Myasthenic Sera Research Articles

Serum acetylcholine receptor antibody measurement in myasthenia gravis: Some trials to background radioactivity in radioimmunoassay

Anti-acetylcholine receptor (AchR) antibodies in 19 non-myasthenic and 9 myasthenic sera were determined by radioimmunoassay, using [ 125I]α-bungarotoxin-complexed receptors of human and denervated rat muscles (standard assay). Anti-AchR antibodies in the same sera blocked by antihuman IgG were assayed in parallel to measure background radioactivity (blank assay). The activity of blank assay did not differ significantly from that of standard assay in non-myasthenic sera. There was no remarkable difference in blank assay titers between non-myasthenic and myasthenic sera. The corrected titers, obtained by subtracting blank assay titers from standard assay titers, were in excess of those of non-myasthenic sera in 6 of 9 myasthenic sera with the denervated rat AchR and 8 of 9 myasthenic sera with human AchR. These findings suggest that this method is useful for the detection of the anti-AchR antibody in myasthenic sera.

Monoclonal anti-torpedo receptor antibodies used to study antibody heterogeneity in myasthenic sera

Monoclonal antibodies against Torpedo acetylcholine receptor were used to define the binding regions of cross-reacting, anti-receptor antibodies from sera of myasthenic patients. Cross-reacting antibodies were directed mainly against the toxin-binding region of the receptor and a region remote from the acetylcholine-binding site. Few patients had antibodies against the acetylcholine-binding site region. The monoclonal antibodies provide probes for defining the heterogeneity of anti-receptor antibodies in myasthenic sera.

Recent advances in the molecular mechanisms of human and animal models of myasthenia gravis.

The receptor-channel molecule is a dynamic system which exists in multiple conformations and that is the way we should think of it when we study antibody interaction with the molecule. The results presented here suggest that some antibodies may affect receptor function by occupying sites other than the receptor site. Some of these sites may by exposed only in certain conformations, and occupation of some site by antibodies may effect conformational changes. These small but perhaps important differences in cholinergic channel properties of the myasthenic muscle from the normal one are revealed by studying the effect of myasthenic sera on drug interactions with the channel sites. The sera of myasthenics are able to react with certain channel conformations and are able to affect the interaction of channel antagonists such as H12HTX and QNB. The sera appear to act preferentially with the open conformation of the channel. As a consequence of such an effect, important conformational changes of the channel may fail to occur upon activation.

Immunochemical properties of junctional and extrajunctional acetylcholine receptor

Immunological assays were performed to compare two distinct forms of the nicotinic acetylcholine receptor (AChR): junctional (JR) and extrajunctional receptor (EJR). Antibodies from myasthenia gravis patients' sera inhibited the binding of [ 125I]α-bungarotoxin (BGT), to EJR more effectively than binding to JR. Immunological differences between JR and EJR were confirmed by other assay methods. In all cases, EJR appeared to have antigenic determinants not found on JR. It was established that enzymatic removal of carbohydrates from EJR caused it to more closely resemble JR. Thus differences between JR and EJR may be due, in part, to carbohydrate residues found on EJR that are absent on JR. The extent of antibody binding to EJR was examined by gel filtration methods. Immunochemical studies of bands from SDS gels showed that antibodies are present in myasthenic serum which react with the 3 subunits (42, 53, 64 kdaltons) of AChR to varying degrees.

An assessment of radioimmunoassay procedures for determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis.

A reproducible radioimmunoassay procedure for the determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis is described and examined in detail. The assay combines features of a number of methods previously outlined and allows repeat determinations of antibody titre in a given myasthenic serum sample with coefficient of variation 6%. The mean +/- standard deviation for normal human serum anti-acetylcholine receptor antibodies was found by this procedure to be 0.024 +/- 0.033 nmol/l alpha-bungarotoxin binding sites whereas the range for myasthenic patients was 0-139.14 nmol/l with a mean value of 7.55 nmol/l alpha-bungarotoxin binding sites.

Passive transfer of human myasthenia gravis to rats: 1. Electrophysiology of the developing neuromuscular block.

Adult female Lewis rats were rendered immunologically tolerant to human gamma globulin, and were given a single intravenous injection of human myasthenic or normal control serum containing 7.5 to 12 mg of immunoglobulin G. The mean amplitude of miniature endplate potentials (MEPPs) in the forelimb flexor digitorum longus muscles from treated animals did not differ from control values during the first 24 hours after serum injection. Subsequently, MEPP amplitude was reduced in muscles from animals that had received myasthenic serum; maximum reduction was reached by 6 days after transfer. Mean MEPP amplitude at maximum reduction was 30 to 40% below the amplitude of controls and returned to control values 14 weeks after transfer. Similar reductions in endplate potential amplitudes were found in immunologically tolerant animals receiving myasthenic serum. No significant reduction of MEPP amplitude was seen in recipients that were not immunologically tolerant or that had received cobra venom factor to reduce complement activity. The delayed development of reduced MEPP amplitude indicated that the defect of neuromuscular transmission produced by myasthenic serum was not due entirely to a simple curare-like block of the acetylcholine receptor site by an IgG antibody.

Experimental myasthenia gravis: Isolation and quantitative assay of anti-acetylcholine receptor antibody protein concentration in sera of rabbits immunized with Narke acetylcholine receptor

Antibody to acetylcholine receptor from Narke electroplax japonica was isolated from serum of rabbits with experimental autoimmune myasthenia gravis by affinity chromatography on a column of torpedo AChR-agarose conjugates. Sixty-three to eight hundred and ninety-six micrograms of antibody protein were obtained per milliliter myasthenic serum. Serum concentrations of antibody protein correlated with the intensity of the disease and were in exact proportion to the antibody titers of the same samples measured by double immunoprecipitant method. This study showed that anti-AChR antibody played a major role in experimental autoimmune myasthenia gravis and provided the first direct, quantitative evidence that the titers measured by Lindstrom's method could be quite a reliable index of antibody protein concentration in the serum of experimental myasthenia gravis subjects.

Myasthenia gravis

Serum antibodies to acetylcholine receptors (AChR) in myasthenia gravis were surveyed by radioimmunoassay, using 125I-alpha-bungarotoxin-complexed receptors of human muscles and denervated rat muscles. Antibodies were detected more frequently with human AChR than with rat AChR. Furthermore, antibody titers to human AChR were not always parallel to titers observed in the rat receptors, indicating the heterogeneity of antibody specificities in different individuals. Correlation between antibody titers and clinical severity was better when the antibody concentration was determined with human AChR. About 90% of the myasthenic sera contained antibodies to AChR, and 40% also contained other antibodies such as thyroid autoantibodies. These findings suggest that myasthenia gravis is a multiple immunopathy.

Serum factor in myasthenia gravis inhibits induction of “active” E by carbachol

It has been reported that incubation with an α-adrenergic agonist ( l-phenylephrine hydrochloride) or cholinergic agonist (carbachol) increased T-lymphocyte “active” E-rosette formation. In the present work, we found that preincubation with myasthenic sera significantly inhibited active E induction by carbachol, while control sera, obtained mostly from those with neuromuscular disorders, had no effect. On the other hand, l-phenylephrine-induced active E formation was not affected by either myasthenic or control sera pretreatment. Presence of such a serum inhibitor in myasthenic patients was associated with the disease activity. Furthermore, a dose-response study showed that this inhibitor was competitive for the effect of carbachol. Heating the sera at 56°C for 30 min or dialysis against 0.9% NaCl at 4°C for 2 days did not abolish the activity. After Sephadex G-200 gel filtration of the crude sera, the same degree of inhibitory activity as was seen in the whole sera was retained at the second peak which consisted mainly of IgG.

Antibodies from patients with myasthenia gravis recognize determinants unique to extrajunctional acetylcholine receptors.

We have examined the interaction between sera from patients with myasthenia gravis and acetylcholine receptor (AcChoR) purified from normal and denervated rat skeletal muscles [junctional receptor (JR) and extrajunctional receptor (EJR), respectively]. Eight of ten myasthenic sera had titers against EJR that were significantly higher (1.1-2.4 times) than their titers against JR. The antireceptor titers of these sera ranged from 2 to 102 nM. Although activities of three other sera were too low (less than 1 nM) to allow accurate titrations, provisional measurements with these sera gave titers against EJR that were at least as high (1.0-1.4 times) as those against JR. Competition experiments with myasthenic sera demonstrated two classes of determinants on rat AcChoR: those that are common to JR and EJR and those that are present or exposed only on EJR. Myasthenic sera did not recognize any determinants unique to JR. Several antisera raised to purified AcChoR from eel or Torpedo electric organs or denervated rat skeletal muscle had equal titers against the two forms of receptor. Treatment of JR and EJR by various enzymatic or chemical procedures designed to alter prosthetic groups on the proteins failed to affect their antigenic reactivity. AcChoR from embryonic rats was indistinguishable immunologically from EJR of adult muscle.

Serum factor blocks neuromuscular transmission in myasthenia gravis

We studied the effects of normal and myasthenic sera on the miniature endplate potential (MEPP) and resting membrane potential (RP) of rat muscle in vitro by conventional intracellular microelectrode techniques. Normal sera had little or no effect on either the amplitude or frequency of MEPP or RP. On the other hand, MEPP amplitude was reduced in each of nine muscles during exposure to myasthenic sera; five of these muscles showed a significant difference, by student's t-test, from the values in a control solution. The half decay time of diminished MEPP remained unchanged. MEPP frequency and RP were not affected by myasthenic sera. The reduced amplitude of the MEPP was almost completely restored when the muscle was washed with a control solution for more than 30 minutes. These observations indicate that myasthenic sera contain factors that bind reversibly with the acetylcholine receptor and reduce postsynaptic responses to acetylcholine.

Myasthenic antibodies cross-link acetylcholine receptors to accelerate degradation.

The decrease of acetylcholine receptors at neuromuscular junctions of myasthenic patients has been attributed to an antibody-mediated autoimmune process that accelerates receptor degradation. We studied the mechanism of this process in skeletal-muscle cultures, using intact antibodies and antibody fragments. Addition of myasthenic IgG or its divalent fragment, F(ab')2, to cultures accelerated the rate of acetylcholine-receptor degradation threefold. By contrast, the monovalent fragment, Fab, from myasthenic serum had no effect on degradation, although it bound to acetylcholine receptors. Addition of a second, "piggyback" antibody to cross-link the Fab:receptor complexes resulted in a threefold increase of the degradation rate. Similarly, when acetylcholine receptors with bound alpha-bungarotoxin were cross-linked by the addition of specific antibody against alpha-bungarotoxin, the degradation rate increased approximately threefold. The effect of myasthenic patients' antibodies in accelerating degradation of acetylcholine receptors is attributed to their ability to cross-link the receptors.

Human myasthenic sera reduce acetylcholine sensitivity of human muscle cells in tissue culture.

MYASTHENIA gravis is a muscle disease of man characterised by impaired neuromuscular transmission during repetitive motor nerve activity. Recent studies suggest that the defect in myasthenia gravis is post-synaptic and that myasthenic end-plates have a lower density of functional acetylcholine (ACh) receptors than normal end-plates. Thus end-plates from myasthenic patients show unusually small miniature end-plate potentials1,2, are less sensitive than normal end-plates to iontophoretically applied ACh (refs 2, 3) and bind reduced amounts of α-bungarotoxin4,5—a snake neurotoxin which is considered to bind specifically to nicotinic ACh receptors (see ref. 5). The presence of antibodies against ACh receptors in the sera of myasthenic but not of control patients6–10 suggests that anti-receptor antibodies might act in some way to decrease the number of functional receptors at the motor end-plate. Previous investigations, however, have failed to show conclusively that myasthenic sera contain a factor that reduces muscle ACh sensitivity3,11–13. Here we show that myasthenic sera can reduce the ACh sensitivity of tissue cultured human muscle cells.

Accelerated degradation of acetylcholine receptor from cultured rat myotubes with myasthenia gravis sera and globulins.

Altered geometry of the neuromuscular junction and a decreased number of acetylcholine receptors appear responsible for the defect of neuromuscular transmission in myasthenia gravis. We have used cultured rat myotubes as a model to study in vitro the potential role of myasthenic globulins in the pathological process. Acetylcholine receptor content was assayed by the extent of 125I-labeled alpha-bungarotoxin binding, and acetylcholine receptor function was assayed by the sensitivity to acetylcholine iontophoresis. The half-life of the acetylcholine receptor was 18.5 hr in the presence or absence of control sera. Myasthenic sera and globulins produced a gradual reduction in acetylcholine receptors, as assessed by biochemical and electrophysiological techniques. The half-life in the presence of myasthenic sera was 6 hr. The accelerated turnover was unaffected by puromycin but was slowed by lowered temperature (18-20 degrees), interference with energy metabolism (2,4-dinitrophenol), and interference with cytoskeletal structures (colchicine and cytochalasin B). We found no electrophysiological evidence to suggest globulin blockade of acetylcholine access to the acetylcholine receptor. Our observations suggest that circulating globulins in myasthenia gravis may contribute to the functional defects of neuromuscular transmission by accelerating the rate of internationalization and degradation of surface membrane acetylcholine receptors.

Myasthenia gravis serum reduces acetylcholine sensitivity in cultured rat myotubes.

MYASTHENIA gravis is a neuromuscular disorder characterised by muscle weakness and fatigability. Muscle fibres from myasthenic patients have reduced amplitudes of endplate potentials and miniature endplate potentials1,2. Early studies concluded that the disorder was presynaptic in origin because the postsynaptic chemosensitivity of the myasthenic muscle fibres was found to be normal1, but recent evidence indicates that myasthenia gravis is an autoimmune disease directed against the postsynaptic acetylcholine receptors3,4. Myasthenic serum globulin has been shown to bind to the acetylcholine receptors5,6, myasthenic muscle contains only 11–30% of α-bungarotoxin binding sites compared with normal muscle7, and iontophoretic studies showed that the acetylcholine sensitivity of the postsynaptic region of myasthenic muscle was markedly reduced2. But the failure of in vitro application of myasthenic serum to reduce the junctional or extrajunctional sensitivity of human, rat or frog muscle8 has raised doubt on the involvement of the serum factor in the disease. In this report we show that the acetylcholine sensitivity of cultured rat myotubes is greatly reduced by in vitro application of myasthenic serum.

Effects of normal and myasthenic serum factors on innervated and chronically denervated mammalian muscles.

Effects of normal and myasthenic serum factors on innervated and chronically denervated mammalian muscles.

Effects of myasthenic sera on cultured muscle

Serum from 17 myasthenic patients was added to mouse somite cultures containing mature cross-striated muscle fibers. Myolysis occurred after 24 hr exposure to the serum of three patients. These findings are discussed in relationship to the pathogenesis of myasthenic gravis.

Interaction of myasthenic serum globulin with the acetylcholine receptor

A serum factor from patients with myasthenia gravis which inhibited the binding of 125I-labeled α-bungarotoxin to acetylcholine receptor extracted with Triton X100 from rat muscle has been studied in detail. The inhibitory activity was localized to the IgG fraction based upon the fractionations by sodium sulfate precipitation and DEAE chromatography as well as reaction with anti-IgG globulin. The myasthenic globulin inhibited toxin binding to receptors extracted from degenerated muscle but did not inhibit toxin binding to normal junctional receptors. At saturation levels of myasthenic globulin, the number of denervated acetylcholine receptors available for toxin binding was reduced approx. 50%. The myastehnic globulin was found to bind to denervated acetylcholine receptors but not to normal acetylcholine receptors by a radioimmunoassay technique in which myasthenic globulin incubated with 125I-labeled α-bungarotoxin-receptor complexes was precipitated by anti-IgG serum. The globulin binding was saturable over the same range as inhibition of toxin binding. The data suggest that the myasthenic IgC binds to a site on the receptor complex juxtaposed to the acetylcholine receptor site. The myasthenic globulin appears to be a useful probe for investigating differences between acetylcholine receptors extracted from normal and denervated muscle and for investigating the pathogenesis of myasthenia gravis.

Studies in Myasthenia Gravis: Active Antigenic Sites Related to Humoral Antibody Induction

Abstract Purification of human muscle proteins by 0.5 M KCl extraction, differential solubility and centrifugation produced fractions which reacted with human myasthenic sera. Absorption with either actomyosin or myosin removed all activity shown by the indirect immunofluorescent assay. Quantitative complement fixation showed that active fractions included the initial crude preparations, highly purified actomyosin and myosin. Actin preparations had no antigenic activity. Myosin contained at least 50 times more activity than actomyosin in both the complement fixation and the absorption studies. These data suggest that myosin contains the antigenic sites related to humoral antibody induction in myasthenia gravis.

"Trophic" influences of nerve on muscle.

"Trophic" influences of nerve on muscle.