- Email: [email protected]
Attempted isolation of viruses from myasthenia gravis thymus
287
Journal of Neuroimmunology, 11 (1986) 287-299 Elsevier
JNI 00363
Attempted Isolation of Viruses from Myasthenia Gravis Thymus L.S. Klavinskis
I,*, H.N.A. Willcox I,**, J.E. Richmond and J. Newsom-Davis 1,3
2
’ Depariment of Neurological Science, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, ’ Central Public Health Laboratory, Colindale Avenue, London N W9 5HT, and 3 National Hospitalfor Nervous Diseases, London WC1 (0: K.) (Received 15 October, 1984) (Revised, received 7 October, 1985) (Accepted 12 October, 1985)
Summary
A systematic study of thymus homogenates and cell suspensions from 13 patients with myasthenia gravis (MG) of recent onset, and 6 non-myasthenic controls, has failed to detect or isolate virus by cell culture with ‘rescue techniques’, electron microscopy, or intracerebral inoculation into neonatal mice. These results do not support the case for persistent viral infection in the thymus, and impose constraints on hypotheses of a viral aetiology of MG. Key words: Myasthenia
gravis
-
Persistent
virus -
Thymus
-
Viruses and auto-
immunity
Introduction
Myasthenia gravis (MG) is a disorder of neuromuscular transmission mediated by autoantibody to the acetylcholine receptor (AChR) at the motor endplate. Cellular
* Present address: Department of Immunology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037 (U.S.A.). ** To whom reprint requests should be addressed. Abbreviations: CMV, cytomegalovirus; CPE, cytopathic effect(s); EM, electron microscopy; FCS, foetal calf serum; HAI, haemagglutination inhibition; HSV, herpes simplex virus; i.c., intracerebral; IEM, immunoelectron microscopy; IF, immunofluorescence; p.i., post-inoculation; RSV, respiratory syncytial virus; s.c., subcutaneous; VZ, varicella zoster.
0165-5728/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
288 and humoral immune mechanisms have been extensively studied in myasthenics, but there are few clinical or epidemiological clues as to what initiates the autoimmune process: in rare cases, however, usually with HLA-DR-1 (Garlepp et al. 1983). penicillamine therapy may be causative. Where the autoimmune process begins in MG is uncertain, but circumstantial evidence suggests that it may not be in muscle. Inflammatory changes in muscle are infrequent and slight; lymphorrhages have occasionally been recognised in some MG patients (usually those with thymoma), but they are not preferentially sited at the motor endplate (Oosterhuis et al. 1968). By contrast, thymic changes are often striking and it has been suggested that it is within the thymus that loss of self-tolerance to the AChR is initiated (Wekerle and Ketelsen 1977). For example. about 10% of MG cases have thymic turnours, and these express antigens shared with skeletal muscle; almost all these cases have antibodies to striational and other muscle antigens in addition to those directed against the AChR (e.g. Gilhus et al. 1984). Thus it appears likely that, in this subgroup, the autosensitising agent may be the thymic tumour itself. Furthermore, hyperplasia of the thymus with medullary germinal centres is frequently found in the major subgroup of myasthenics those in whom the disease starts before the age of 40, and who show a 3 : 1 female bias, strong HLA-B8 and -DR3 associations, and an increased risk of several other autoimmune diseases (e.g. Compston et al. 1980). The myasthenia often improves after thymectomy, perhaps implying some central role for the thymus in the pathogenesis of the MG in this subgroup. Moreover, thymocytes from many of these cases spontaneously synthesise anti-AChR antibody in culture which may have a specific activity lo-100 times greater than in serum (Scadding et al. 1981) and appears to be mainly derived from the germinal centres (Willcox et al. 1983). In addition, these thymocytes can stimulate anti-AChR production by autologous blood lymphocytes, perhaps because they include specific antigen-presenting cells (Newsom-Davis et al. 1981). The connecting link between these patients and the thymoma subgroup may be the presence of muscle autoantigens - including AChR - in the thymus. Rare ’ myoid’ or muscle-like cells have been recognised in both the normal and myasthenic medulla, which express AChR in culture (Kao and Drachman 1977) and other cell types there may do so too. The thymic medulla is rich in antigen-presenting cells (Janossy et al. 1980) and, if these took up self-antigens such as AChR, they might present them in such a highly immunogenic form as to autosensitise newly developing T cells instead of rendering them tolerant. ultimately leading to germinal centre formation. It is conceivable that such a chain reaction might be initiated by extrinsic agents including viruses, perhaps through damage to AChR-bearing cells, or activation of antigen-presenting cells (Bottazzo et al. 1983). Viruses have often been suspected as initiating factors in autoimmune diseases. Autoantibodies to host antigens, e.g. DNA (Catalan0 et al. 1980). erythrocytes and platelets (Datta and Schwartz 1979) myelin (Panitch et al. 1980) smooth muscle, lymphocytes and immunoglobulins (Daugharty et al. 1979) have been observed after viral infections. In addition, type I diabetes in mice has been experimentally induced with Coxsackie B4, meningoencephalomyocarditis and reoviruses (Yoon et al. 1978;
289 Craighead and McLane 1968; Onodera et al. 1978) and C-type retroviruses have been implicated in the systemic lupus erythematosus of NZB X NZW mice (Datta and Schwartz 1976). It is therefore not surprising, in view of the thymic abnormalities and strong HLA associations in young female myasthenics, that persistent viral infection of the thymus has been proposed as an initiating event in MG (Datta and Schwartz 1974). There is no clear evidence for this as yet, however, and viral antibody studies have given conflicting results. Antibodies against cytomegalovirus (CMV) were found to be increased in myasthenics in one study (Tindall et al. 1978) though we have been unable to confirm this in a large survey with carefully matched controls, and have found no increase in incidence or titre of antibody to 8 other common viruses (Klavinskis et al. 1985). Alternative approaches are clearly necessary to detect possible extrinsic provoking agents in MG. We report here the results of a systematic study to detect and/or isolate virus from the thymuses of 13 patients with MG of recent onset and of 6 non-myasthenic controls.
Materials and Methods Patients Thymus tissue was analysed from 12 MG patients, selected because of a short time between onset (or relapse in case 13) and thymectomy (I 2 years). Case 1 was
TABLE
1
THE MYASTHENIA Cases 1-12 thymus.
showed
Case No.
Sex
1 2 3 4 5 6 I 8 9 10 11 12 13
F F F F F F F F F F F F F
CRAWS thymic
PATIENTS
hyperplasia
STUDIED
with medullary
germinal
centres,
and case 13 had an atrophic
Clinical grade ’
Plasma anti-AChR
(years)
Duration of symptoms before thymectomy (months)
22 20 15 21 24 37 24 22 22 31 24 18 4
60 19 6 14 7 24 11 10 17 11 10 23 120 (7) b
IIB IIB IIB IIA IIA IIB III IIA IIA IIB IIA IIB IIA
1190 621 316 258 96 56 54 50 38 31.2 32.8 18.2 3.0
Age at onset
a Clinical grading after Osserman and Genkins (1971): IIA = mild generalised; alised; III = acute, severe disease. b Remission from the age of 9 until 7 months before thymectomy.
IIB = moderate
gener-
290 TABLE
2
ANTIVIRAL
ANTIBODY
TITRES
Complement
fixing antibody
titres
Case No. 1 2 3h 4 5 6 7 x 9 10 I1 12 13
Influenza
Influenza
type A
type B
8 1: <8 32 <8 16 8 16 8 32 8 8
32 <8 8 8 8 (8 16 8 8 8 8 <8 ~8
OF THE MG PATIENTS
Adenovirus
RSV
STUDIED
Measles
Mumps
Rubella
a
(V) <8 18 8 32 8 32 16 8 8 <8
8 <8 16 16 8 16 16 8 8 8 18 8 <8
16 16 16 8 128 8 18 64 128 8 12x 8 16
16 18 8 16 16 8 8 8 16 8 16 8 64
240 156 2 849 176 687 176 394 446 505 289 166 348 200
” Rubella antibodies assayed by single radial haemolysis (IU/ml). h Case 3 presented with Still’s disease 6 years prior to onset of MG at which time levels of anti-rubella IgG (but not IgM) were also high, and anti-AChR antibody was undetectable. ’ Anti-reovirus antibodies assayed by HAI.
also selected because of very high thymocyte anti-AChR antibody production in culture and plasma titre. Clinical details are summarised in Table 1, where the cases are ranked according to their plasma anti-AChR titres: their antibody titres to 14 common infectious agents are given in Table 2. Control thymus was obtained from 6 non-myasthenic adults undergoing thoracic surgery. Antibody titres (Table 2) Sera obtained at the time of thymectomy were assayed by complement fixation (Bradstreet and Taylor 1962) for antibody to 9 viruses prevalent in the community, as well as to Mycoplasma hominis, psittacosis, and Coxiellu burnetii. In addition rubella IgG antibodies were assayed by radial haemolysis (Clarke et al. 1977) with reference to the British Standard antiserum for anti-rubella 69/60, and expressed in terms of international units of rubella potency/ml. Rubella IgM antibody was assayed using the Rubenz ‘M’ commercial kit (Northumbria Biologicals). Antireovirus type 3 HA1 antibodies were kindly assayed by Dr. E.H. Boxall (East Birmingham Hospital). Anti-AChR antibody was assayed by radioimmunoassay using human AChR as antigen (Newsom-Davis et al. 1978). Isolution procedures (a) General strategy We attempted to isolate virus by direct inoculation of thymus range of cell lines, into embryonated hens’ eggs and by intracerebral
extracts onto a inoculation of
291
HSV
CMV
vz
Reovirus
’
Mycoplasma
Psittacosis
Coxiella burn&i
8 18 <8 8 8 <8 ~8 <8 16 ~8 8 8 32
32 ~8 16 8 8 (8 ~8 16 <8 8 (8 8 ~8
<8 <8 <8 ~8 8 ~8 18 ~8 18 ~8 ~8 8 8
type 3 <8 <8 ~8 32 8 18 16 <8 18 8 <8 (8 (8
TABLE
8 18 8 32 64 18 (8 8 16 <8 8 ~8 64
32 8 64 16 16 ~8 8 32 32 8 32 8 32
64 256 32 64 32 64 256 32 64 32 32 128 32
3
THE CELL LINES AND CONDITIONS Cell line
MRC-5 HEP-2 (Cincinnati line) HEK
RK-13 Vero
PBK
USED
Cell type
Culture
conditions
Growth
medium a,’
Human fibroblasts Human laryngeal carcinoma (epithelial) Primary human foetal kidney (epithelial) Rabbit kidney (epithelial) African green monkey kidney (epithelial) Primary baboon kidney (epithelial)
MEM + 5% FCS + 0.04% NaHCO,
Maintenance
medium’
MEM + 2% FCS + 0.04% NaHCO,
MEM + 5% FCS + 0.013% NaHCO,
MEM + 2% FCS + 0.013% NaHCO,
MEM + 10% FCS
MEM + 2% FCS
MEM + 5% FCS + 0.013% NaHCO,
MEM + 2% RS + 0.013% NaHCO,
MEM + 4% FCS + 0.013% NaHCO, + 0.1% glucose
MEM + 2% FCS + 0.013% NaHCO, + 0.1% glucose
Medium 199 + 10% FCS + 0.013% NaHCO,
Medium NaHCO,
199 + 2% FCS + 0.013%
a MEM (minimal essential medium)+ Earle’s salts + 15 mM Hepes (Gibco, U.K.)+100 U/ml penicillin+ 100 ug/ml streptomycin. h Medium 199 + Earle’s modified salts+ 1.25 g/l NaHCO, (Gibco, U.K.)+ 100 U/ml penicillin + 100 ,ug/ml streptomycin. ’ RS = rabbit serum (Wellcome Laboratories).
292 neonatal mice; cell suspensions
also by co-cultivation of untreated with tissue culture monolayers.
and mitogen-stimulated
thymic
(6) Preparation of tissue homogenates and cell suspensions Homogenates were prepared (from about 20% of the total thymus) in an MSE blender and diluted to 10% w/v of original tissue with Hanks’ lactalbumin hydrolysate (Flow Laboratories) containing antibiotics, 0.35 g/l NaHCO,, 2% bovine serum albumin, and 15% sorbitol, before storage at - 70°C. Cell suspensions (from a further 20% of thymus) were prepared with proteolytic enzymes and stored in liquid nitrogen until used (Willcox et al. 1983). (c) Direct inoculation of cell cultures with thymus homogenates The cell lines and culture conditions used are summarised in Table 3. Confluent cell monolayers in 2 ml flat-bottomed (Linbro) wells were washed twice, and inoculated in triplicate with 0.1 ml of neat and lop2 dilutions of homogenates. After 2 h at 37°C (or 34°C for RK-13 cells), monolayers were washed and re-fed. Cultures were observed for CPE and passaged 3 times at lo-14-day intervals by trypsinization and adsorption onto fresh monolayers for 2 h (as above), prior to washing and re-feeding. PBK cells were cultured in tubes on roller apparatus at 33°C. In addition, concentrates of thymus homogenates from cases 3, 4 and 9, and of brain homogenate from a mouse inoculated i.c. with patient 4 thymus homogenate, were inoculated onto PBK cells and passed at least twice in the Virus Reference Laboratory, Cohndale. (d) Co-cultivation of thymus cell suspensions Subconfluent cultures of all the cell lines were washed, and seeded in triplicate with 1-3 X lo6 thymic cells in 2 ml wells. To some monolayers, cell suspensions pre-incubated for 4 days with pokeweed mitogen (Gibco, Grand Island, NY). at 10 ~1 per ml in RPMI/lS% FCS were added. All cultures were re-fed at day 7 and subcultured (2-3 X ) at lo-14 days. (e) Virus identification Four methods were employed in attempts to detect the presence of virus in cell cultures: (1) haemadsorption: inoculated monolayers (of all the cell lines in Table 3) were overlayed with human ‘O’, rhesus monkey (Flow Labs., U.S.A.) and fowl erythrocytes prior to subculture or passage; (2) immunofluorescence (IF): inoculated or co-cultured cell lines (RK-13, MRC-5 and Vero) were tested for viral antigens after the first and third subculture (see below) and PBK and HEK cells after the first subculture only; (3) interference: 7 days after the final passage, inoculated monolayers of every cell line were challenged with ECHO virus type 11, for which they were assayed by haemadsorption; (4) electron microscopy (EM): HEK monolayers inoculated with MG or control homogenates were fixed, processed and thin-sectioned for EM by standard procedures (Patterson and Bingham 1976); sonicates of cultured HEK cells were also pelleted, negatively stained and examined by EM.
293 (f) Egg inoculation Embryonated hens’ eggs were inoculated (in triplicate) chorioallantoic routes with 0.1 ml neat or 10e2 diluted thymocytes/O.l ml. Membranes were examined for lesions, for haemagglutinin with human ‘O’, rhesus monkey or fowl and 37°C (Grist et al. 1979b). Non-haemagglutinating fluids re-tested.
by the amniotic and homogenates, or lo6 and fluids were tested erythrocytes at +4”C were re-passaged and
(g) Suckling mouse inoculation Homogenate (0.05 ml of 10%) was injected i.c. and S.C. into newborn BKTO mice < 24 h old (Grist et al. 1979a). Daily observations of these ‘first passage’ mice were made for 25 days p.i. Deaths occurring within 48 h were considered to be due to inoculation trauma, and disregarded. Mice showing signs of disease were killed, the brains removed and 10% suspensions of these inoculated onto HEK, HEP-2, MRC-5 and Vero cells, and i.c. into ‘second passage’ mice.
(h) Immunoji’uorescence (IF) To detect viral antigens that might be present in cell cultures or thymus sections, we used indirect IF with polyclonal antisera to HSV types 1 and 2 (kindly provided by Dr. A. Minson), VZ (kindly provided by Prof. R. Heath), adenovirus, measles, rubella (Wellcome Laboratories), influenza A and B viruses, and pooled monoclonal antibodies (ascitic fluid) to CMV (kindly provided by Dr. L. Pereira), as well as autologous plasma taken at the time of thymectomy. Uninoculated cells and cells inoculated with the appropriate virus served as negative and positive controls. In addition, cryostat sections (6 pm) were prepared from thymus blocks and stained as described by Janossy et al. (1980).
(i) Electron microscopy of thymus homogenates Thymus homogenates from the MG patients were clarified at 1500 X g for 10 min and then the supernatants were centrifuged at 48000 X g for 2 h. The resulting pellets were negatively stained with 3% phosphotungstic acid pH 6.3 and examined in a JEOL 100 CX electron microscope. Brain extracts from mice inoculated i.c. with Coxsackie B3, or reovirus, and brain extracts from ‘first’ and ‘second passage’ mice showing signs of disease after inoculation with patient 4 thymus homogenate, were similarly prepared and examined.
0) Immunoelectron microscopy (IEM) Thymus homogenates from 8 MG patients (including patient 4) and brain extracts from mice inoculated with Coxsackie B3, or with patient 4 thymus homogenate, were examined by IEM (Richmond et al. 1984), using a hyperimmune Coxsackie B3 antiserum, or autologous serum obtained at the time of thymectomy.
294 Results (a) Positive controls Prior to our isolation experiments with MG thymus, we tested each of the cell lines for their sensitivity to a wide spectrum of viruses, and were able to detect HSV types 1 and 2, CMV (AD/169), VZ, measles, rubella, adenovirus type 10, Coxsackie A3, B3 and B5 viruses, ECHO 11, parainfluenza 3, reovirus types 1, 2 and 3, and influenza A and B. The last two viruses were also consistently harvested from amniotic fluids, and herpes lesions were readily detected on chorioallantoic membranes. Results of infection of mice by i.c. inoculation with Coxsackie B3 virus were as follows: 5 x lo4 TCID,, - complete paralysis then death of 4 of 4 mice by day 3; 5 X lo3 TCID,, - paralysis in 6 of 8 mice by day 3; and 5 X lo2 TCID,, ~ death or severe paralysis in all mice by day 6. Picornavirus particles were observed by EM and IEM in the brain extracts of 1 of 3 mice inoculated with Coxsackie B3. Brain histopathology in the sick mice showed mild acute inflammatory changes consistent with an infectious process. There was perivascular cuffing, a mononuclear infiltrate and phagocytic tissue breakdown, plus meningeal inflammation. Reovirus particles were detected by EM in brain extracts of 1 of 2 mice after i.c. inoculation with reovirus. Using the appropriate antisera, nuclear or cytoplasmic IF (depending on the virus) was observed in virus-infected but not control cells. In interference tests, cells preincubated with 10 TCID,, of HSV were resistant to subsequent challenge with ECHO 11 virus. Finally, in haemadsorption and haemagglutination tests, measles, ECHO 11 and influenza-A viruses gave positive results. (6) Inoculation of cultures with MC and control thymus homogenates, and co-cultivation with thymic cell suspensions Each thymic homogenate was inoculated onto all of the cell lines listed in Table 3. CPE attributable to viral agents were not detected in any culture from MC or control thymus, even in PBK and HEK cells. Transient toxic effects of some MG and control homogenates on the HEP-2 cells were occasionally seen but disappeared 24 h after the medium was changed. IF was not detected in any of the cells with any of the antibodies used: nor could we detect any viruses by EM in the inoculated HEK cells. Even when we used proteolytically prepared thymocyte suspensions (from all patients and controls), containing various stromal and macrophage-related cell types in addition to lymphocytes, we were still unable to detect any sign of virus from using PWM-stimulated co-cultures (even with PBK cells). ‘Rescue’ techniques, thymocytes co-cultivated with several cell lines, also failed to yield infectious virus or viral antigens detectable by IF, haemadsorption, or interference. Cell monolayers inoculated with thymus extracts from patients 2, 4, 8 and 13 showed weak nuclear IF when stained with the patients’ own plasma; as the same was seen in uninoculated cells, antinuclear antibodies were probably responsible. No lesions or haemagglutinating virus were detected in inoculated eggs.
295
(c) Inoculation of neonatal mice with thymus homogenates No signs of neurotropic infections, paralysis or growth retardation
were observed in neonatal mice by 25 days p.i. with 12 of 13 MG or 6 of 6 control thymus extracts, and there were only sporadic deaths. Some of the mice injected with homogenate from patient 4, however, developed oily coats, generalised weakness and growth retardation. In total, 7 of 14 mice died at days 2-12 in the first litter; the only gross abnormality found at post-mortem was cerebral atrophy with much intracranial fluid (approximately 0.2-0.3 ml). The remaining mice were killed at day 25, 2 being stunted (but otherwise healthy) and 5 of normal stature. In 1 of 3 further litters, 7 of the 12 mice were sick by day 9, but only 2 of the 14 mice in the other 2 litters became ill up to day 18. We failed to isolate virus from brain extracts from the sick, disease-free and control mice (using HEK, HEP-2, MRC-5 and Vero cells); viral antigens could not be detected by IF in any of the cell lines inoculated, and virus particles were found neither by EM nor IEM using serum from patient 4. Three of 10 ‘second passage’ mice inoculated with extract of brains from sick mice developed weakness, growth retardation and died at days 3, 20 and 26 p.i. One of 11 recipients of extract from apparently disease-free ‘first passage’ mice also died and the post-mortem findings in all 4 were as above. Brain sections from the sick mice were reviewed by an experienced neuropathologist. In the extreme examples, the appearances were of degeneration with focal collections of activated microglial cells, sparse mononuclear meningeal infiltrate, and no obvious perivascular cuffing or ependymitis. These changes were consistent with a delayed degenerative reaction to some toxic, traumatic or ischaemic effect of the inoculum, and there was no obvious sign of an infection or inflammatory process.
(d) EM and IEM of thymus homogenates and IF staining of thymus sections Virus particles could not be detected by EM in high-speed pellets of thymus homogenates from any of the patients, nor from the extracts of 8 MG thymuses (including that from case 4) examined by IEM. We were also unable to detect viral antigens in cryostat sections of any MG thymus by IF with any of the antisera used.
Discussion Despite rigorous efforts, this study has failed to detect any signs of persistent virus or viral antigens in the patients with MG, nor, indeed, have we found increased antiviral antibodies in MG sera (Klavinskis et al. 1985). We cannot, however, exclude a viral aetiology for a number of reasons. Our methods might not have been sufficiently sensitive to detect latent or small amounts of virus. Alternatively, host antibody to a virus could interfere with its detection, though this seems unlikely since we used washed cell suspensions as well as thymic extracts for the isolation work, and we inoculated the cell lines with high concentrations of extracts to maximise the sensitivity. Viral genes can be identified at much greater sensitivity by hybridisation to suitable probes, but these methods are only appropriate in seeking well-studied and highly specific gene sequences. It is obviously premature to apply
296
these techniques in the present studies, in the absence of any clues as to which virus to focus upon. Our negative observations might reflect the fastidious nature or defective expression of certain viruses in culture, which might therefore escape detection. To minimise these difficulties, we used a wide range of cell lines and attempted to ‘rescue’ viruses from minority cell types by preparing the cell suspensions with collagenase and dispase, and/or stimulating with pokeweed mitogen. In case defecwith the tive or non-cytocidal viruses were present, we tested for interference replication of a challenging picornavirus, a method that can detect rubella virus with great sensitivity (Parkman et al. 1964). In addition, concentrates of thymus homogenates were examined by EM and IEM and extracts were inoculated i.c. into neonatal mice. One might have expected the patients’ own sera to detect early antigens, even of fastidious viruses, in cell culture. There was intracranial pathology in some of the mice inoculated with patient 4 thymic homogenate, and in a few of the ‘second passage’ recipients. The pathological findings could have been late effects of viral ependymitis, which can lead to blockage of CSF flow and hydrocephalus. and can be caused by, for example, adeno-, myxo-, polyoma or type 1 reoviruses (summarised by Tardieu and Weiner 1982). Of these, perhaps the most likely to have been overlooked is the reovirus. Repeated isolation attempts, however, failed to show any CPE in any cultures (including PBK cells). Furthermore, this patient’s antibody titre to reovirus type 3 was similar to the titres of the other 12 patients and 6 control siblings. Although we cannot completely rule out a viral cause of the somewhat non-specific findings in some of the inoculated mice, it is equally possible that some toxic factor in the homogenate may have interfered with CSF resorption or circulation. Other agents, e.g. mycoplasma, chlamydiae or even a ‘novel’ (MG) virus analogous to SMON in multiple sclerosis (Melnick et al. 1982) or RA-1 virus in rheumatoid arthritis (Simpson et al. 1984), might conceivably be involved, for which our culture conditions or cell lines may have been unsuitable. Epidemiological without any studies argue against that, however, since MG occurs worldwide obvious regional differences or temporal fluctuations in incidence which might suggest an infectious aetiology (Kurtzke 1978). A final difficulty might be that virus is only present at an early stage in the disease (perhaps months or years before the onset of symptoms). or in some extra-thymic site. For both this and the sero-epidemiological study (Klavinskis et al. 1985) we therefore selected cases with the shortest duration of symptoms. A delay of several months between onset and thymectomy is almost inevitable: the incubation period prior to the onset of symptoms might well be even greater. The case for concentrating on the thymus, however, is strong and there are no comparable indications for testing other tissues. A similar isolation study by Aoki et al. (1985) has also failed to show any sign of viruses in MG thymus. Taken together. these negative sero-epidemiological and isolation studies considerably weaken the case for a viral aetiology in MG, especially in the subgroup of patients with thymic hyperplasia, the subjects of the current study. Each of our cases showed serological evidence of previous infection with
297 several common viruses (almost none to exaggerated titres) although no such virus was isolated. These studies therefore do not support the hypothesis of a viral aetiology in MG. Perhaps autoreactive T helper cells may be recruited by a transient viral infection, and then proliferate and induce autoantibody production long after the virus has been eliminated. Such a ‘hit-and-run’ effect could conceivably result from a crossreaction between virus and self (Allison 1977; Weigle 1977), from cross-reactions between AChR and viral anti-idiotypes (Cooke et al. 1983; Plotz 1983) or from a virus associating with self-antigen to provide a ‘helper determinant’ (Bromberg et al. 1982). The aetiology of autoimmune diseases has long been a challenging problem. Viruses are inevitably hard to exclude, though the evidence in their favour is scarcely stronger now than 20 years ago and, even in the best-documented examples, is not conclusive. These include type I diabetes mellitus, in which Coxsackie B viruses are implicated and there are seasonal peaks of incidence (e.g. Gamble 1980). It is not yet clear whether the Coxsackie viruses are merely precipitating factors, or play some more fundamental role. Even in the NZB X NZW mouse model of lupus erythematosus, in which retroviruses have been implicated, it now appears that they are not essential for the characteristic autoimmune responses (e.g. Datta and Schwartz 1979). An important alternative to viral-induced autoimmunity is that some chance unprovoked aberration in immunological homeostasis initiates the response. Many possibilities exist, e.g. inappropriate expression of Ia antigens on myoid cells, as may happen with thyroid epithelial cells (Bottazzo et al. 1983) over-officious antigen-presenting cells (Dausset and Contu 1980) loss of suppressor T cells, interference in tolerance induction of T helper cells, or imbalances in the idiotypic network. The association of several quite diverse immunological diseases with the same HLA B8-DR3 haplotype may well prove to reflect some general property of Ia antigens or the cells bearing them.
Acknowledgements
We are grateful to Prof. R. Heath, Drs. A.C. Minson, L. Pereira and M.S. Pereira for generous gifts of polyclonal and monoclonal antibodies and viruses, to Dr. P. Fryer and Mrs. J. Gross for invaluable help in screening material by thin-section EM, to Dr. E.H. Boxall for the anti-reovirus assays, to Messrs. B. Megson and P. Cunningham of the Virus Reference Laboratory, Colindale, and to Mr. M. Sturridge, FRCS, and Dr. L. Loh, FFA, RCS, for help in obtaining thymic tissue. We are also grateful to Drs. J.S. Oxford and P. Griffiths and Mr. M. Ross for help and advice, and to Dr. J. McCloughlin for his neuropathological expertise. This work was supported by the Muscular Dystrophy Group of Great Britain and the Sir Jules Thorn Charitable Trust.
29X References Allison. A.C., Autoimmune diseases: concepts of pathogenesis and control. In: N. Talal (Ed.). Autoimmunity: Genetic, Immunologic, Virologic and Clinical Aspects. Academic Press, New York, 1977. pp. 91-139. Aoki, T.. D.B. Drachman. D.M. Asher, C.J. Gibbs. S. Bahmanyar and J.S. Wolinsky. Attempts to implicate viruses in myasthenia gravis, Neurology, 35 (1985) 185-192. Bottazzo, G.F., R. Pujol-Borrell, T. Hanafusa and M. Feldmann, Role of aberrant HLA-DR expression and antigen presentation in induction of endocrine autoimmunity, Lancet, ii (1983) 1115-1119 Bradstreet. C.M. and C.E. Taylor, Technique of complement fixation test applicable to the diagnosis of virus diseases. Monthly Bull. Minist. Hlth. (London). 21 (1962) 966104. Bromberg, J.S., P. Lake and M. Brunswick, Viral antigens act as helper determinants for antibody responses to cell surface antigens, J. Immunol., 129 (1982) 683-688. Catalano, M.A., D.A. Carson, J.C. Niederman, P. Feorino and J.H. Vaughan, Antibody to the rheumatoid arthritis nuclear antigen. Its relationship to in viva Epstein-Barr virus infection. J. Clin. Invest.. 65 (1980) 1238-1242. Clarke, M., J. Bonstred. V. Seagroatt and G.C. Schild, The use of single radial haemolysis for rubella antibody studies. J. Hyg., 79 (1977) 355-364. Compston, D.A.S., A. Vincent, J. Newsom-Davis and J.R. Batchelor. Clinical, pathological, HLA antigen and immunological evidence for disease heterogeneity in myasthenia gravis, Brain. 103 (1980) 5799601. Cooke. A., P.M. Lydyard and 1.M. Roitt, Mechanisms of autoimmunity: a role of cross reactive idiotypes, Immunol. Today, 4 (1983) 170-174. Craighead, J.E. and N.F. McLane. Diabetes mellitus: induction in mice by encephalomyocarditis virus. Science, 162 (1968) 913-916. Datta, S.K. and R.S. Schwartz, Infectious myasthenia? N. Engl. J. Med., 291 (1974) 130441305. Datta. S.K. and R.S. Schwartz, Genetics of expression of xenotropic virus and autoimmunity in NZB mice. Nature (London), 263 (1976) 412-414. Datta, SK. and R.S. Schwartz, Genetic basis of autoimmune disease: the New Zealand mouse model. In: N.R. Proffitt (Ed.), Virus Lymphocytic Interactions: Implications for Disease. Elsevier-North Holland, New York, 1979. pp. 71-82. Da&arty, H., K. Kelly, C. Moore and T. Hersh, Autoimmune implications of immune complexes in clinical variants of hepatitis B, Clin. Exp. Immunol., 37 (1979) 2133220. Dausset. J. and L. Contu. The MHC and immune response in man. In: M. Fougereau and J. Dausset (Eds.), Progress in Immunology, Vol. IV, Academic Press, London, 1980, pp. 513-529. Gamble, D.R., The epidemiology of insulin dependent diabetes, with particular reference to the relationship of virus infection to its aetiology, Am. J. Epidemiol., 2 (1980) 49-70. Garlepp. M.I., R.L. Dawkins and F.T. Christiansen, HLA antigens and acetylcholine receptor anttbodies in penicillamine induced myasthenia gravis, Br. Med. J.. 286 (1983) 338-340. Gilhus, N.E.. J.A. Aarli, B. Christensson and R. Matre, Rabbit antiserum to a citric acid extract of human skeletal muscle staining thymomas from myasthenia gravis patients. J. Neuroimmunol., 7 (1984) 55-64. Grist, N.R.. E.J. Bell, E.A.C. Follet and G.E.D. Urquhart, Chapter 5, Animal and egg techniques. In: Diagnostic Methods in Clinical Virology, Blackwell Scientific Publications, Oxford, lY79a. pp. 44459. Grist, N.R., E.J. Bell, E.A.C. Follet and G.E.D. Urquhart, Chapter 9, Haemagglutination and haemagglutination inhibition tests. In: Diagnostic Methods in Clinical Virology, Blackwell Scientific Publications. Oxford, 1979b, pp. 116-128. Janossy, G.. J.A. Thomas, F.J. Bollum, S. Granger, G. Pizzolo, K.F. Bradstock, L. Wang, A. McMichael. K. Ganeshaguru and A.V. Hoffbrand, The human thymic environment: an immunologic study, J. Immunol., 125 (1980) 2022212. Kao, I. and D.B. Drachman, Thymic muscle cells bear acetylcholine receptors: possible relationship to myasthenia gravis, Science, 195 (1977) 74-75. Klavinskis, L.S., H.N.A. Willcox, J.S. Oxford and J. Newsom-Davis. Antivirus antibodies in myasthenia gravis. Neurology. 35 (1985) 13X1-1384.
299 Kurtzke, J.F., Epidemiology of myasthenia gravis, Adv. Nemo]., 19 (1978) 545-566. Melnick, J.L., E. Seidel, Y.K. Inoue and Y. Nishibe, Isolation of virus from the spinal fluid of three patients with multiple sclerosis and one with amyotrophic lateral sclerosis, Lancet, i (1982) 830-833. Newsom-Davis, J., A.J. Pinching, A. Vincent and S.G. Wilson, Function of circulating antibody to acetylcholine receptor in myasthenia gravis investigated by plasma exchange, Neurology (Minneap.), 28 (1978) 266-272. Newsom-Davis, J., H.N.A. Willcox and L. Calder, Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine receptor antibody by autologous blood lymphocytes, N. Engl. J. Med., 305 (1981) 1313-1318. Onodera, T., A.B. Janson, J.W. Yoon and A.L. Notkins, Virus induced diabetes mellitus: reovirus infection of pancreatic B cells in mice, Science, 201 (1978) 529-531. Oosterhuis, H.J.G.H., J. Bethlem and T.E.W. Feltkamp, Muscle pathology, thymoma and immunological abnormalities in patients with myasthenia gravis, J. Neurol. Neurosurg. Psychiatry, 31 (1968) 460-463. Osserman, K.E. and G. Genkins, Studies in myasthenia gravis: review of a twenty-year experience in over 1200 patients, Mt. Sinai J. Med., 38 (1971) 497-537. Panitch, H.S., C.J. Hooper and K.P. Johnson, CSF antibody to myelin basic protein; measurement in patients with multiple sclerosis and SSPE, Arch. Neurol., 37 (1980) 206-209. Parkman, P.D., E.L. Buescher, M.S. Artenstein, J.M. McCown, F.K. Mundon and A.D. Druzd, Studies of rubella, I. Properties of the virus, J. Immunol., 93 (1964) 595-607. Patterson, S. and R.W. Bingham, Electron microscope observations on the entry of avian infectious bronchitis virus into susceptible cells, Arch. Virol., 52 (1976) 191-200. Plotz, P.H., Autoantibodies are anti-idiotype antibodies to antiviral antibodies, Lancet, ii (1983) 824-826. Richmond, J.E., J.V. Parry and S.D. Gardner, Characterisation of a polyomavirus in two foetal rhesus monkey kidney cell lines used for the growth of hepatitis A virus, Arch. Viral., 80 (1984) 131-146. Scadding, G.K., A. Vincent, J. Newsom-Davis and K. Henry, Acetylcholine receptor antibody synthesis by thymic lymphocytes: correlation with thymic histology, Neurology, 31 (1981) 935-943. Simpson, R.W., L. McGinty, L. Simon, G.A. Smith, C.W. Godzeski and R.J. Boyd, Association of parvoviruses with rheumatoid arthritis of humans, Science, 223 (1984) 1425-1428. Tardieu, M. and H.L. Weiner, Viral receptors on isolated murine and human ependymal cells, Science, 215 (1982) 419-421. Tindall, R.S.A., R. Cloud, J. Lucky and R.N. Rosenberg, Serum antibodies to cytomegalovirus in myasthenia gravis: effects of thymectomy and steroids, Neurology (Minneap.), 28 (1978) 273-277. Weigle, W.O., Cellular events in experimental autoimmune thyroiditis, allergic encephalomyelitis and tolerance to self. In: N. Talal (Ed.), Autoimmunity: Genetic, Immunologic, Virologic and Clinical Aspects, Academic Press, New York, 1977, pp. 140-170. Wekerle, H. and U.P. Ketelsen, Intrathymic pathogenesis and dual genetic control of myasthenia gravis, Lancet, i (1977) 678-680. Willcox. H.N.A., J. Newsom-Davis and L.R. Calder, Greatly increased antibody production in myasthenia gravis by thymocyte suspensions prepared with proteolytic enzymes, Clin. Exp. Immunol., 54 (1983) 3788386. Yoon, J.W., T. Onodera and A.L. Notkins, Virus induced diabetes mellitus. XV. Beta cell damage and insulin-dependent hyperglycaemia in mice infected with Coxsackie virus B4, J. Exp. Med., 148 (1978) 1068-1080.
Journal of Neuroimmunology, 11 (1986) 287-299 Elsevier
JNI 00363
Attempted Isolation of Viruses from Myasthenia Gravis Thymus L.S. Klavinskis
I,*, H.N.A. Willcox I,**, J.E. Richmond and J. Newsom-Davis 1,3
2
’ Depariment of Neurological Science, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, ’ Central Public Health Laboratory, Colindale Avenue, London N W9 5HT, and 3 National Hospitalfor Nervous Diseases, London WC1 (0: K.) (Received 15 October, 1984) (Revised, received 7 October, 1985) (Accepted 12 October, 1985)
Summary
A systematic study of thymus homogenates and cell suspensions from 13 patients with myasthenia gravis (MG) of recent onset, and 6 non-myasthenic controls, has failed to detect or isolate virus by cell culture with ‘rescue techniques’, electron microscopy, or intracerebral inoculation into neonatal mice. These results do not support the case for persistent viral infection in the thymus, and impose constraints on hypotheses of a viral aetiology of MG. Key words: Myasthenia
gravis
-
Persistent
virus -
Thymus
-
Viruses and auto-
immunity
Introduction
Myasthenia gravis (MG) is a disorder of neuromuscular transmission mediated by autoantibody to the acetylcholine receptor (AChR) at the motor endplate. Cellular
* Present address: Department of Immunology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037 (U.S.A.). ** To whom reprint requests should be addressed. Abbreviations: CMV, cytomegalovirus; CPE, cytopathic effect(s); EM, electron microscopy; FCS, foetal calf serum; HAI, haemagglutination inhibition; HSV, herpes simplex virus; i.c., intracerebral; IEM, immunoelectron microscopy; IF, immunofluorescence; p.i., post-inoculation; RSV, respiratory syncytial virus; s.c., subcutaneous; VZ, varicella zoster.
0165-5728/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
288 and humoral immune mechanisms have been extensively studied in myasthenics, but there are few clinical or epidemiological clues as to what initiates the autoimmune process: in rare cases, however, usually with HLA-DR-1 (Garlepp et al. 1983). penicillamine therapy may be causative. Where the autoimmune process begins in MG is uncertain, but circumstantial evidence suggests that it may not be in muscle. Inflammatory changes in muscle are infrequent and slight; lymphorrhages have occasionally been recognised in some MG patients (usually those with thymoma), but they are not preferentially sited at the motor endplate (Oosterhuis et al. 1968). By contrast, thymic changes are often striking and it has been suggested that it is within the thymus that loss of self-tolerance to the AChR is initiated (Wekerle and Ketelsen 1977). For example. about 10% of MG cases have thymic turnours, and these express antigens shared with skeletal muscle; almost all these cases have antibodies to striational and other muscle antigens in addition to those directed against the AChR (e.g. Gilhus et al. 1984). Thus it appears likely that, in this subgroup, the autosensitising agent may be the thymic tumour itself. Furthermore, hyperplasia of the thymus with medullary germinal centres is frequently found in the major subgroup of myasthenics those in whom the disease starts before the age of 40, and who show a 3 : 1 female bias, strong HLA-B8 and -DR3 associations, and an increased risk of several other autoimmune diseases (e.g. Compston et al. 1980). The myasthenia often improves after thymectomy, perhaps implying some central role for the thymus in the pathogenesis of the MG in this subgroup. Moreover, thymocytes from many of these cases spontaneously synthesise anti-AChR antibody in culture which may have a specific activity lo-100 times greater than in serum (Scadding et al. 1981) and appears to be mainly derived from the germinal centres (Willcox et al. 1983). In addition, these thymocytes can stimulate anti-AChR production by autologous blood lymphocytes, perhaps because they include specific antigen-presenting cells (Newsom-Davis et al. 1981). The connecting link between these patients and the thymoma subgroup may be the presence of muscle autoantigens - including AChR - in the thymus. Rare ’ myoid’ or muscle-like cells have been recognised in both the normal and myasthenic medulla, which express AChR in culture (Kao and Drachman 1977) and other cell types there may do so too. The thymic medulla is rich in antigen-presenting cells (Janossy et al. 1980) and, if these took up self-antigens such as AChR, they might present them in such a highly immunogenic form as to autosensitise newly developing T cells instead of rendering them tolerant. ultimately leading to germinal centre formation. It is conceivable that such a chain reaction might be initiated by extrinsic agents including viruses, perhaps through damage to AChR-bearing cells, or activation of antigen-presenting cells (Bottazzo et al. 1983). Viruses have often been suspected as initiating factors in autoimmune diseases. Autoantibodies to host antigens, e.g. DNA (Catalan0 et al. 1980). erythrocytes and platelets (Datta and Schwartz 1979) myelin (Panitch et al. 1980) smooth muscle, lymphocytes and immunoglobulins (Daugharty et al. 1979) have been observed after viral infections. In addition, type I diabetes in mice has been experimentally induced with Coxsackie B4, meningoencephalomyocarditis and reoviruses (Yoon et al. 1978;
289 Craighead and McLane 1968; Onodera et al. 1978) and C-type retroviruses have been implicated in the systemic lupus erythematosus of NZB X NZW mice (Datta and Schwartz 1976). It is therefore not surprising, in view of the thymic abnormalities and strong HLA associations in young female myasthenics, that persistent viral infection of the thymus has been proposed as an initiating event in MG (Datta and Schwartz 1974). There is no clear evidence for this as yet, however, and viral antibody studies have given conflicting results. Antibodies against cytomegalovirus (CMV) were found to be increased in myasthenics in one study (Tindall et al. 1978) though we have been unable to confirm this in a large survey with carefully matched controls, and have found no increase in incidence or titre of antibody to 8 other common viruses (Klavinskis et al. 1985). Alternative approaches are clearly necessary to detect possible extrinsic provoking agents in MG. We report here the results of a systematic study to detect and/or isolate virus from the thymuses of 13 patients with MG of recent onset and of 6 non-myasthenic controls.
Materials and Methods Patients Thymus tissue was analysed from 12 MG patients, selected because of a short time between onset (or relapse in case 13) and thymectomy (I 2 years). Case 1 was
TABLE
1
THE MYASTHENIA Cases 1-12 thymus.
showed
Case No.
Sex
1 2 3 4 5 6 I 8 9 10 11 12 13
F F F F F F F F F F F F F
CRAWS thymic
PATIENTS
hyperplasia
STUDIED
with medullary
germinal
centres,
and case 13 had an atrophic
Clinical grade ’
Plasma anti-AChR
(years)
Duration of symptoms before thymectomy (months)
22 20 15 21 24 37 24 22 22 31 24 18 4
60 19 6 14 7 24 11 10 17 11 10 23 120 (7) b
IIB IIB IIB IIA IIA IIB III IIA IIA IIB IIA IIB IIA
1190 621 316 258 96 56 54 50 38 31.2 32.8 18.2 3.0
Age at onset
a Clinical grading after Osserman and Genkins (1971): IIA = mild generalised; alised; III = acute, severe disease. b Remission from the age of 9 until 7 months before thymectomy.
IIB = moderate
gener-
290 TABLE
2
ANTIVIRAL
ANTIBODY
TITRES
Complement
fixing antibody
titres
Case No. 1 2 3h 4 5 6 7 x 9 10 I1 12 13
Influenza
Influenza
type A
type B
8 1: <8 32 <8 16 8 16 8 32 8 8
32 <8 8 8 8 (8 16 8 8 8 8 <8 ~8
OF THE MG PATIENTS
Adenovirus
RSV
STUDIED
Measles
Mumps
Rubella
a
(V) <8 18 8 32 8 32 16 8 8 <8
8 <8 16 16 8 16 16 8 8 8 18 8 <8
16 16 16 8 128 8 18 64 128 8 12x 8 16
16 18 8 16 16 8 8 8 16 8 16 8 64
240 156 2 849 176 687 176 394 446 505 289 166 348 200
” Rubella antibodies assayed by single radial haemolysis (IU/ml). h Case 3 presented with Still’s disease 6 years prior to onset of MG at which time levels of anti-rubella IgG (but not IgM) were also high, and anti-AChR antibody was undetectable. ’ Anti-reovirus antibodies assayed by HAI.
also selected because of very high thymocyte anti-AChR antibody production in culture and plasma titre. Clinical details are summarised in Table 1, where the cases are ranked according to their plasma anti-AChR titres: their antibody titres to 14 common infectious agents are given in Table 2. Control thymus was obtained from 6 non-myasthenic adults undergoing thoracic surgery. Antibody titres (Table 2) Sera obtained at the time of thymectomy were assayed by complement fixation (Bradstreet and Taylor 1962) for antibody to 9 viruses prevalent in the community, as well as to Mycoplasma hominis, psittacosis, and Coxiellu burnetii. In addition rubella IgG antibodies were assayed by radial haemolysis (Clarke et al. 1977) with reference to the British Standard antiserum for anti-rubella 69/60, and expressed in terms of international units of rubella potency/ml. Rubella IgM antibody was assayed using the Rubenz ‘M’ commercial kit (Northumbria Biologicals). Antireovirus type 3 HA1 antibodies were kindly assayed by Dr. E.H. Boxall (East Birmingham Hospital). Anti-AChR antibody was assayed by radioimmunoassay using human AChR as antigen (Newsom-Davis et al. 1978). Isolution procedures (a) General strategy We attempted to isolate virus by direct inoculation of thymus range of cell lines, into embryonated hens’ eggs and by intracerebral
extracts onto a inoculation of
291
HSV
CMV
vz
Reovirus
’
Mycoplasma
Psittacosis
Coxiella burn&i
8 18 <8 8 8 <8 ~8 <8 16 ~8 8 8 32
32 ~8 16 8 8 (8 ~8 16 <8 8 (8 8 ~8
<8 <8 <8 ~8 8 ~8 18 ~8 18 ~8 ~8 8 8
type 3 <8 <8 ~8 32 8 18 16 <8 18 8 <8 (8 (8
TABLE
8 18 8 32 64 18 (8 8 16 <8 8 ~8 64
32 8 64 16 16 ~8 8 32 32 8 32 8 32
64 256 32 64 32 64 256 32 64 32 32 128 32
3
THE CELL LINES AND CONDITIONS Cell line
MRC-5 HEP-2 (Cincinnati line) HEK
RK-13 Vero
PBK
USED
Cell type
Culture
conditions
Growth
medium a,’
Human fibroblasts Human laryngeal carcinoma (epithelial) Primary human foetal kidney (epithelial) Rabbit kidney (epithelial) African green monkey kidney (epithelial) Primary baboon kidney (epithelial)
MEM + 5% FCS + 0.04% NaHCO,
Maintenance
medium’
MEM + 2% FCS + 0.04% NaHCO,
MEM + 5% FCS + 0.013% NaHCO,
MEM + 2% FCS + 0.013% NaHCO,
MEM + 10% FCS
MEM + 2% FCS
MEM + 5% FCS + 0.013% NaHCO,
MEM + 2% RS + 0.013% NaHCO,
MEM + 4% FCS + 0.013% NaHCO, + 0.1% glucose
MEM + 2% FCS + 0.013% NaHCO, + 0.1% glucose
Medium 199 + 10% FCS + 0.013% NaHCO,
Medium NaHCO,
199 + 2% FCS + 0.013%
a MEM (minimal essential medium)+ Earle’s salts + 15 mM Hepes (Gibco, U.K.)+100 U/ml penicillin+ 100 ug/ml streptomycin. h Medium 199 + Earle’s modified salts+ 1.25 g/l NaHCO, (Gibco, U.K.)+ 100 U/ml penicillin + 100 ,ug/ml streptomycin. ’ RS = rabbit serum (Wellcome Laboratories).
292 neonatal mice; cell suspensions
also by co-cultivation of untreated with tissue culture monolayers.
and mitogen-stimulated
thymic
(6) Preparation of tissue homogenates and cell suspensions Homogenates were prepared (from about 20% of the total thymus) in an MSE blender and diluted to 10% w/v of original tissue with Hanks’ lactalbumin hydrolysate (Flow Laboratories) containing antibiotics, 0.35 g/l NaHCO,, 2% bovine serum albumin, and 15% sorbitol, before storage at - 70°C. Cell suspensions (from a further 20% of thymus) were prepared with proteolytic enzymes and stored in liquid nitrogen until used (Willcox et al. 1983). (c) Direct inoculation of cell cultures with thymus homogenates The cell lines and culture conditions used are summarised in Table 3. Confluent cell monolayers in 2 ml flat-bottomed (Linbro) wells were washed twice, and inoculated in triplicate with 0.1 ml of neat and lop2 dilutions of homogenates. After 2 h at 37°C (or 34°C for RK-13 cells), monolayers were washed and re-fed. Cultures were observed for CPE and passaged 3 times at lo-14-day intervals by trypsinization and adsorption onto fresh monolayers for 2 h (as above), prior to washing and re-feeding. PBK cells were cultured in tubes on roller apparatus at 33°C. In addition, concentrates of thymus homogenates from cases 3, 4 and 9, and of brain homogenate from a mouse inoculated i.c. with patient 4 thymus homogenate, were inoculated onto PBK cells and passed at least twice in the Virus Reference Laboratory, Cohndale. (d) Co-cultivation of thymus cell suspensions Subconfluent cultures of all the cell lines were washed, and seeded in triplicate with 1-3 X lo6 thymic cells in 2 ml wells. To some monolayers, cell suspensions pre-incubated for 4 days with pokeweed mitogen (Gibco, Grand Island, NY). at 10 ~1 per ml in RPMI/lS% FCS were added. All cultures were re-fed at day 7 and subcultured (2-3 X ) at lo-14 days. (e) Virus identification Four methods were employed in attempts to detect the presence of virus in cell cultures: (1) haemadsorption: inoculated monolayers (of all the cell lines in Table 3) were overlayed with human ‘O’, rhesus monkey (Flow Labs., U.S.A.) and fowl erythrocytes prior to subculture or passage; (2) immunofluorescence (IF): inoculated or co-cultured cell lines (RK-13, MRC-5 and Vero) were tested for viral antigens after the first and third subculture (see below) and PBK and HEK cells after the first subculture only; (3) interference: 7 days after the final passage, inoculated monolayers of every cell line were challenged with ECHO virus type 11, for which they were assayed by haemadsorption; (4) electron microscopy (EM): HEK monolayers inoculated with MG or control homogenates were fixed, processed and thin-sectioned for EM by standard procedures (Patterson and Bingham 1976); sonicates of cultured HEK cells were also pelleted, negatively stained and examined by EM.
293 (f) Egg inoculation Embryonated hens’ eggs were inoculated (in triplicate) chorioallantoic routes with 0.1 ml neat or 10e2 diluted thymocytes/O.l ml. Membranes were examined for lesions, for haemagglutinin with human ‘O’, rhesus monkey or fowl and 37°C (Grist et al. 1979b). Non-haemagglutinating fluids re-tested.
by the amniotic and homogenates, or lo6 and fluids were tested erythrocytes at +4”C were re-passaged and
(g) Suckling mouse inoculation Homogenate (0.05 ml of 10%) was injected i.c. and S.C. into newborn BKTO mice < 24 h old (Grist et al. 1979a). Daily observations of these ‘first passage’ mice were made for 25 days p.i. Deaths occurring within 48 h were considered to be due to inoculation trauma, and disregarded. Mice showing signs of disease were killed, the brains removed and 10% suspensions of these inoculated onto HEK, HEP-2, MRC-5 and Vero cells, and i.c. into ‘second passage’ mice.
(h) Immunoji’uorescence (IF) To detect viral antigens that might be present in cell cultures or thymus sections, we used indirect IF with polyclonal antisera to HSV types 1 and 2 (kindly provided by Dr. A. Minson), VZ (kindly provided by Prof. R. Heath), adenovirus, measles, rubella (Wellcome Laboratories), influenza A and B viruses, and pooled monoclonal antibodies (ascitic fluid) to CMV (kindly provided by Dr. L. Pereira), as well as autologous plasma taken at the time of thymectomy. Uninoculated cells and cells inoculated with the appropriate virus served as negative and positive controls. In addition, cryostat sections (6 pm) were prepared from thymus blocks and stained as described by Janossy et al. (1980).
(i) Electron microscopy of thymus homogenates Thymus homogenates from the MG patients were clarified at 1500 X g for 10 min and then the supernatants were centrifuged at 48000 X g for 2 h. The resulting pellets were negatively stained with 3% phosphotungstic acid pH 6.3 and examined in a JEOL 100 CX electron microscope. Brain extracts from mice inoculated i.c. with Coxsackie B3, or reovirus, and brain extracts from ‘first’ and ‘second passage’ mice showing signs of disease after inoculation with patient 4 thymus homogenate, were similarly prepared and examined.
0) Immunoelectron microscopy (IEM) Thymus homogenates from 8 MG patients (including patient 4) and brain extracts from mice inoculated with Coxsackie B3, or with patient 4 thymus homogenate, were examined by IEM (Richmond et al. 1984), using a hyperimmune Coxsackie B3 antiserum, or autologous serum obtained at the time of thymectomy.
294 Results (a) Positive controls Prior to our isolation experiments with MG thymus, we tested each of the cell lines for their sensitivity to a wide spectrum of viruses, and were able to detect HSV types 1 and 2, CMV (AD/169), VZ, measles, rubella, adenovirus type 10, Coxsackie A3, B3 and B5 viruses, ECHO 11, parainfluenza 3, reovirus types 1, 2 and 3, and influenza A and B. The last two viruses were also consistently harvested from amniotic fluids, and herpes lesions were readily detected on chorioallantoic membranes. Results of infection of mice by i.c. inoculation with Coxsackie B3 virus were as follows: 5 x lo4 TCID,, - complete paralysis then death of 4 of 4 mice by day 3; 5 X lo3 TCID,, - paralysis in 6 of 8 mice by day 3; and 5 X lo2 TCID,, ~ death or severe paralysis in all mice by day 6. Picornavirus particles were observed by EM and IEM in the brain extracts of 1 of 3 mice inoculated with Coxsackie B3. Brain histopathology in the sick mice showed mild acute inflammatory changes consistent with an infectious process. There was perivascular cuffing, a mononuclear infiltrate and phagocytic tissue breakdown, plus meningeal inflammation. Reovirus particles were detected by EM in brain extracts of 1 of 2 mice after i.c. inoculation with reovirus. Using the appropriate antisera, nuclear or cytoplasmic IF (depending on the virus) was observed in virus-infected but not control cells. In interference tests, cells preincubated with 10 TCID,, of HSV were resistant to subsequent challenge with ECHO 11 virus. Finally, in haemadsorption and haemagglutination tests, measles, ECHO 11 and influenza-A viruses gave positive results. (6) Inoculation of cultures with MC and control thymus homogenates, and co-cultivation with thymic cell suspensions Each thymic homogenate was inoculated onto all of the cell lines listed in Table 3. CPE attributable to viral agents were not detected in any culture from MC or control thymus, even in PBK and HEK cells. Transient toxic effects of some MG and control homogenates on the HEP-2 cells were occasionally seen but disappeared 24 h after the medium was changed. IF was not detected in any of the cells with any of the antibodies used: nor could we detect any viruses by EM in the inoculated HEK cells. Even when we used proteolytically prepared thymocyte suspensions (from all patients and controls), containing various stromal and macrophage-related cell types in addition to lymphocytes, we were still unable to detect any sign of virus from using PWM-stimulated co-cultures (even with PBK cells). ‘Rescue’ techniques, thymocytes co-cultivated with several cell lines, also failed to yield infectious virus or viral antigens detectable by IF, haemadsorption, or interference. Cell monolayers inoculated with thymus extracts from patients 2, 4, 8 and 13 showed weak nuclear IF when stained with the patients’ own plasma; as the same was seen in uninoculated cells, antinuclear antibodies were probably responsible. No lesions or haemagglutinating virus were detected in inoculated eggs.
295
(c) Inoculation of neonatal mice with thymus homogenates No signs of neurotropic infections, paralysis or growth retardation
were observed in neonatal mice by 25 days p.i. with 12 of 13 MG or 6 of 6 control thymus extracts, and there were only sporadic deaths. Some of the mice injected with homogenate from patient 4, however, developed oily coats, generalised weakness and growth retardation. In total, 7 of 14 mice died at days 2-12 in the first litter; the only gross abnormality found at post-mortem was cerebral atrophy with much intracranial fluid (approximately 0.2-0.3 ml). The remaining mice were killed at day 25, 2 being stunted (but otherwise healthy) and 5 of normal stature. In 1 of 3 further litters, 7 of the 12 mice were sick by day 9, but only 2 of the 14 mice in the other 2 litters became ill up to day 18. We failed to isolate virus from brain extracts from the sick, disease-free and control mice (using HEK, HEP-2, MRC-5 and Vero cells); viral antigens could not be detected by IF in any of the cell lines inoculated, and virus particles were found neither by EM nor IEM using serum from patient 4. Three of 10 ‘second passage’ mice inoculated with extract of brains from sick mice developed weakness, growth retardation and died at days 3, 20 and 26 p.i. One of 11 recipients of extract from apparently disease-free ‘first passage’ mice also died and the post-mortem findings in all 4 were as above. Brain sections from the sick mice were reviewed by an experienced neuropathologist. In the extreme examples, the appearances were of degeneration with focal collections of activated microglial cells, sparse mononuclear meningeal infiltrate, and no obvious perivascular cuffing or ependymitis. These changes were consistent with a delayed degenerative reaction to some toxic, traumatic or ischaemic effect of the inoculum, and there was no obvious sign of an infection or inflammatory process.
(d) EM and IEM of thymus homogenates and IF staining of thymus sections Virus particles could not be detected by EM in high-speed pellets of thymus homogenates from any of the patients, nor from the extracts of 8 MG thymuses (including that from case 4) examined by IEM. We were also unable to detect viral antigens in cryostat sections of any MG thymus by IF with any of the antisera used.
Discussion Despite rigorous efforts, this study has failed to detect any signs of persistent virus or viral antigens in the patients with MG, nor, indeed, have we found increased antiviral antibodies in MG sera (Klavinskis et al. 1985). We cannot, however, exclude a viral aetiology for a number of reasons. Our methods might not have been sufficiently sensitive to detect latent or small amounts of virus. Alternatively, host antibody to a virus could interfere with its detection, though this seems unlikely since we used washed cell suspensions as well as thymic extracts for the isolation work, and we inoculated the cell lines with high concentrations of extracts to maximise the sensitivity. Viral genes can be identified at much greater sensitivity by hybridisation to suitable probes, but these methods are only appropriate in seeking well-studied and highly specific gene sequences. It is obviously premature to apply
296
these techniques in the present studies, in the absence of any clues as to which virus to focus upon. Our negative observations might reflect the fastidious nature or defective expression of certain viruses in culture, which might therefore escape detection. To minimise these difficulties, we used a wide range of cell lines and attempted to ‘rescue’ viruses from minority cell types by preparing the cell suspensions with collagenase and dispase, and/or stimulating with pokeweed mitogen. In case defecwith the tive or non-cytocidal viruses were present, we tested for interference replication of a challenging picornavirus, a method that can detect rubella virus with great sensitivity (Parkman et al. 1964). In addition, concentrates of thymus homogenates were examined by EM and IEM and extracts were inoculated i.c. into neonatal mice. One might have expected the patients’ own sera to detect early antigens, even of fastidious viruses, in cell culture. There was intracranial pathology in some of the mice inoculated with patient 4 thymic homogenate, and in a few of the ‘second passage’ recipients. The pathological findings could have been late effects of viral ependymitis, which can lead to blockage of CSF flow and hydrocephalus. and can be caused by, for example, adeno-, myxo-, polyoma or type 1 reoviruses (summarised by Tardieu and Weiner 1982). Of these, perhaps the most likely to have been overlooked is the reovirus. Repeated isolation attempts, however, failed to show any CPE in any cultures (including PBK cells). Furthermore, this patient’s antibody titre to reovirus type 3 was similar to the titres of the other 12 patients and 6 control siblings. Although we cannot completely rule out a viral cause of the somewhat non-specific findings in some of the inoculated mice, it is equally possible that some toxic factor in the homogenate may have interfered with CSF resorption or circulation. Other agents, e.g. mycoplasma, chlamydiae or even a ‘novel’ (MG) virus analogous to SMON in multiple sclerosis (Melnick et al. 1982) or RA-1 virus in rheumatoid arthritis (Simpson et al. 1984), might conceivably be involved, for which our culture conditions or cell lines may have been unsuitable. Epidemiological without any studies argue against that, however, since MG occurs worldwide obvious regional differences or temporal fluctuations in incidence which might suggest an infectious aetiology (Kurtzke 1978). A final difficulty might be that virus is only present at an early stage in the disease (perhaps months or years before the onset of symptoms). or in some extra-thymic site. For both this and the sero-epidemiological study (Klavinskis et al. 1985) we therefore selected cases with the shortest duration of symptoms. A delay of several months between onset and thymectomy is almost inevitable: the incubation period prior to the onset of symptoms might well be even greater. The case for concentrating on the thymus, however, is strong and there are no comparable indications for testing other tissues. A similar isolation study by Aoki et al. (1985) has also failed to show any sign of viruses in MG thymus. Taken together. these negative sero-epidemiological and isolation studies considerably weaken the case for a viral aetiology in MG, especially in the subgroup of patients with thymic hyperplasia, the subjects of the current study. Each of our cases showed serological evidence of previous infection with
297 several common viruses (almost none to exaggerated titres) although no such virus was isolated. These studies therefore do not support the hypothesis of a viral aetiology in MG. Perhaps autoreactive T helper cells may be recruited by a transient viral infection, and then proliferate and induce autoantibody production long after the virus has been eliminated. Such a ‘hit-and-run’ effect could conceivably result from a crossreaction between virus and self (Allison 1977; Weigle 1977), from cross-reactions between AChR and viral anti-idiotypes (Cooke et al. 1983; Plotz 1983) or from a virus associating with self-antigen to provide a ‘helper determinant’ (Bromberg et al. 1982). The aetiology of autoimmune diseases has long been a challenging problem. Viruses are inevitably hard to exclude, though the evidence in their favour is scarcely stronger now than 20 years ago and, even in the best-documented examples, is not conclusive. These include type I diabetes mellitus, in which Coxsackie B viruses are implicated and there are seasonal peaks of incidence (e.g. Gamble 1980). It is not yet clear whether the Coxsackie viruses are merely precipitating factors, or play some more fundamental role. Even in the NZB X NZW mouse model of lupus erythematosus, in which retroviruses have been implicated, it now appears that they are not essential for the characteristic autoimmune responses (e.g. Datta and Schwartz 1979). An important alternative to viral-induced autoimmunity is that some chance unprovoked aberration in immunological homeostasis initiates the response. Many possibilities exist, e.g. inappropriate expression of Ia antigens on myoid cells, as may happen with thyroid epithelial cells (Bottazzo et al. 1983) over-officious antigen-presenting cells (Dausset and Contu 1980) loss of suppressor T cells, interference in tolerance induction of T helper cells, or imbalances in the idiotypic network. The association of several quite diverse immunological diseases with the same HLA B8-DR3 haplotype may well prove to reflect some general property of Ia antigens or the cells bearing them.
Acknowledgements
We are grateful to Prof. R. Heath, Drs. A.C. Minson, L. Pereira and M.S. Pereira for generous gifts of polyclonal and monoclonal antibodies and viruses, to Dr. P. Fryer and Mrs. J. Gross for invaluable help in screening material by thin-section EM, to Dr. E.H. Boxall for the anti-reovirus assays, to Messrs. B. Megson and P. Cunningham of the Virus Reference Laboratory, Colindale, and to Mr. M. Sturridge, FRCS, and Dr. L. Loh, FFA, RCS, for help in obtaining thymic tissue. We are also grateful to Drs. J.S. Oxford and P. Griffiths and Mr. M. Ross for help and advice, and to Dr. J. McCloughlin for his neuropathological expertise. This work was supported by the Muscular Dystrophy Group of Great Britain and the Sir Jules Thorn Charitable Trust.
29X References Allison. A.C., Autoimmune diseases: concepts of pathogenesis and control. In: N. Talal (Ed.). Autoimmunity: Genetic, Immunologic, Virologic and Clinical Aspects. Academic Press, New York, 1977. pp. 91-139. Aoki, T.. D.B. Drachman. D.M. Asher, C.J. Gibbs. S. Bahmanyar and J.S. Wolinsky. Attempts to implicate viruses in myasthenia gravis, Neurology, 35 (1985) 185-192. Bottazzo, G.F., R. Pujol-Borrell, T. Hanafusa and M. Feldmann, Role of aberrant HLA-DR expression and antigen presentation in induction of endocrine autoimmunity, Lancet, ii (1983) 1115-1119 Bradstreet. C.M. and C.E. Taylor, Technique of complement fixation test applicable to the diagnosis of virus diseases. Monthly Bull. Minist. Hlth. (London). 21 (1962) 966104. Bromberg, J.S., P. Lake and M. Brunswick, Viral antigens act as helper determinants for antibody responses to cell surface antigens, J. Immunol., 129 (1982) 683-688. Catalano, M.A., D.A. Carson, J.C. Niederman, P. Feorino and J.H. Vaughan, Antibody to the rheumatoid arthritis nuclear antigen. Its relationship to in viva Epstein-Barr virus infection. J. Clin. Invest.. 65 (1980) 1238-1242. Clarke, M., J. Bonstred. V. Seagroatt and G.C. Schild, The use of single radial haemolysis for rubella antibody studies. J. Hyg., 79 (1977) 355-364. Compston, D.A.S., A. Vincent, J. Newsom-Davis and J.R. Batchelor. Clinical, pathological, HLA antigen and immunological evidence for disease heterogeneity in myasthenia gravis, Brain. 103 (1980) 5799601. Cooke. A., P.M. Lydyard and 1.M. Roitt, Mechanisms of autoimmunity: a role of cross reactive idiotypes, Immunol. Today, 4 (1983) 170-174. Craighead, J.E. and N.F. McLane. Diabetes mellitus: induction in mice by encephalomyocarditis virus. Science, 162 (1968) 913-916. Datta, S.K. and R.S. Schwartz, Infectious myasthenia? N. Engl. J. Med., 291 (1974) 130441305. Datta. S.K. and R.S. Schwartz, Genetics of expression of xenotropic virus and autoimmunity in NZB mice. Nature (London), 263 (1976) 412-414. Datta, SK. and R.S. Schwartz, Genetic basis of autoimmune disease: the New Zealand mouse model. In: N.R. Proffitt (Ed.), Virus Lymphocytic Interactions: Implications for Disease. Elsevier-North Holland, New York, 1979. pp. 71-82. Da&arty, H., K. Kelly, C. Moore and T. Hersh, Autoimmune implications of immune complexes in clinical variants of hepatitis B, Clin. Exp. Immunol., 37 (1979) 2133220. Dausset. J. and L. Contu. The MHC and immune response in man. In: M. Fougereau and J. Dausset (Eds.), Progress in Immunology, Vol. IV, Academic Press, London, 1980, pp. 513-529. Gamble, D.R., The epidemiology of insulin dependent diabetes, with particular reference to the relationship of virus infection to its aetiology, Am. J. Epidemiol., 2 (1980) 49-70. Garlepp. M.I., R.L. Dawkins and F.T. Christiansen, HLA antigens and acetylcholine receptor anttbodies in penicillamine induced myasthenia gravis, Br. Med. J.. 286 (1983) 338-340. Gilhus, N.E.. J.A. Aarli, B. Christensson and R. Matre, Rabbit antiserum to a citric acid extract of human skeletal muscle staining thymomas from myasthenia gravis patients. J. Neuroimmunol., 7 (1984) 55-64. Grist, N.R.. E.J. Bell, E.A.C. Follet and G.E.D. Urquhart, Chapter 5, Animal and egg techniques. In: Diagnostic Methods in Clinical Virology, Blackwell Scientific Publications, Oxford, lY79a. pp. 44459. Grist, N.R., E.J. Bell, E.A.C. Follet and G.E.D. Urquhart, Chapter 9, Haemagglutination and haemagglutination inhibition tests. In: Diagnostic Methods in Clinical Virology, Blackwell Scientific Publications. Oxford, 1979b, pp. 116-128. Janossy, G.. J.A. Thomas, F.J. Bollum, S. Granger, G. Pizzolo, K.F. Bradstock, L. Wang, A. McMichael. K. Ganeshaguru and A.V. Hoffbrand, The human thymic environment: an immunologic study, J. Immunol., 125 (1980) 2022212. Kao, I. and D.B. Drachman, Thymic muscle cells bear acetylcholine receptors: possible relationship to myasthenia gravis, Science, 195 (1977) 74-75. Klavinskis, L.S., H.N.A. Willcox, J.S. Oxford and J. Newsom-Davis. Antivirus antibodies in myasthenia gravis. Neurology. 35 (1985) 13X1-1384.
299 Kurtzke, J.F., Epidemiology of myasthenia gravis, Adv. Nemo]., 19 (1978) 545-566. Melnick, J.L., E. Seidel, Y.K. Inoue and Y. Nishibe, Isolation of virus from the spinal fluid of three patients with multiple sclerosis and one with amyotrophic lateral sclerosis, Lancet, i (1982) 830-833. Newsom-Davis, J., A.J. Pinching, A. Vincent and S.G. Wilson, Function of circulating antibody to acetylcholine receptor in myasthenia gravis investigated by plasma exchange, Neurology (Minneap.), 28 (1978) 266-272. Newsom-Davis, J., H.N.A. Willcox and L. Calder, Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine receptor antibody by autologous blood lymphocytes, N. Engl. J. Med., 305 (1981) 1313-1318. Onodera, T., A.B. Janson, J.W. Yoon and A.L. Notkins, Virus induced diabetes mellitus: reovirus infection of pancreatic B cells in mice, Science, 201 (1978) 529-531. Oosterhuis, H.J.G.H., J. Bethlem and T.E.W. Feltkamp, Muscle pathology, thymoma and immunological abnormalities in patients with myasthenia gravis, J. Neurol. Neurosurg. Psychiatry, 31 (1968) 460-463. Osserman, K.E. and G. Genkins, Studies in myasthenia gravis: review of a twenty-year experience in over 1200 patients, Mt. Sinai J. Med., 38 (1971) 497-537. Panitch, H.S., C.J. Hooper and K.P. Johnson, CSF antibody to myelin basic protein; measurement in patients with multiple sclerosis and SSPE, Arch. Neurol., 37 (1980) 206-209. Parkman, P.D., E.L. Buescher, M.S. Artenstein, J.M. McCown, F.K. Mundon and A.D. Druzd, Studies of rubella, I. Properties of the virus, J. Immunol., 93 (1964) 595-607. Patterson, S. and R.W. Bingham, Electron microscope observations on the entry of avian infectious bronchitis virus into susceptible cells, Arch. Virol., 52 (1976) 191-200. Plotz, P.H., Autoantibodies are anti-idiotype antibodies to antiviral antibodies, Lancet, ii (1983) 824-826. Richmond, J.E., J.V. Parry and S.D. Gardner, Characterisation of a polyomavirus in two foetal rhesus monkey kidney cell lines used for the growth of hepatitis A virus, Arch. Viral., 80 (1984) 131-146. Scadding, G.K., A. Vincent, J. Newsom-Davis and K. Henry, Acetylcholine receptor antibody synthesis by thymic lymphocytes: correlation with thymic histology, Neurology, 31 (1981) 935-943. Simpson, R.W., L. McGinty, L. Simon, G.A. Smith, C.W. Godzeski and R.J. Boyd, Association of parvoviruses with rheumatoid arthritis of humans, Science, 223 (1984) 1425-1428. Tardieu, M. and H.L. Weiner, Viral receptors on isolated murine and human ependymal cells, Science, 215 (1982) 419-421. Tindall, R.S.A., R. Cloud, J. Lucky and R.N. Rosenberg, Serum antibodies to cytomegalovirus in myasthenia gravis: effects of thymectomy and steroids, Neurology (Minneap.), 28 (1978) 273-277. Weigle, W.O., Cellular events in experimental autoimmune thyroiditis, allergic encephalomyelitis and tolerance to self. In: N. Talal (Ed.), Autoimmunity: Genetic, Immunologic, Virologic and Clinical Aspects, Academic Press, New York, 1977, pp. 140-170. Wekerle, H. and U.P. Ketelsen, Intrathymic pathogenesis and dual genetic control of myasthenia gravis, Lancet, i (1977) 678-680. Willcox. H.N.A., J. Newsom-Davis and L.R. Calder, Greatly increased antibody production in myasthenia gravis by thymocyte suspensions prepared with proteolytic enzymes, Clin. Exp. Immunol., 54 (1983) 3788386. Yoon, J.W., T. Onodera and A.L. Notkins, Virus induced diabetes mellitus. XV. Beta cell damage and insulin-dependent hyperglycaemia in mice infected with Coxsackie virus B4, J. Exp. Med., 148 (1978) 1068-1080.