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Skeletal muscle antibodies in patients with a thymic tumour but without myasthenia gravis
Journal of Neuroimmunology, 8 (1985) 69-78
69
Elsevier JNI 00244
Skeletal Muscle Antibodies in Patients with a Thymic Tumour but Without Myasthenia Gravis Nils Erik Gilhus 1.2, Johan A. Aarli i Rudolf W.C. Janzen 3, Herwart F. Otto 4, E. Fasske 5 and Roald Matre 2 t Department of Neurology and ' Broegelmann Research Laboratory for Microbiology, University of Bergen, Bergen (Norway)," 3 Department of Neurology, University of HamburN Hamburg, 4 Department of Pathology, University of Heidelberg, Heidelberg, and 5 Division of Pathology, Research Institute Borstel, Borstel (F.R.G.) (Received 10 April, 1984) (Revised, received 21 August, 1984) (Accepted 21 August, 1984)
Summa~ Sera from 9 patients with a primary thymic tumour but without myasthenia gravis (MG) at the time of thymectomy were examined for skeletal muscle antibodies. Antibodies to a citric acid extract (CA), associated with the presence of a thymic lymphoepithelioma in MG patients, were detected in 3 sera. AChR antibodies were detected in 5 sera, the concentration markedly elevated in 1 of them. Sarcolemmal and cross-striational antibodies were detected in 2 and 3 sera, respectively. Four of the 5 patients with thymic tumours other than a lymphoepithelioma had AChR antibodies, and 3 of them also had CA antibodies. Key words:
M y a s t h e n i a graois - S k e l e t a l muscle antibodies - T h y m i c tumour
Introduction
Lymphoepithelioma is the most common primary thymic tumour (Henry 1981). One-third of the patients with a lymphoepithelioma have myasthenia gravis (MG), whereas MG occurs very rarely together with thymic tumours which are not derived N.E. Gilhus was a Research Fellow of the Norwegian Cancer Society when this study was undertaken. The study was supported by a grant from the Norwegian Research Council for Science and the Humanities. Reprint requests: N.E. Gilhus, Department, of :Neurology, N-5016 Haukeland Sykehus, Bergen, Norway. 0165-5728/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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71 from the epithelial cells (Lisak and Barchi 1982). Most patients with MG have circulating antibodies to antigens in skeletal muscle, especially to the acetylcholine receptor (AChR) region. We have recently reported that antibodies to a citric acid extract (CA) of skeletal muscle are closely associated with the presence of a thymic lymphoepithelioma in MG patients (Aarli et al. 1981; Gilhus et al. 1984). In the present study, we have collected serum from 9 patients with various types of primary thymic turnouts, but without any myasthenic symptoms at the time of thymectomy. The sera were examined for antibodies to CA, AChR and to other skeletal muscle antigens to determine whether these antibodies are linked to the combination thymoma - - MG, or to the thymoma per se. Materials and Methods Patients
Preoperative sera were obtained from 9 patients with primary thymic tumours and without any clinical evidence of neuromuscular disorder. The sex and age of the patients, and the growth and histology of their tumours are shown in Table 1. Preoperative sera were also obtained from 9 control patients with a thymic tumour and MG. All these patients had non-invasive lymphoepitheliomas. They were comparable with the non-MG patients regarding sex and age. Tissue
Human skeletal muscle was obtained from amputated limbs. One part was homogenized in isotonic phosphate-buffered saline, pH 7.2 (PBS), washed and extracted with acetone. The CA antigen was extracted from the sediment by 0.05 M citric acid, as previously described (Aarli et al. 1981). Another part of the same muscle was homogenized in 0.05 M Tris-HC1, 0.1 M NaCI, 0.001 M EDTA, pH 7.4, with the addition of aprotinin to 106 Kallikrein inhibitory units/1 (Trasylol, Bayer AG, Leverkusen, F.R.G.). Membrane-bound AChR was extracted with the same buffer containing 1.5% Triton X-100, as described by Lefvert et al. (1978). Suitable pieces of fresh muscle were snap-frozen in liquid nitrogen and mounted on specimen holders. The frozen tissue was sectioned in 4 - 6 ~m thick sections on a cryostat, and then placed on cover glasses. CA antibodies
CA antibodies were detected by an indirect haemagglutination test as previously described (Aarli et al. 1981). Tannic acid-treated sheep erythrocytes (SE) were incubated with CA, washed and made up to a 0.75% suspension of CA-coated SE in PBS. The sera to be tested were 2-fold diluted using microtitre equipment. CA-coated SE were added to each well. The titre was defined as the reciprocal of the highest dilution of serum giving agglutination after 4 h. Titres of 32 or higher were regarded as positive. A ChR antibodies
AChR antibodies were determined according to Lefvert et al. (1978) with minor
72 modifications. In brief, the Triton extract was incubated with [t25i]a_bungarotoxin (New England Nuclear, Boston, MA, U.S.A.) for 1 h at 37°C. To remove excess toxin, the labelled toxin-receptor complex was separated by gel filtration on Sephacryi S-200 superfine at room temperature. The column was equilibrated with the same buffer as used for AChR extraction, except that Triton X-100 was in this instance used at 0.5% concentration. Five/xl of serum were incubated with 200 ~1 of the toxin-receptor complex for 16 h at 4°C. Rabbit antiserum to human IgG (Behringwerke AG, Marburg/Lahn, F.R.G.), 200/zl, was added and the tubes kept at room temperature for 3 h. The tubes were centrifuged at 8 000 x g for 5 min, the precipitate washed once with 1 ml of the same buffer and the radioactivity of the precipitate was then determined. Titres of AChR antibodies were expressed as moles of [125I]a-bungarotoxin binding sites precipitated per liter of serum (M). The normal serum, which was used in all determinations, was pooled from 50 healthy blood donors. Values > 0.4 M were recorded as positive.
Sarcolemmal and cross-striational antibodies Sections of skeletal muscle cut both longitudinally and transversally were incubated with the sera diluted 1 in 32 for 30 min at room temperature, as previously described (Gilhus et al. 1983, 1984). After washing in PBS, the tissue sections were incubated with fluorescein isothiocyanate-conjugated F(ab')2 fragments of goat IgG antibodies to the F(ab')2 part of human IgG, purchased from Nordic Immunological Lab., Tilburg, The Netherlands (code no. 24-581, protein concentration 10 mg/mi, molar F / P ratio between 1 and 4). The conjugate was diluted 1 in 20 in PBS. After a final washing in PBS, the sections were mounted and examined under a Leitz orthoplane microscope equipped with an Osram HBO mercury lamp. To determine the titre, serum which showed fluorescence was diluted 2-fold in PBS until there was no reaction.
Absorption experiments Serum from 2 patients without MG, which contained CA and AChR antibodies, was repeatedly absorbed with packed, tanned CA-coated SE, packed tanned SE, and homogenized human skeletal muscle, liver and kidney as previously described (Gilhus et al. 1984).
Control experiments The specificity of the positive reactions observed in the indirect haemagglutination and indirect immunofluorescence tests was determined as has been described previously (Aarli et al. 1981; Gilhus et al. 1983, 1984). IgG F(ab')2 fragments of positive sera were used, and positive and negative human control sera were tested in parallel experiments.
Results
Non-MG patients Sera from 3 of the 9 patients contained CA antibodies in high titres, whereas 6
73
Fig. 1. Thymic T lymphoblast lymphoma, patient 1. Some pyknotic tumour cells, only few convoluted nuclei. Giemsa, orig. magn. X 350.
s e r a d i d n o t ( T a b l e 2). T h e p o s i t i v e s e r a w e r e d e r i v e d f r o m 3 p a t i e n t s w i t h t h e following thymic tumours: Patient 1 was o p e r a t e d for a large t u m o u r of the anterior m e d i a s t i n u m f o u n d on X-ray. Histologic examination revealed a T lymphoblast lymphoma of convoluted
TABLE 2 TITRES OF VARIOUS SKELETAL MUSCLE ANTIBODIES IN SERA FROM 9 PATIENTS WITHOUT MG The histology of the thymic tumours is stated. Patient
1 2 3 4 5 6 7 8 9
Antibodies
Histology
CA
AChR ( × 10 -9 M)
Sarcolemmal
Crossstriational
16 384 2048 1024 < 32 < 32 < 32 < 32 < 32 < 32
58.6 0.5 0.8 2.2 < 0.4 1.5 < 0.4 < 0.4 < 0.4
256 32 < 32 < 32 < 32 < 32 < 32 < 32 < 32
128 < 32 < 32 < 32 < 32 64 32 < 32 < 32
Non-Hodgldn lymphoma Hamartoma Mesenchymal tumour Non-Hodgkin lymphoma Malignant histiocytoma Unknown Lymphoepithelioma Lymphoepithelioma Lymphoepithelioma
74
Fig. 2. Thymic Castleman tumour, vascular-hyaline type, patient 2. H & E, orig. magn. × 120.
Fig. 3. Thymic malignant mesenchymal tumour, patient 3. H & E, orig. magn. × 80.
75 cell type, whereas normal thymus tissue was not seen (Fig. 1). He had multiple metastases to regional lymph nodes, and later developed a lymphoblast leukemia. Patient 2 had a symptomless mediastinal tumour detected by X-ray. At operation a large mediastinal tumour weighing 150 g was found. Histologic examination revealed giant lymph node hyperplasia (Castleman's disease) of the vascular-hyaline type (Fig. 2). Patient 3 had an invasive mediastinal tumour where biopsy revealed a malignant mesenchymal tumour of the anterior mediastinum with metastatic infiltration of the thorax muscles and skin. This tumour was either a sarcoma or a lymphoma (Fig. 3). There were 3 n o n - M G patients with lymphoepitheliomas. None of them had CA antibodies. Patient 4 had a non-Hodgkin lymphoma. Her serum contained A C h R antibodies, but not CA antibodies (Table 2). AChR antibodies were detected in the 3 sera which contained CA antibodies. In addition, the patient with a thymic tumour of unknown histology had an increased A C h R antibody concentration. The A C h R antibody concentration was highest in the patient with a T lymphoblast lymphoma. None of the 3 patients with a lymphoepithelioma had A C h R antibodies, whereas only 1 of the patients with other tumours did not have these antibodies. Sarcolemmal antibodies were detected in sera from 2 of the 3 patients with CA antibodies. The antibodies stained the surface of skeletal muscle cells in indirect immunofluorescence. The 7 remaining sera did not stain the muscle cell surface. Staining of cross-striational double-bands with a central non-fluorescent zone was seen when sera from 3 of the 9 patients were incubated with sections of skeletal muscle. Only one of the sera with cross-striational antibodies contained CA antibodies. This serum had all types of skeletal muscle antibodies in high concentrations. The CA antibody titres of the 2 sera to be absorbed were 16 384 and 2084 before any absorptions. After absorption with either CA-coated SE or homogenized muscle the titres were less than 32. Absorption with CA-coated SE or homogenized muscle also removed the cell surface staining. Absorption with homogenized muscle reduced the A C h R antibody concentration to less than 0.4 x 10 -9 M and removed the TABLE 3 TITRES OF VARIOUSSKELETALMUSCLE ANTIBODIESIN SERA FROM 9 PATIENTSWITH BOTH A THYMIC LYMPHOEPITHELIOMAAND MG Patient
CA
AChR ( x 10- 9 M)
Sarcolemmal
Crossstriational
1 2 3 4 5 6 7 8 9
32768 16384 16384 8192 8192 8192 4096 4096 1024
64.0 9.4 8.2 28.5 7.3 36.2 26.4 16.0 21.1
1024 512 128 128 32 < 32 512 64 32
512 64 32 32 < 32 < 32 512 64 32
76 cross-striational staining, whereas absorption with CA-coated SE did not affect these reactions. Absorption with tanned SE, liver and kidney did not reduce the reactivity of the sera against skeletal muscle.
MG control patients All the sera from patients with both a thymoma and MG contained CA antibodies in high titres (Table 3). The serum concentration of AChR antibodies was also markedly elevated in all 9 sera. Eight of the sera contained antibodies to the skeletal muscle cell surface, and 7 sera contained antibodies to the cross-striational bands.
Discussion
The results of this study show that the presence of a thymic tumour in non-MG patients is not strongly associated with circulating CA antibodies, since only 3 of our 9 patients without MG had such antibodies. In contrast, nearly all patients with both MG and a thymic tumour have CA antibodies (Aarli et al. 1981; Gilhus et al. 1984), and all 9 patients with MG and such a tumour included in the present study had high-titred CA antibodies. Patients with a thymic tumour but without MG can also have other types of skeletal muscle antibodies. Cross-striational antibodies were detected in 3 of our 9 non-MG patients and have previously been reported in about 25% of such patients (McFarlin et al. 1966; Oosterhuis et al. 1976; Limburg et al. 1983). Sarcolemmal antibodies were found in serum from 2 of our patients, who also had CA antibodies, and have previously been reported in one patient with a thymoma but without MG (Beutner et al. 1966). Sarcolemmal antibodies are ~ssociated with the CA antibodies (Gilhus et al. 1983). Nearly all our control patients with both thymoma and MG had sarcolemmal and cross-striational muscle antibodies, in accordance with previous studies of MG patients with thymoma (Oosterhuis et al. 1976; Gilhus et al. 1983). Skeletal muscle antibodies usually do not occur in patients with secondary malignant involvement of the thymus (Oosterhuis et al. 1976). Also, AChR antibodies can be found in patients with a thymic tumour but without any muscular weakness. Such antibodies were found in 5 of our patients without MG. In 3 of them, the concentrations were only slightly above the normal level, whereas 2 patients had a markedly increased AChR antibody level. Cu6noud et al. (1980) reported AChR antibodies in 3 of 11 non-MG patients with a thymoma, and Limburg et al. (1983) found AChR antibodies in 2 of 11 similar patients. In 2 of these 5 non-MG thymoma patients with AChR antibodies, the AChR antibody concentration was very high. The 9 thymoma patients with MG in the present study had markedly elevated AChR antibody concentrations, as have such patients in general (Lefvert et al. 1978; Limburg et al. 1983). Our non-MG patients with skeletal muscle antibodies had thyrnic tumours of various histological types. The patients with a lymphoepithelioma had less antibodies than those with other thymic tumours; none of the 3 patients with a lymphoe-
77 pithelioma had CA antibodies, whereas 3 of the 5 patients with other tumours had such antibodies in high titres. Nearly all patients with M G together with a thymic tumour have a lymphoepithelioma (Henry 1981; Lisak and Barchi 1982). The present study shows that skeletal muscle antibody production can also occur together with other thymic tumours such as lymphomas, sarcomas and hamartomas. M G has only rarely been reported together with such tumours (Null et al. 1977; Yuasa et al. 1980; Otto et al. 1982; Hansen et al. 1983). Thus, our results, combined with previous knowledge, indicate that although patients with thymic tumours other than lymphoepithelioma may have circulating muscle antibodies, they usually do not develop symptoms of MG. A reason why a high concentration of A C h R antibodies does not necessarily lead to M G could be that the antibodies do not bind to the appropriate region of the A C h R molecule. Another possibility is that a circulating factor in addition to the A C h R antibodies is required to induce the muscular weakness in MG. Our results indicate that such a possible additional factor often occurs together with a thymic lymphoepithelioma, since most patients with this tumour have MG, whereas patients with a lymphoepithelioma but without M G usually lack A C h R antibodies. This possible additional factor rarely operates in the presence of other thymic tumours, since most patients with such tumours do not have M G although they may have A C h R antibodies. The postulated factor for the development of M G may be a hormone secreted by thymic epithelial cells (Dalakas et al. 1981; Olanow and Roses
1981). The relationship between a thymic tumour, skeletal muscle antibodies and M G is far from understood. It is generally accepted that thymus pathology as well as A C h R antibodies participate in the pathogenesis of MG. However, onset of M G years after the removal of a thymoma and adjacent thymus has been reported (Janzen et al. 1977; N a m b a et al. 1978), and M G can also be seen without detectable AChR antibodies (Oosterhuis et al. 1983). Conversely, the present study shows that a thymic tumour as well as A C h R antibodies can be present without MG. A C h R antibodies have been detected in sera from elderly patients with cerebrovascular disease and from patients with Down's syndrome, without any symptoms of M G (Tanaka and Miyatake 1983). Additional factors most probably participate in the development of the muscular weakness in MG.
References Aarli, J.A., A.-K. Lefvert and O. Tender, Thymoma-specific antibodies in sera from patients with
myasthenia gravis demonstrated by indirect ha~magglutination,J. Neuroimmunol., 1 (1981) 421-427. Beutner, E.H., G. Fazekas, A. Scott and E. Witebsky, Direct fluorescent antibody studies of gamma globuhn localization in muscle of patients with myasthenia gravis, Ann. N.Y. Acad. Sci., 135 (1966) 588-600. Cu6noud, M., T.E.W. Feltkarnp, B.W. Fulpius and H.J.G.H. Oosterhuis, Antibodies to acetylcholine receptor in patients with thymoma but without myasthenia gravis, Neurology, 30 (1980) 201-203. Dalakas, M.C., W.K. Engel, J.E. McClure, A.L. Goldstein and V. Askanas, Identification of human thymic epithelial cells with antibodies to thymosin a I in myasthenia gravis, Ann. N.Y. Acad. Sci., 377 (1981) 477-485.
78 Gilhus, N.E., J.A. Aarli and R. Matre, Myasthenia gravis - - Antibodies to skeletal muscle cell surface antigens, J. Neuroimmunol., 5 (1983) 239-249. Gilhus, N.E., J.A. Aarli and R. Matre, Myasthenia gravis - - The difference between thymoma-associated antibodies and cross-striational staining skeletal muscle antibodies, Neurology, 34 (1984) 246-249. Hansen, B.A., P.S. Sorensen, M.J. Lauritzen, J. Iversen, S. Laulund, J.E. Petrera, J.B. Nielsen and O.B. Paulson, A case of malignant lymphoma and myasthenia gravis, Scand. J. Haematol., 31 (1983) 155-160. Henry, K., The human thymus in disease with particular emphasis on thymitis and thymoma. In: M.D. Kendall (Ed.), The Thymus Gland, Academic Press, London, 1981, pp. 85-111. Janzen, R.W.C., L. Lachenmayer and K. Fischer, Probleme der Postthymektomiesyndrome--Am Beispiel der myasthenischen Reaktion, Med. Klin., 72 (1977) 1931-1937. Lefvert, A.K., K. Bergstrom, G. Matell, P.O. Osterman and R. Pirskanen, Determination of acetylcholine receptor antibody in myasthenia g r a v i s - Clinical usefulness and pathogenetic implications, J. Neurol. Neurosurg. Psychiat., 41 (1978) 394-403. Limburg, P.C., T.H. The, E. HummeI-Tappel and H.J.G.H. Oosterhuis, Anti-acetylcholine receptor antibodies in myasthenia gravis, Part 1 (Relation to clinical parameters in 250 patients), J. Neurol. Sci., 58 (1983) 357-370. Lisak, R.P. and R.L. Barchi, Myasthenia Gravis, W.B. Saunders Company, Philadelphia, 1982. McFarlin, D.E., M. Barlow and A.J.L. Strauss, Antibodies to muscle and thymus in nonmyasthenic patients with thymoma - - Clinical evaluation, N. Engl. J. Med., 275 (1966) 1321-1326. Namba, T., N.G. Brunner and D. Grob, Myasthenia gravis in patients with thymoma, with particular reference to onset after thymectomy, Medicine (Baltimore), 57 (1978) 411-433. Null, J.A., V.A. LiVolsi and W.W.L. Glenn, Hodgkin's disease of the thymus (granulomatous thymoma) and myasthenia gravis - - A unique association, Amer. J. Clin. Path., 67 (1977) 521-525. Olanow, C.W. and A.D. Roses, The pathogenesis of myasthenia gravis - - A hypothesis, Med. Hypotheses, 7 (1981) 957-968. Oosterhuis, H.J.G.H., T.E.W. Feltkamp, A.L. Van Rossum, P.M. Van den Berg-Loonen and L.E. Nijenhuis, HL-A antigens, autoantibody production, and associated diseases in thymoma patients, with and without myasthenia gravis, Ann. N.Y. Acad. Sci., 274 (1976) 468-474. Oosterhuis, H.J.G.H., P.C. Limburg, E. Hummel-Tappel and T.H. The, Anti-acetylcholine receptor antibodies in myasthenia gravis, Part 2 (Clinical and serological follow-up of individual patients), J. Neurol. Sci., 58 (1983) 371-385. Otto, H.F., T. LOning, L. Lachenmayer, R.W.C. Janzen, K~F. Giartler and K. Fischer, Thymolipoma in association with myasthenia gravis, Cancer, 50 (1982) 1623-1628. Tanaka, M. and T. Miyatake, Anti-acetylcholine receptor antibody in aged individuals and in patients with Down's syndrome, J. Neuroimmunol., 4 (1983) 17-24. Yuasa, T., M. Hanano, F. Ohshima and T. Tsubaki, The association of myasthenia gravis with multiple hamartoma syndrome (Cowden disease), Ann. Neurol., 7 (1980) 591.
69
Elsevier JNI 00244
Skeletal Muscle Antibodies in Patients with a Thymic Tumour but Without Myasthenia Gravis Nils Erik Gilhus 1.2, Johan A. Aarli i Rudolf W.C. Janzen 3, Herwart F. Otto 4, E. Fasske 5 and Roald Matre 2 t Department of Neurology and ' Broegelmann Research Laboratory for Microbiology, University of Bergen, Bergen (Norway)," 3 Department of Neurology, University of HamburN Hamburg, 4 Department of Pathology, University of Heidelberg, Heidelberg, and 5 Division of Pathology, Research Institute Borstel, Borstel (F.R.G.) (Received 10 April, 1984) (Revised, received 21 August, 1984) (Accepted 21 August, 1984)
Summa~ Sera from 9 patients with a primary thymic tumour but without myasthenia gravis (MG) at the time of thymectomy were examined for skeletal muscle antibodies. Antibodies to a citric acid extract (CA), associated with the presence of a thymic lymphoepithelioma in MG patients, were detected in 3 sera. AChR antibodies were detected in 5 sera, the concentration markedly elevated in 1 of them. Sarcolemmal and cross-striational antibodies were detected in 2 and 3 sera, respectively. Four of the 5 patients with thymic tumours other than a lymphoepithelioma had AChR antibodies, and 3 of them also had CA antibodies. Key words:
M y a s t h e n i a graois - S k e l e t a l muscle antibodies - T h y m i c tumour
Introduction
Lymphoepithelioma is the most common primary thymic tumour (Henry 1981). One-third of the patients with a lymphoepithelioma have myasthenia gravis (MG), whereas MG occurs very rarely together with thymic tumours which are not derived N.E. Gilhus was a Research Fellow of the Norwegian Cancer Society when this study was undertaken. The study was supported by a grant from the Norwegian Research Council for Science and the Humanities. Reprint requests: N.E. Gilhus, Department, of :Neurology, N-5016 Haukeland Sykehus, Bergen, Norway. 0165-5728/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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71 from the epithelial cells (Lisak and Barchi 1982). Most patients with MG have circulating antibodies to antigens in skeletal muscle, especially to the acetylcholine receptor (AChR) region. We have recently reported that antibodies to a citric acid extract (CA) of skeletal muscle are closely associated with the presence of a thymic lymphoepithelioma in MG patients (Aarli et al. 1981; Gilhus et al. 1984). In the present study, we have collected serum from 9 patients with various types of primary thymic turnouts, but without any myasthenic symptoms at the time of thymectomy. The sera were examined for antibodies to CA, AChR and to other skeletal muscle antigens to determine whether these antibodies are linked to the combination thymoma - - MG, or to the thymoma per se. Materials and Methods Patients
Preoperative sera were obtained from 9 patients with primary thymic tumours and without any clinical evidence of neuromuscular disorder. The sex and age of the patients, and the growth and histology of their tumours are shown in Table 1. Preoperative sera were also obtained from 9 control patients with a thymic tumour and MG. All these patients had non-invasive lymphoepitheliomas. They were comparable with the non-MG patients regarding sex and age. Tissue
Human skeletal muscle was obtained from amputated limbs. One part was homogenized in isotonic phosphate-buffered saline, pH 7.2 (PBS), washed and extracted with acetone. The CA antigen was extracted from the sediment by 0.05 M citric acid, as previously described (Aarli et al. 1981). Another part of the same muscle was homogenized in 0.05 M Tris-HC1, 0.1 M NaCI, 0.001 M EDTA, pH 7.4, with the addition of aprotinin to 106 Kallikrein inhibitory units/1 (Trasylol, Bayer AG, Leverkusen, F.R.G.). Membrane-bound AChR was extracted with the same buffer containing 1.5% Triton X-100, as described by Lefvert et al. (1978). Suitable pieces of fresh muscle were snap-frozen in liquid nitrogen and mounted on specimen holders. The frozen tissue was sectioned in 4 - 6 ~m thick sections on a cryostat, and then placed on cover glasses. CA antibodies
CA antibodies were detected by an indirect haemagglutination test as previously described (Aarli et al. 1981). Tannic acid-treated sheep erythrocytes (SE) were incubated with CA, washed and made up to a 0.75% suspension of CA-coated SE in PBS. The sera to be tested were 2-fold diluted using microtitre equipment. CA-coated SE were added to each well. The titre was defined as the reciprocal of the highest dilution of serum giving agglutination after 4 h. Titres of 32 or higher were regarded as positive. A ChR antibodies
AChR antibodies were determined according to Lefvert et al. (1978) with minor
72 modifications. In brief, the Triton extract was incubated with [t25i]a_bungarotoxin (New England Nuclear, Boston, MA, U.S.A.) for 1 h at 37°C. To remove excess toxin, the labelled toxin-receptor complex was separated by gel filtration on Sephacryi S-200 superfine at room temperature. The column was equilibrated with the same buffer as used for AChR extraction, except that Triton X-100 was in this instance used at 0.5% concentration. Five/xl of serum were incubated with 200 ~1 of the toxin-receptor complex for 16 h at 4°C. Rabbit antiserum to human IgG (Behringwerke AG, Marburg/Lahn, F.R.G.), 200/zl, was added and the tubes kept at room temperature for 3 h. The tubes were centrifuged at 8 000 x g for 5 min, the precipitate washed once with 1 ml of the same buffer and the radioactivity of the precipitate was then determined. Titres of AChR antibodies were expressed as moles of [125I]a-bungarotoxin binding sites precipitated per liter of serum (M). The normal serum, which was used in all determinations, was pooled from 50 healthy blood donors. Values > 0.4 M were recorded as positive.
Sarcolemmal and cross-striational antibodies Sections of skeletal muscle cut both longitudinally and transversally were incubated with the sera diluted 1 in 32 for 30 min at room temperature, as previously described (Gilhus et al. 1983, 1984). After washing in PBS, the tissue sections were incubated with fluorescein isothiocyanate-conjugated F(ab')2 fragments of goat IgG antibodies to the F(ab')2 part of human IgG, purchased from Nordic Immunological Lab., Tilburg, The Netherlands (code no. 24-581, protein concentration 10 mg/mi, molar F / P ratio between 1 and 4). The conjugate was diluted 1 in 20 in PBS. After a final washing in PBS, the sections were mounted and examined under a Leitz orthoplane microscope equipped with an Osram HBO mercury lamp. To determine the titre, serum which showed fluorescence was diluted 2-fold in PBS until there was no reaction.
Absorption experiments Serum from 2 patients without MG, which contained CA and AChR antibodies, was repeatedly absorbed with packed, tanned CA-coated SE, packed tanned SE, and homogenized human skeletal muscle, liver and kidney as previously described (Gilhus et al. 1984).
Control experiments The specificity of the positive reactions observed in the indirect haemagglutination and indirect immunofluorescence tests was determined as has been described previously (Aarli et al. 1981; Gilhus et al. 1983, 1984). IgG F(ab')2 fragments of positive sera were used, and positive and negative human control sera were tested in parallel experiments.
Results
Non-MG patients Sera from 3 of the 9 patients contained CA antibodies in high titres, whereas 6
73
Fig. 1. Thymic T lymphoblast lymphoma, patient 1. Some pyknotic tumour cells, only few convoluted nuclei. Giemsa, orig. magn. X 350.
s e r a d i d n o t ( T a b l e 2). T h e p o s i t i v e s e r a w e r e d e r i v e d f r o m 3 p a t i e n t s w i t h t h e following thymic tumours: Patient 1 was o p e r a t e d for a large t u m o u r of the anterior m e d i a s t i n u m f o u n d on X-ray. Histologic examination revealed a T lymphoblast lymphoma of convoluted
TABLE 2 TITRES OF VARIOUS SKELETAL MUSCLE ANTIBODIES IN SERA FROM 9 PATIENTS WITHOUT MG The histology of the thymic tumours is stated. Patient
1 2 3 4 5 6 7 8 9
Antibodies
Histology
CA
AChR ( × 10 -9 M)
Sarcolemmal
Crossstriational
16 384 2048 1024 < 32 < 32 < 32 < 32 < 32 < 32
58.6 0.5 0.8 2.2 < 0.4 1.5 < 0.4 < 0.4 < 0.4
256 32 < 32 < 32 < 32 < 32 < 32 < 32 < 32
128 < 32 < 32 < 32 < 32 64 32 < 32 < 32
Non-Hodgldn lymphoma Hamartoma Mesenchymal tumour Non-Hodgkin lymphoma Malignant histiocytoma Unknown Lymphoepithelioma Lymphoepithelioma Lymphoepithelioma
74
Fig. 2. Thymic Castleman tumour, vascular-hyaline type, patient 2. H & E, orig. magn. × 120.
Fig. 3. Thymic malignant mesenchymal tumour, patient 3. H & E, orig. magn. × 80.
75 cell type, whereas normal thymus tissue was not seen (Fig. 1). He had multiple metastases to regional lymph nodes, and later developed a lymphoblast leukemia. Patient 2 had a symptomless mediastinal tumour detected by X-ray. At operation a large mediastinal tumour weighing 150 g was found. Histologic examination revealed giant lymph node hyperplasia (Castleman's disease) of the vascular-hyaline type (Fig. 2). Patient 3 had an invasive mediastinal tumour where biopsy revealed a malignant mesenchymal tumour of the anterior mediastinum with metastatic infiltration of the thorax muscles and skin. This tumour was either a sarcoma or a lymphoma (Fig. 3). There were 3 n o n - M G patients with lymphoepitheliomas. None of them had CA antibodies. Patient 4 had a non-Hodgkin lymphoma. Her serum contained A C h R antibodies, but not CA antibodies (Table 2). AChR antibodies were detected in the 3 sera which contained CA antibodies. In addition, the patient with a thymic tumour of unknown histology had an increased A C h R antibody concentration. The A C h R antibody concentration was highest in the patient with a T lymphoblast lymphoma. None of the 3 patients with a lymphoepithelioma had A C h R antibodies, whereas only 1 of the patients with other tumours did not have these antibodies. Sarcolemmal antibodies were detected in sera from 2 of the 3 patients with CA antibodies. The antibodies stained the surface of skeletal muscle cells in indirect immunofluorescence. The 7 remaining sera did not stain the muscle cell surface. Staining of cross-striational double-bands with a central non-fluorescent zone was seen when sera from 3 of the 9 patients were incubated with sections of skeletal muscle. Only one of the sera with cross-striational antibodies contained CA antibodies. This serum had all types of skeletal muscle antibodies in high concentrations. The CA antibody titres of the 2 sera to be absorbed were 16 384 and 2084 before any absorptions. After absorption with either CA-coated SE or homogenized muscle the titres were less than 32. Absorption with CA-coated SE or homogenized muscle also removed the cell surface staining. Absorption with homogenized muscle reduced the A C h R antibody concentration to less than 0.4 x 10 -9 M and removed the TABLE 3 TITRES OF VARIOUSSKELETALMUSCLE ANTIBODIESIN SERA FROM 9 PATIENTSWITH BOTH A THYMIC LYMPHOEPITHELIOMAAND MG Patient
CA
AChR ( x 10- 9 M)
Sarcolemmal
Crossstriational
1 2 3 4 5 6 7 8 9
32768 16384 16384 8192 8192 8192 4096 4096 1024
64.0 9.4 8.2 28.5 7.3 36.2 26.4 16.0 21.1
1024 512 128 128 32 < 32 512 64 32
512 64 32 32 < 32 < 32 512 64 32
76 cross-striational staining, whereas absorption with CA-coated SE did not affect these reactions. Absorption with tanned SE, liver and kidney did not reduce the reactivity of the sera against skeletal muscle.
MG control patients All the sera from patients with both a thymoma and MG contained CA antibodies in high titres (Table 3). The serum concentration of AChR antibodies was also markedly elevated in all 9 sera. Eight of the sera contained antibodies to the skeletal muscle cell surface, and 7 sera contained antibodies to the cross-striational bands.
Discussion
The results of this study show that the presence of a thymic tumour in non-MG patients is not strongly associated with circulating CA antibodies, since only 3 of our 9 patients without MG had such antibodies. In contrast, nearly all patients with both MG and a thymic tumour have CA antibodies (Aarli et al. 1981; Gilhus et al. 1984), and all 9 patients with MG and such a tumour included in the present study had high-titred CA antibodies. Patients with a thymic tumour but without MG can also have other types of skeletal muscle antibodies. Cross-striational antibodies were detected in 3 of our 9 non-MG patients and have previously been reported in about 25% of such patients (McFarlin et al. 1966; Oosterhuis et al. 1976; Limburg et al. 1983). Sarcolemmal antibodies were found in serum from 2 of our patients, who also had CA antibodies, and have previously been reported in one patient with a thymoma but without MG (Beutner et al. 1966). Sarcolemmal antibodies are ~ssociated with the CA antibodies (Gilhus et al. 1983). Nearly all our control patients with both thymoma and MG had sarcolemmal and cross-striational muscle antibodies, in accordance with previous studies of MG patients with thymoma (Oosterhuis et al. 1976; Gilhus et al. 1983). Skeletal muscle antibodies usually do not occur in patients with secondary malignant involvement of the thymus (Oosterhuis et al. 1976). Also, AChR antibodies can be found in patients with a thymic tumour but without any muscular weakness. Such antibodies were found in 5 of our patients without MG. In 3 of them, the concentrations were only slightly above the normal level, whereas 2 patients had a markedly increased AChR antibody level. Cu6noud et al. (1980) reported AChR antibodies in 3 of 11 non-MG patients with a thymoma, and Limburg et al. (1983) found AChR antibodies in 2 of 11 similar patients. In 2 of these 5 non-MG thymoma patients with AChR antibodies, the AChR antibody concentration was very high. The 9 thymoma patients with MG in the present study had markedly elevated AChR antibody concentrations, as have such patients in general (Lefvert et al. 1978; Limburg et al. 1983). Our non-MG patients with skeletal muscle antibodies had thyrnic tumours of various histological types. The patients with a lymphoepithelioma had less antibodies than those with other thymic tumours; none of the 3 patients with a lymphoe-
77 pithelioma had CA antibodies, whereas 3 of the 5 patients with other tumours had such antibodies in high titres. Nearly all patients with M G together with a thymic tumour have a lymphoepithelioma (Henry 1981; Lisak and Barchi 1982). The present study shows that skeletal muscle antibody production can also occur together with other thymic tumours such as lymphomas, sarcomas and hamartomas. M G has only rarely been reported together with such tumours (Null et al. 1977; Yuasa et al. 1980; Otto et al. 1982; Hansen et al. 1983). Thus, our results, combined with previous knowledge, indicate that although patients with thymic tumours other than lymphoepithelioma may have circulating muscle antibodies, they usually do not develop symptoms of MG. A reason why a high concentration of A C h R antibodies does not necessarily lead to M G could be that the antibodies do not bind to the appropriate region of the A C h R molecule. Another possibility is that a circulating factor in addition to the A C h R antibodies is required to induce the muscular weakness in MG. Our results indicate that such a possible additional factor often occurs together with a thymic lymphoepithelioma, since most patients with this tumour have MG, whereas patients with a lymphoepithelioma but without M G usually lack A C h R antibodies. This possible additional factor rarely operates in the presence of other thymic tumours, since most patients with such tumours do not have M G although they may have A C h R antibodies. The postulated factor for the development of M G may be a hormone secreted by thymic epithelial cells (Dalakas et al. 1981; Olanow and Roses
1981). The relationship between a thymic tumour, skeletal muscle antibodies and M G is far from understood. It is generally accepted that thymus pathology as well as A C h R antibodies participate in the pathogenesis of MG. However, onset of M G years after the removal of a thymoma and adjacent thymus has been reported (Janzen et al. 1977; N a m b a et al. 1978), and M G can also be seen without detectable AChR antibodies (Oosterhuis et al. 1983). Conversely, the present study shows that a thymic tumour as well as A C h R antibodies can be present without MG. A C h R antibodies have been detected in sera from elderly patients with cerebrovascular disease and from patients with Down's syndrome, without any symptoms of M G (Tanaka and Miyatake 1983). Additional factors most probably participate in the development of the muscular weakness in MG.
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