B. Eymard

Association of arthrogryposis multiplex congenita with maternal antibodies inhibiting fetal acetylcholine receptor function.

Arthrogryposis multiplex congenita (AMC), characterized by multiple joint contractures developing in utero, results from lack of fetal movement. Some cases are genetically determined, but AMC occasionally complicates pregnancy in patients with myasthenia gravis (MG) suggesting involvement of circulating maternal antibodies. We previously demonstrated antibodies that inhibited the function of fetal acetylcholine receptor (AChR) in one healthy woman with an obstetric history of recurrent AMC. Here we study sera from this woman, from one other with a similar history, and from three (one asymptomatic) whose babies had neonatal MG and AMC. All five maternal sera had high titers of antibodies that inhibited alpha-Bungarotoxin (alpha-BuTx) binding to fetal AChR, and their sera markedly inhibited fetal AChR function with little effect on adult AChR function. Moreover, in a further survey, 3 of 20 sera from anti-AChR negative AMC mothers inhibited fetal AChR function significantly at 1:100 dilution. These results demonstrate the role of antibodies to fetal AChR and perhaps other muscle antigens in some cases of AMC. More generally, they suggest that placental transfer of antibodies directed at fetal antigens should be considered as a cause of other recurrent fetal or perinatal disorders.

Neonatal myasthenia gravis A new clinical and immunologic appraisal on 30 cases

Anti-acetylcholine receptor (AChR) antibody titers, toxin binding blocking antibody, functional activity of serum on rat myotube cultures, IgG subclasses, and clinical data were studied in relation to the onset of neonatal myasthenia gravis (NMG) in 30 children of myasthenic mothers. Fourteen had NMG, including 4 atypical cases. Anti-AChR antibody titer was the best indication of NMG onset. NMG in a previous baby was also predictive. Pattern of IgG subclasses, presence of toxin-binding blocking antibodies, and serum functional activity were less predictive, but cast light on the mechanism of anti-AChR antibody pathogenicity.

Association of neonatal myasthenia gravis with antibodies against the fetal acetylcholine receptor.

The specificities of autoantibodies directed against the acetylcholine receptor (AChR) for embryonic and adult muscle AChR were studied in 22 mothers with myasthenia gravis (MG) and in their newborns using human fetus and normal adult muscle AChR preparations. 12 mothers had transmitted MG to their neonates with, in three cases, antenatal injury. A clear correlation was found between occurrence of neonatal MG (NMG) and the high overall level of anti-AChR antibodies (embryonic or adult muscle AChR). However, a strong correlation was also found between occurrence of NMG and the ratio of anti-embryonic AChR to anti-adult muscle (Te/Ta) AChR antibodies (P < 0.0002). Taken together, these data suggest that autoantibodies directed against the embryonic form of the AChR could play a predominant role in the pathogenesis of NMG. Paradoxically, the three cases with antenatal injury presumably the most severe form of NMG, were not associated with high Te/Ta. At the clinical level, these observations could prove helpful in the prediction of transmission of NMG.

The fetal/adult acetylcholine receptor antibody ratio in mothers with myasthenia gravis as a marker for transfer of the disease to the newborn

High anti-fetal/anti-adult muscle anti-acetylcholine receptor (AChR) antibody (Ab) titer ratio is predictive of the occurrence of neonatal myasthenia gravis in a first child. The aim of the present study was to determine whether the ratio between the levels of antibodies is an intrinsic property of the mothers sera or varies with physiologic status such as pregnancy. We performed a longitudinal study of the levels of Ab directed against both fetal and adult AChRs and the ratio between them in 11 mothers with myasthenia gravis (MG). Sera were taken during, before, and after pregnancy. Absolute levels of Ab varied considerably during the time of observation as indicated by analyzing the maximum change between any two sample times during the study (adult mean percentage change 45.9 +/- 26.4; fetal 42.51 +/- 22.05). In contrast to this, the anti-fetal/anti-adult muscle AChR Ab titer ratio was much less variable (mean percentage change 16.66 +/- 10.11; p < 0.0033). The levels of the two Ab types yielded a correlation coefficient of 0.918, consistent with the stability of the ratio between them. This stability of ratio has practical value in the management of pregnancy and infant care in mothers with MG because the ratio at any time, before or during pregnancy, will predict whether the child will contract neonatal MG. We determined this for the first child, but further studies are necessary to establish if this remains true for subsequent pregnancies. NEUROLOGY 1997;48: 50-54

Anti-acetylcholine receptor antibodies in neonatal myasthenia gravis: Heterogeneity and pathogenic significance

Precipitating, blocking and modulating anti-AChR antibodies and their capacity to recognize embryo or adult muscle were investigated in parallel in maternal and neonatal sera of fifty-two newborns and their myasthenic mothers. Twenty-four babies presented neonatal myasthenia gravis (NMG) with a common expression in twenty cases and foetal involvement in four cases. Occurrence of NMG was clearly related to levels of maternal precipitating, decamethonium blocking (DC blocking) and modulating antibodies (respectively P less than 0.001, P less than 0.02, P less than 0.01, Mann-Whitney test), these three parameters being interrelated. The only significant difference between mothers with severely affected babies and others with mild NMG newborns concerned DC blocking antibodies. Transfer fraction of DC blocking antibodies was significantly higher in NMG than in asymptomatic babies. Our data suggest that (1) the amount of anti-AChR antibodies, whatever the tested category, is involved in the pathogeny of NMG in a more direct manner than in adult MG where the correlation with their levels is poor, and (2) among antibodies, those with blocking effects could be preponderant for triggering NMG and be involved in the severity of the disease in the child.

Effect of myasthenic patient sera on the number and distribution of acetylcholine receptors in muscle and nerve-muscle cultures from rat: Correlations with clinical state

We studied the functional activities (FA) of sera obtained from 83 myasthenic patients on rat muscle cultures. Using the same sets of cultures, two parameters were evaluated after exposure to sera: residual fraction (RF) of acetylcholine receptors (AChR) coupled to 125I-labelled alpha-bungarotoxin (alpha Bgt) (81 sera) and the number of rhodamine labelled clusters (56 sera). Two types of culture were assayed: muscle alone and nerve-muscle cocultures (12 cases). In all combinations (fluorescence, radiolabelling, muscle alone and nerve-muscle cocultures), we found a significant correlation between FA and antibody (Ab) titre, and no correlation between FA and clinical severity: only sera with a high or intermediate Ab titre were effective, whatever the clinical severity of disease. With active sera, AChR loss was about 50% whereas the disappearance of AChR clusters was quite complete, which suggests AChR redistribution induced by MG sera.

Alpha-bungarotoxin blocking antibodies in neonatal myasthenia gravis: frequency and selectivity.

We have studied antibodies to the acetylcholine receptor (AChR) that inhibit alpha-bungarotoxin binding in 22 mothers with myasthenia gravis (MG) and in their 23 newborns. Of the 13 showing neonatal MG, seven of the mothers had detectable direct blocking antibodies, all 13 had decamethonium-dependent (DC) blocking, and nine had high titres of precipitating antibodies (greater than 40 nM). In those with symptom-free newborns, the corresponding figures were 2/10, 8/10 and 2/10; the mean titres of DC blocking and of precipitating antibodies were 5- and 3-fold lower than in the mothers of affected babies. Thus, blocking antibodies, in addition to high total antibody levels, may help to predict the occurrence of neonatal MG. However, the antibodies appear not to cross the placenta to the same extent in each case. 86, 69 and 84% of the maternal antibodies with precipitating, direct and DC blocking activities, respectively, were found in the myasthenic neonates versus 65, 28 and 44% in the unaffected. These data suggest (1) involvement of blocking antibodies in the pathogenesis of MG, and (2) variable placental transfer of anti-AChR antibodies, which makes neonatal affliction more difficult to predict.

Cellular aspects of myasthenia gravis

Several cellular aspects were investigated in a large series of patients with MG. First, non-Ag-specific proliferation was tested by measuring the response to r-IL2. Thymocytes from most MG patients showed hyperactivity to r-IL2. Peripheral blood lymphocytes (PBL) from some patients also showed a high response to r-IL2. These responding patients were generally those tested before thymectomy, presenting a high anti-AChR Ab titer and a severe form of the disease. Second, Ag-specific proliferation of MG PBL was assayed using 8 synthetic peptides corresponding to selected domains of torpedo or human AChR. Only 2 peptides gave a positive response in a significant number of patients, essentially in those presenting high anti-AChR Ab titer. The first is located near the alpha-bungarotoxin binding site and the second is in a cytoplasmic domain, according to models predicting the AChR transmembrane orientation. The positive results were essentially obtained with the human peptides; the corresponding torpedo peptides were positive in very few patients. Both human and torpedo peptides which include a part of the alpha-bungarotoxin binding site were negative. Finally, although morphological abnormalities were clearly visible in thymic hyperplasia, no correlation could be established between the thymus type and the cellular proliferation either to r-IL2, or to the peptides. Overall, our data indicate that cell-dependent mechanisms participate in the pathogenesis of MG, but the level of their involvement deserves further investigation.

Effect of sera from myasthenia gravis patients and of α-bungarotoxin on acetylcholinesterase during in vitro neuromuscular synaptogenesis

Myasthenia gravis (MG) is mediated by circulating antibodies directed against acetylcholine receptor (AChR) but the antibody titre is poorly correlated with the clinical severity of the disease. We analysed acetylcholinesterase (AChE) activity, molecular forms and distribution during in vitro synaptogenesis, in the presence of sera from MG patient. We observed that the formation of AChE patches is inhibited in proportion to the anti-AChR antibody titre, whatever the clinical severity of the disease. The total activity and the proportion of the different molecular forms were unchanged suggesting that AChE level and distribution are controlled by independent mechanisms. To clarify the relationship between the mechanisms of AChE concentration during synaptogenesis and AChR concentration, we compared the effect of MG sera (receptors are internalised and degraded) and of the acetylcholine antagonist alpha-bungarotoxin (non-functional receptors are still present in the muscular membrane). In the presence of alpha-bungarotoxin, the number of AChR clusters, and AChE activity and concentration were equivalent to control values. The comparison of the results obtained with antibodies and alpha-bungarotoxin suggests that the presence and/or concentration of AChR is a necessary condition for normal concentration of AChE during synaptogenesis.

Combined effects of a thymic peptide, thymopoietin and myasthenic patient sera in rat myotube culture

We investigated in a rat myotube assay the combined effect of 26 myasthenic (MG) patient sera and a thymic peptide, thymopoietin (Tpo) which had previously been shown to bind Torpedo and human AChR and to compete with alpha-bungarotoxin (alpha-Bgt) binding. Cultures were first exposed to Tpo alone for 3 h (0.3, 7.5, 15 nM), then MG sera (5% final dilution) were added for an additional 18 h. Reduction in the amount of 125I-alpha-Bgt binding sites in the presence of various concentrations of Tpo were similar with control sera and in all the patients with low or undetectable anti-AChR Ab (11 cases). In cultures exposed to Tpo and sera with high anti-AChR Ab titre (15 cases), Tpo and anti-AChR Ab have an additive capacity to reduce the number of alpha-Bgt binding sites. The results are compatible with the hypothesis that anti-AChR Ab and Tpo could impair neuromuscular transmission by complementary mechanisms.