96 HUMORAL IMMUNITY IN MYASTHENIA GRAVIS: RELATIONSHIP TO DISEASE SEVERITY AND STEROID TREATMENT
SiR,—There is convincing evidence that myasthenia gravis
(M.G.) is an autoimmune disease in that most M.G. patients have a serum-IgG which binds to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction.’-3 We have measured this antibody at different times in the same M.G. patients and during drug-induced remission to see if there is any correlation with the severity of the disease. A radioimmunoassay (R.I.A.) was used with AChR from rat as the specific antigen. The AChR was purified from denervated rat muscle by a-toxin affinity chromatography, ion-exchange chromatography on ’DEAE Sephadex’, and gel filtration on ’Sepharose 6B’. The specific activity of this protein was approximately 7pmol [’z5I]-bungarotoxin bound per fLg AChR protein. [1151]-bungarotoxin-AChR complexes were prepared by incubating an excess of [125 I]-bungarotoxin with AChR for 1 h at room temperature and separation on a column of ’CM-50 Sephadex’. ANTIBODY TITRES AGAINST THE
AChR
FOR A SERIES OF M.G.
PATIENTS AND CONTROLS WITH OTHER NEUROMUSCULAR DISEASES
symptom-free after five weeks on anticholinesterase and prednisone the antibody titre was still 1 - 24 runola. It is, therefore, possible that the mode of action of steroids in M.G. may not pertain to the production of a specific antibody. Rather, steroids may directly affect the neuromuscular junctionb or, possibly, interfere with complement activation or some other cellular component of the immune system. Complement activation plays a major role in the aetiology of experimental autoimmune M.G. in the rat, and the C3 component of complement binds at the neuromuscular junction in M.G. patients.8 Under certain conditions steroids interfere with the complement-induced lysis of tumour cells.9 However, the role of complement in the aetiology of M.G. has yet to be demonstrated. The high average antibody titre which we have found in these patients who were recently symptom-free, leads us to consider the possibility that the antibody may be necessary, but not sufficient, to explain the autoimmune basis of the disease, because a patient can have very high antibody levels and no symptoms. R. J. BRADLEY* D. DWYER B. J. MORLEY Neuroscience Program G. ROBINSON and Department of Neurology, Medical Center, G. E. KEMP University of Albama in Birmingham, S. J. OH Birmingham, Alabama 35294, U.S.A. *
On leave
at
II
Physiologisches Institut,
Universitat des
Saarlandes, D6650
Homburg/Saar, Germany.
TOLUENE DIISOCYANATE his colleagues discuss the respiratory of toluene diisocyanate (T.D.I.) exposure. They conclude that the current threshold limit value (T.L.V.) of 0.02 parts per million is sufficient to protect the vast majority of workers. We believe that,there is insufficient evidence to support this. There are three separate problems which are often confused: (1) non-specific mucosal irritation in most of those exposed to high concentrations ofT.D.L; (2) the rate of decline in forced expiratory volume in 1 s (F.E.V.1) with age in symptom-free exposed workers, which some investigators have suggested is greater than that expected in unexposed subjects; and (3) the dose required to sensitise workers, resulting in occupational asthma. The current T.L.v. is below the irritant concentration of T.D.I. It is not clear, however, whether, if this T.L.V. is adhered to, those exposed will still be at risk either from an excessive rate of decline of F.E.V.1 or from the development of T.D.I.- induced bronchial asthma. Studies of the rate of decline of F.E.V.1 with age in exposed workers are conflicting. Adams2 found no excess fall in 180 symptom-free workers, but excluded all those with symptoms. Wegman et aI.3 concluded that 0.003 p.p.m. Of T.D.I. produces excess falls in F.E.V.!. However this study included only 57 of the original 112 workers at the start of the study and contained no control group. Butcher et al:4 found no excess fall in F.E.V.! in a group often exposed to levels above 0-02 p.p.m., but again omitted sensitised workers.
SiR,—Dr Adams and
problems
The
R.I.A. was then done by incubating the radiactive complex with varying amounts of serum for 24 h at 4°C. Sufficient goat anti-human IgG was then added to preciptate the total IgG in the assay, and incu-
bation was continued for 12 h at 4°C. This assay is similar to others4 and we obtain titres comparable to those obtained when human AChR has been used as the antigen.s Additionally, we obtain the same titres with both AChR from human muscle and denervated rat muscle when
subjected to our purification procedure. As others have found, 73% of our M.G. patients had the antibody but controls with other neuromuscular diseases did not. The average titre increased with the severity of the disease (see table). Repeated measurements on the same patients showed that the antibody titre increased as the disease became more severe. A Pearson correlation of M.G. classification with antibody titre gave a value of 0-4. In the table group i is patients who only have ocular M.G.; classification iic is equivalent to the Osserman classification of III and iv. Group 0 refers to two patients who were symptom-free and currently not under medication. Groups 10 and m0 are M.G. patients who had lately become symptom-free after treatment with anticholinesterase and prednisone. These patients were still under medication when assays were conducted. 10 out of 11 patients in the symptom-free groups had a measurable antibody titre. In fact, the mean value for group m0 was almost as high as group IIB. For example, 1 female patient classified as IIA had a serum antibody titre of 1.26 nmol/1 but when she became serum
1. Almon, R. R., Andrew, D. G., Appel, S. H. Science, 1974, 186, 55. 2. Aharonov, A., Tarrab-Hazdai, R., Abramsky, O., Fuchs, S. Lancet, 1975, ii, 340. 3. Bender, A. M., Ringel, S. P., Engel, W. K., Daniels, M. P., Vogel, F. ibid. 1975, ii, 607. 4. Monnier, V. M., Fulpius, B. W. Clin exp. Immun. 1977, 29, 16. 5. DeGrousaz, G., Fulpius, B. W. Lancet, 1978, i, 41.
Bradley, R. J., Dreyer, F., Müller, K. -D., Peper, K., Sterz, R. Pflügers Arch. 1978, 373, 226. 7. Lennon, V. A., Seybold, M. E., Lindstrom, J. M., Cochrane, C., Ulevitch, R. J. exp. Med. 1978, 147, 973. 8. Engel, A. G., Lambert, E. H., Howard, F. M. Mayo Clin. Proc. 1977, 52,
6.
267. 9. 1. 2. 3. 4.
Schlager, S. I., Ohanian, S. H., Borsos, T. Cancer Res. 1977, 37, 765. Adams, W. G. F, Carney, I. F., Chamberlain, J. D., Paddle, G. M. Lancet, 1978, i, 1308. Adams, W. G. F. Br J. ind. Med. 1975, 32, 72. Wegman, D. H., Peters, J. M., Pagnotto, L., Fine, L. J ibid. 1977, 34, 195. Butcher, B. T., Jones, R. N., O’Neil, C. E., Glindmeyer, H. W, Deim, J. E., Dharmarajan, V., Weill, H., Salvaggio, J. E. Am. Rev. resp. Dis. 1977, 116, 411
SiR,—There is convincing evidence that myasthenia gravis
(M.G.) is an autoimmune disease in that most M.G. patients have a serum-IgG which binds to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction.’-3 We have measured this antibody at different times in the same M.G. patients and during drug-induced remission to see if there is any correlation with the severity of the disease. A radioimmunoassay (R.I.A.) was used with AChR from rat as the specific antigen. The AChR was purified from denervated rat muscle by a-toxin affinity chromatography, ion-exchange chromatography on ’DEAE Sephadex’, and gel filtration on ’Sepharose 6B’. The specific activity of this protein was approximately 7pmol [’z5I]-bungarotoxin bound per fLg AChR protein. [1151]-bungarotoxin-AChR complexes were prepared by incubating an excess of [125 I]-bungarotoxin with AChR for 1 h at room temperature and separation on a column of ’CM-50 Sephadex’. ANTIBODY TITRES AGAINST THE
AChR
FOR A SERIES OF M.G.
PATIENTS AND CONTROLS WITH OTHER NEUROMUSCULAR DISEASES
symptom-free after five weeks on anticholinesterase and prednisone the antibody titre was still 1 - 24 runola. It is, therefore, possible that the mode of action of steroids in M.G. may not pertain to the production of a specific antibody. Rather, steroids may directly affect the neuromuscular junctionb or, possibly, interfere with complement activation or some other cellular component of the immune system. Complement activation plays a major role in the aetiology of experimental autoimmune M.G. in the rat, and the C3 component of complement binds at the neuromuscular junction in M.G. patients.8 Under certain conditions steroids interfere with the complement-induced lysis of tumour cells.9 However, the role of complement in the aetiology of M.G. has yet to be demonstrated. The high average antibody titre which we have found in these patients who were recently symptom-free, leads us to consider the possibility that the antibody may be necessary, but not sufficient, to explain the autoimmune basis of the disease, because a patient can have very high antibody levels and no symptoms. R. J. BRADLEY* D. DWYER B. J. MORLEY Neuroscience Program G. ROBINSON and Department of Neurology, Medical Center, G. E. KEMP University of Albama in Birmingham, S. J. OH Birmingham, Alabama 35294, U.S.A. *
On leave
at
II
Physiologisches Institut,
Universitat des
Saarlandes, D6650
Homburg/Saar, Germany.
TOLUENE DIISOCYANATE his colleagues discuss the respiratory of toluene diisocyanate (T.D.I.) exposure. They conclude that the current threshold limit value (T.L.V.) of 0.02 parts per million is sufficient to protect the vast majority of workers. We believe that,there is insufficient evidence to support this. There are three separate problems which are often confused: (1) non-specific mucosal irritation in most of those exposed to high concentrations ofT.D.L; (2) the rate of decline in forced expiratory volume in 1 s (F.E.V.1) with age in symptom-free exposed workers, which some investigators have suggested is greater than that expected in unexposed subjects; and (3) the dose required to sensitise workers, resulting in occupational asthma. The current T.L.v. is below the irritant concentration of T.D.I. It is not clear, however, whether, if this T.L.V. is adhered to, those exposed will still be at risk either from an excessive rate of decline of F.E.V.1 or from the development of T.D.I.- induced bronchial asthma. Studies of the rate of decline of F.E.V.1 with age in exposed workers are conflicting. Adams2 found no excess fall in 180 symptom-free workers, but excluded all those with symptoms. Wegman et aI.3 concluded that 0.003 p.p.m. Of T.D.I. produces excess falls in F.E.V.!. However this study included only 57 of the original 112 workers at the start of the study and contained no control group. Butcher et al:4 found no excess fall in F.E.V.! in a group often exposed to levels above 0-02 p.p.m., but again omitted sensitised workers.
SiR,—Dr Adams and
problems
The
R.I.A. was then done by incubating the radiactive complex with varying amounts of serum for 24 h at 4°C. Sufficient goat anti-human IgG was then added to preciptate the total IgG in the assay, and incu-
bation was continued for 12 h at 4°C. This assay is similar to others4 and we obtain titres comparable to those obtained when human AChR has been used as the antigen.s Additionally, we obtain the same titres with both AChR from human muscle and denervated rat muscle when
subjected to our purification procedure. As others have found, 73% of our M.G. patients had the antibody but controls with other neuromuscular diseases did not. The average titre increased with the severity of the disease (see table). Repeated measurements on the same patients showed that the antibody titre increased as the disease became more severe. A Pearson correlation of M.G. classification with antibody titre gave a value of 0-4. In the table group i is patients who only have ocular M.G.; classification iic is equivalent to the Osserman classification of III and iv. Group 0 refers to two patients who were symptom-free and currently not under medication. Groups 10 and m0 are M.G. patients who had lately become symptom-free after treatment with anticholinesterase and prednisone. These patients were still under medication when assays were conducted. 10 out of 11 patients in the symptom-free groups had a measurable antibody titre. In fact, the mean value for group m0 was almost as high as group IIB. For example, 1 female patient classified as IIA had a serum antibody titre of 1.26 nmol/1 but when she became serum
1. Almon, R. R., Andrew, D. G., Appel, S. H. Science, 1974, 186, 55. 2. Aharonov, A., Tarrab-Hazdai, R., Abramsky, O., Fuchs, S. Lancet, 1975, ii, 340. 3. Bender, A. M., Ringel, S. P., Engel, W. K., Daniels, M. P., Vogel, F. ibid. 1975, ii, 607. 4. Monnier, V. M., Fulpius, B. W. Clin exp. Immun. 1977, 29, 16. 5. DeGrousaz, G., Fulpius, B. W. Lancet, 1978, i, 41.
Bradley, R. J., Dreyer, F., Müller, K. -D., Peper, K., Sterz, R. Pflügers Arch. 1978, 373, 226. 7. Lennon, V. A., Seybold, M. E., Lindstrom, J. M., Cochrane, C., Ulevitch, R. J. exp. Med. 1978, 147, 973. 8. Engel, A. G., Lambert, E. H., Howard, F. M. Mayo Clin. Proc. 1977, 52,
6.
267. 9. 1. 2. 3. 4.
Schlager, S. I., Ohanian, S. H., Borsos, T. Cancer Res. 1977, 37, 765. Adams, W. G. F, Carney, I. F., Chamberlain, J. D., Paddle, G. M. Lancet, 1978, i, 1308. Adams, W. G. F. Br J. ind. Med. 1975, 32, 72. Wegman, D. H., Peters, J. M., Pagnotto, L., Fine, L. J ibid. 1977, 34, 195. Butcher, B. T., Jones, R. N., O’Neil, C. E., Glindmeyer, H. W, Deim, J. E., Dharmarajan, V., Weill, H., Salvaggio, J. E. Am. Rev. resp. Dis. 1977, 116, 411
