,ssat. bor mneasle.s antitihodl' Antibodies to measles virus were assessed by a micro-
A Ch receptor antibodies in myasthenic dogs
adaptation of a standard haemagglutination inhibition assay (Katz & Enders, 1969). RESULTS Anti-AChR The titres of anti-AChR in the sera of the myasthenic and eleven normal dogs are shown in Table 2. Sera from dogs 1, 2 and 3 showed definite anti-AChR activity. The 'anti-AChR titres of dogs 4-12 were of the same order as those of their mother and the eleven normal dogs. It should be noted that the serum from dog 3 was taken toward the end of the disease period. Figure 2 shows the 'anti-AChR titres of a number of sera taken from dog 3 over a period of 9 months since the development of myasthenia gravis. The first sample corresponds to that shown in Table 2 and the remainder were taken during the subsequent period of remission. Low titres of antibody were present at all times. Two attempts were made to reinduce the disease in Table 2. Autoantibodies in myasthenic dogs
Dog
Anti-AChR titre*
1 2 3 4-11 12 13 14-24
496 1-44 1 52
Antibodies to the acetylcholine receptor in myasthenic dogs
M. GARLEPP, B. FARROW*, P. KAY& R. L. DAWKINS Department of Clinical Immunology, Hospital & University Pathology Services, Queen Elizabeth II Medical Centre, Perth, Western Australia and *Department of Veterinary Medicine, University of Sydney
Acceptedfor publication 1 February 1979
Summary. Antibodies to the AChR and anti-striational antibodies have been detected in dogs suffering from myasthenia gravis. The detection of these antibodies adds to the known similarities between the human and canine disease. This animal model will facilitate investigation of agents involved in the induction of spontaneous myasthenia gravis.
regurgitation of food and mega-oesophagus. Administration of anti-cholinesterase leads to rapid but transient recovery (Palmer & Barker, 1974). Although thymomata have been reported in these dogs (Darke et al., 1975) there has been no previous demonstration ofautoantibodies. We report the detection of anti-AChR in three myasthenic dogs and the detection of anti-striational antibody in one of them. We also present some preliminary evidence suggesting that canine myasthenia gravis may provide insights into the development of the anti-AChR.
INTRODUCTION
Myasthenia gravis in man is a disease characterized clinically by excessive muscle fatigue which may be alleviated by administration of anti-cholinesterase. The disease is associated with thymic abnormalities (i.e. thymoma and thymic hyperplasia), anti-striational antibodies (Peers, McDonald & Dawkins, 1977) and antibodies to the acetylcholine receptor (AChR) (Lindstrom, 1977). It is very likely that the anti-AChR interferes with neuromuscular transmission, but little is known concerning the development of the antibody. A similar disease has been reported in a few dogs (Palmer & Barker, 1974; Johnson, Watson, Smith & Cooper, 1975; Darke, McCullagh & Geldart, 1975). Clinical features of the animal disease include muscular weakness after exercise, excessive salivation,
MATERIALS AND METHODS Dogs The clinical features of the dogs diagnosed to be suffering from myasthenia gravis are summarized in Table 1. Dogs 1, 2 and 3 presented with a 2 week history of weakness on exercise, excessive salivation and regurgitation of food. Dog I the most severely affected, showed a poor response to anti-cholinesterase, and died after developing aspiration pneumonia. Dogs 2 and 3 showed a dramatic response to intravenous edrophonium, and underwent spontaneous recovery I month after onset, dog 2 having been maintained on anti-cholinesterase throughout this time. Dog 3 gave a decremental response to repetitive nerve stimulation
Correspondence: Dr R. L. Dawkins, Dept of Clinical Immunology, Hospital & University Pathology Services, Queen Elizabeth II Medical Centre, Perth, Western Austra-
(Fig. 1). Dogs 4-12 were pups from the litter of dog 13. At 12 weeks of age dogs 4-11 developed a neostigmine-responsive weakness, having been vaccinated 3 weeks
lia. 00 19-2805/79/0800-0807$02.00 ©) 1979 Blackwell Scientific Publications
807
808
M. Garlepp et al. Table 1. Clinical features of myasthenic dogs Dog
1 2 3
Age
Diagnosis
21 years Kelpie Airedale I year Kelpie Cross 6 years
Old English Sheepdog 12 Old English Sheepdog 13 Old english Sheepdog 14-24 Various 4-1 1
Sex
12 weeks 12 weeks Unknown Various
M
MG; Clinical F MG; Clinical, Pharmacologic F MG; Clinical, Pharmacologic EMG ?MG; Transient weakness M&F neostigmine responsive M Nil Nil
F
M&F
Normal controls
Triton X-l00 (Rohm-Haas Co., Philadelphia) according to the standard method (Lindstrom, 1977).
Figure 1. Electromyogram from dog 3. The ulnar nerve was stimulated at a rate of 30 s- and recording made from the interosseus muscle.
previously with a combined measles and distemper vaccine. Spontaneous recovery occurred during 2 weeks without treatment. Healthy dogs of various ages and breeds were used to supply normal dog sera. Reagent.s
7-Bungarotoxin (Sigma, St Louis, Missouri) was labelled according to a previously published method (Lindstromn Lennon, Seybold & Whittingham, 1976). Goat anti-dog IgG was prepared in our laboratory by hyperimmunization of a goat with dog IgG in Freund's complete adjuvant (FCA). Benzoquinonium was a gift from Sterling Winthrop Research Institute. i)-Penicillamine was given as 125 mg tablets (DISTA Products Ltd, Speke, Liverpool, England).
Assay of aniti-A ChR Anti-AChR was detected by a modification of the standard assay (Lindstrom, 1977). A mixture of I ml of AChR and I ml of 1 x 10-9 M ot-bungarotoxin were incubated overnight at 4'. Ten microlitres of test serum was then added to tubes in triplicate and after a further overnight incubation sufficient goat anti-dog IgG (usually 100 pl) was added to precipitate the dog IgG in 24 h. The precipitate was washed once with 4 ml of 0 1 M sodium phosphate buffer pH 7 0 and the radioactivity counted in a Packard gamma spectrometer (model 5285). Parallel assays in which the AChR was pre-incubated with 10- M benzoquinonium for 6 h were performed for each serum. The counts precipitated in the presence of benzoquinonium were subtracted from those precipitated in its absence and the anti-AChR activity of each serum was then expressed as the number of moles of o-bungarotoxin binding sites precipitated per litre of serum. .Anlti-.srliationabl anHlthodi'
All sera were tested by indirect immunofluorescence on glycerinated guinea-pig skeletal muscle myofibrils as previously described (Peers et a1., 1977). Rat ACi R Sprague Dawley rat skeletal muscle was extracted as above and purified according to Lindstrom et al. (1976).
Extraction oftA Cli R
Dog AChR was extracted from fresh gastrocnemius muscle of normal dogs using the non-ionic detergent
,ssat. bor mneasle.s antitihodl' Antibodies to measles virus were assessed by a micro-
A Ch receptor antibodies in myasthenic dogs
adaptation of a standard haemagglutination inhibition assay (Katz & Enders, 1969). RESULTS Anti-AChR The titres of anti-AChR in the sera of the myasthenic and eleven normal dogs are shown in Table 2. Sera from dogs 1, 2 and 3 showed definite anti-AChR activity. The 'anti-AChR titres of dogs 4-12 were of the same order as those of their mother and the eleven normal dogs. It should be noted that the serum from dog 3 was taken toward the end of the disease period. Figure 2 shows the 'anti-AChR titres of a number of sera taken from dog 3 over a period of 9 months since the development of myasthenia gravis. The first sample corresponds to that shown in Table 2 and the remainder were taken during the subsequent period of remission. Low titres of antibody were present at all times. Two attempts were made to reinduce the disease in Table 2. Autoantibodies in myasthenic dogs
Dog
Anti-AChR titre*
1 2 3 4-11 12 13 14-24
496 1-44 1 52
