Mechanism of block of nicotinic acetylcholine receptor channels by purified IgG from seropositive patients with myasthenia gravis

OBJECTIVE: To clarify the mechanism of block of nicotinic receptor channels by myasthenic antibodies. BACKGROUND: Nicotinic acetylcholine receptor (nAChR) channel currents are functionally blocked by purified immunoglobulin G (IgG) of patients with myasthenia gravis (MG). METHODS: The molecular mechanism of block of IgG fractions containing antibodies to nAChR channels was tested with the patch-clamp technique in combination with a system for ultrafast solution exchange. For the experiments, outside-out patches from cultured mouse myotubes that express embryonic-type nAChR channels at their surface were used. RESULTS: Incubation of outside-out patches with purified IgG from four myasthenic patients blocked nAChR channel currents activated by the application of 1.0 mM ACh reversibly. The peak current amplitude and the time course of block of nAChR channels decreased with increasing concentrations of IgG. The block became at least partly irreversible if incubation time of outside-out patches exceeded 2 minutes. For the block of nAChR channel currents with a-bungarotoxin, a similar mechanism of block was found. CONCLUSIONS: The reversibility of functional block of nAChR channel currents by myasthenic IgG depended strongly on the incubation time of the receptors with antibodies. Interaction of myasthenic antibodies with nicotinic receptors may proceed in several stages from a low-affinity reversible to a high-affinity irreversible binding.     

Modulation of acetylcholine receptor expression in seronegative myasthenia gravis

In a previous study, we demonstrated a compensatory mechanism for regulating acetylcholine receptor (AChR) gene expression in muscle biopsies from seropositive and seronegative (SN) myasthenia gravis (MG) patients. To further characterize the AChR regulation mechanisms involved in SNMG disease, we investigated the effects of MG sera on nicotinic AChR expression (at the protein and messenger RNA [mRNA] levels) in cultured human muscle cells. Sera from SNMG patients induced an in vitro increase in the level of nicotinic AChR beta-subunit mRNA but did not cause a decrease in AChR protein level. This apparent discrepancy was not due to a higher level of AChR synthesis as demonstrated by analysis of AChR turnover. In SN patients, the increase in beta-subunit mRNA level was followed after 48 hours by a slight increase in the amount of AChR surface protein. This regulation of nicotinic receptor expression was due to the purified IgG-containing fraction. Thus, sera from SNMG patients contain an immunoglobulin that induces an increase in AChR mRNA without causing a decrease in AChR protein level, suggesting an indirect regulatory mechanism involving another surface molecule. This model is therefore useful for defining the targets involved in the pathogenesis of SNMG disease.     

Antibody-secreting cells to acetylcholine receptor and to presynaptic membrane receptor in seronegative myasthenia gravis

Peripheral blood and bone marrow from seronegative and seropositive myasthenics were evaluated for antibody-secreting cells (ASC). Cells secreting antibody to acetylcholine receptor (AchR) and to presynaptic membrane receptor (prsmR) were counted using an immunospot assay. Immunoglobulin G (IgG) anti-AchR ASC were present in peripheral blood lymphocytes (PBL) from nine of 13 seronegative and nine of 12 seropositive myasthenics and in bone marrow lymphocytes (BML) from nine of 13 seronegative and eight of 12 seropositive myasthenics. The mean number of IgG anti-AchR ASC was lower for seronegative than for seropositive patients (P < 0.01 for PBL and P < 0.0001 for BML). In seropositive patients the mean number of IgG anti-AchR ASC was higher for BML than for PBL (P < 0.01); in seronegative patients it was not. IgG anti-prsmR ASC were detected in PBL from four of eight seronegative and six of eight seropositive myasthenics and in BML from three of eight seronegative and five of eight seropositive patients. The mean number of IgG-anti-prsmR ASC did not differ between seronegative and seropositive patients for PBL but for BML the value was higher for seropositive than for seronegative patients (P < 0.01). We conclude that seronegative myasthenia gravis is an autoimmune disease and that ASC to AchR and to prsmR are present both in the blood and the bone marrow in seronegative patients as in seropositive ones. A major difference between the groups lies in the significantly greater number of ASC found in the bone marrow in the seropositive cohort.     

Inhibition of acetylcholine receptor function by seronegative myasthenia gravis non-IgG factor correlates with desensitisation

15% of myasthenia gravis (MG) patients do not have antibodies against the acetylcholine receptor (AChR). Some of these "seronegative" MG patients have antibodies against muscle specific kinase (MuSK), and many have a non-IgG factor that acutely inhibits AChR function in a muscle-like cell line, CN21. Here we show, using mainly one plasma negative for both AChR and MuSK antibodies, that the inhibitory effect of the non-IgG fraction correlates well with the desensitisation caused by 100 microM nicotine, and is found also when AChRs are expressed in a non-muscle cell line (HEK). Moreover, a similar effect was seen with M3C7-a monoclonal antibody against human AChR. The results suggest that, rather than acting indirectly as previously proposed, the SNMG factor may bind directly to an allosteric site that induces or enhances AChR desensitisation.     

Central acetylcholine receptor function in patients with myasthenia gravis

There are some reports on central nervous system involvements in patients with myasthenia gravis, such as abnormal EEG, and memory disturbance. Myasthenia gravis is considered to be an autoimmune disease with antibodies against the skeletal nicotinic acetylcholine receptor (n-AChR). ACh is a neurotransmitter in osmoregulation. Neuronal n-AChR plays an important role in this regulation. In order to investigate the function of neuronal n-AChR in patients with myasthenia gravis, we performed a 5% hypertonic saline infusion test on 9 patients and 9 healthy volunteers. We also carried out an orthostatic stress test (50 degree passive head-up tilt) on 6 patients with myasthenia gravis and 5 healthy controls to evaluate arginine-vasopressin (AVP) release via baroreceptors. Three of the 9 MG patients showed exaggerated plasma AVP secretion, and one revealed a blunt response to hypertonic stimulation. Both patients and controls did not differ significantly in terms of plasma AVP response to orthostatic stress. To conclude, we suggest the possibility that function of neuronal n-AChR in the central nervous system is impaired in patients with myasthenia gravis.     

IgG from "seronegative" myasthenia gravis patients binds to a muscle cell line, TE671, but not to human acetylcholine receptor

Antibodies to acetylcholine receptor (AChR) are found in 85% of patients with myasthenia gravis (seropositive MG [SPMG]) and are thought to be pathogenic; but in 15% of MG patients, the standard immunoprecipitation test for anti-AChR is negative (seronegative MG [SNMG]). Here, we used a novel approach, fluorescence-activated cell sorting analysis, to measure binding of SPMG and SNMG IgG antibodies to rhabdomyosarcoma cell lines that express human adult (TE671-epsilon) or fetal (TE671-gamma) AChR, and to human embryonic kidney (HEK) fibroblasts that express adult human AChR (HEK-AChR). We found that whereas most SPMG antibodies bind to all three cell lines, IgG from 8 of 15 SNMG sera/plasmas bind to the surface of both TE671 cell lines but not to HEK-AChR cells. These results indicate that SNMG antibodies bind to a muscle surface antigen that is not the AChR, which strongly supports previous studies that suggest that SNMG should be considered a distinct subtype of MG.     

HLA and acetylcholine receptor antibody in patients with myasthenia gravis]

Human leucocyte antigen (HLA) and acetylcholine receptor antibody (AchRab) titer in the serum assayed were in 106 and 100 patients with myasthenia gravis (MG), respectively. 93 of the total patients had both HLA and AchRab assays. There is a strong association between HLA Bw46, Cx46 antigens and the patients with MG. The AchRab level in the patients with positive Bw46, Cx46 antigen is significantly lower than that of the patients with negative Bw46 Cx46 antigen. The geometric means (G) of AchRab were 2.99 and 4.74, respectively, P less than 0.05. From our study, We think the patients with MG could be divided into two groups, according to the clinical presentation, the AchRab level and the association with HLA Bw46, Cx46 antigens. The first group is that who present ocular muscular myasthenia, childhood or adolescence onset, lower level of AchRab titer and strong association with HLA Bw46 and Cx46. The second one is that who have general muscular myasthenia, adult onset, higher level of AchRab titer and no association with Bw46 and Cx46.     

The effectiveness of thymectomy on seronegative generalized myasthenia gravis: comparing with seropositive cases

OBJECTIVES: To investigate the efficacy of thymectomy between patients with seronegative myasthenia gravis (SNMG) and seropositive myasthenia gravis (SPMG). METHODS: We present here the first Taiwanese retrospective paired cohort study comparing the effectiveness of thymectomy among 16 seronegative and 32 seropositive MG patients after matching for age-of-onset and time-to-thymectomy, and following up over a mean of 35 +/- 20 (7-86) months. Clinical characteristics and complete stable remission (CSR) rates were compared and analyzed between the groups. RESULTS: There were no major clinical differences between the two groups except for our finding of a lower percentage of SNMG receiving preoperative plasmapheresis or human immunoglobulin than SPMG (31% for SNMG vs 72% for SPMG, P = 0.007). CSR rates calculated using the Kaplan-Meier method were similar in the two groups (38% for SNMG vs 50% for SPMG, P = 0.709). The median time for CSR was 47.4 months for SNMG and 48.2 months for SPMG. Thymic hyperplasia were the most common pathology (69% for SNMG vs 88% for SPMG, P = 0.24). During the follow-up period, we found no group difference on prednisolone or pyridostigmine dosages. Significant postoperative dosage reductions on pyridostigmine, but not on prednisolone, were found in both groups. CONCLUSIONS: Thymectomy has a comparable response among SNMG and SPMG in our study. Thymic hyperplasia is prevalent in our SNMG patients and thymectomy may also be a therapeutic option to increase the probability of remission or improvement in SNMG. More prospective controlled trial will be helpful in the future.     

Cellular response to human acetylcholine receptor in patients with myasthenia gravis

A preparation of human skeletal muscle acetylcholine receptor (AchR) was used in vitro as an antigen to stimulate lymphocytes from patients with myasthenia gravis (MG). Clinical data obtained from the patients included duration and severity of disease; history of steroid treatment or prior thymectomy; and the presence of thymoma. Lymphocytes from patients with MG showed a significantly higher response to human AchR antigen than did lymphocytes from control subjects. Previous studies of cellular response to AchR have used receptor prepared from eel or ray electric organs. By stimulating lymphocytes from MG patients with a preparation of human AchR, we have come one step closer to documenting a possible contribution of a cellular immune response to the pathogenesis of MG.     

Possible implications of dysregulated nicotinic acetylcholine receptor diffusion and nanocluster formation in myasthenia gravis

Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles. The classical form of this autoimmune disease is characterized by the presence of antibodies against the most abundant protein in the neuromuscular junction, the nicotinic acetylcholine receptor. Other variants of the disease involve autoimmune attack of non-receptor scaffolding proteins or enzymes essential for building or maintaining the integrity of this peripheral synapse. This review summarizes the participation of the above proteins in building the neuromuscular junction and the destruction of this cholinergic synapse by autoimmune aggression in myasthenia gravis. The review also covers the application of a powerful biophysical technique, superresolution optical microscopy, to image the nicotinic receptor in live cells and follow its motional dynamics. The hypothesis is entertained that anomalous nanocluster formation by antibody crosslinking may lead to accelerated endocytic internalization and elevated turnover of the receptor, as observed in myasthenia gravis.     

Antibodies in sera from patients with myasthenia gravis do not bind to nicotinic acetylcholine receptors from human brain

Nicotinic acetylcholine receptors (AChRs) from brains of chickens and rats have recently been purified and characterized (Whiting and Lindstrom, Biochemistry, 25 (1986) 2082-2093; J. Neurosci., 6 (1986) 3061-3069; Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 595-599). Using both antisera and monoclonal antibodies prepared to AChRs from rat brain, we have demonstrated the existence of a homologous AChR in human brain. Here we report that antibodies to muscle AChRs in the sera of patients with myasthenia gravis (MG) do not bind to AChRs from human brain. Similarly, there was no binding of sera from patients with Guillain-Barré, amyotrophic lateral sclerosis, multiple sclerosis, or Lambert-Eaton myasthenic syndrome. Additionally, no binding of any of these sera to the alpha-bungarotoxin (alpha-Bgt) binding protein from human brain could be detected. This data is consistent with other data using antibodies to AChRs from muscle and nerve in demonstrating that the AChR in brain is antigenically distinct from the AChR in skeletal muscle AChR, and, together with the lack of central neurological symptoms in MG, suggests that the low concentrations of anti-AChR antibodies in the cerebrospinal fluid of MG patients do not bind to AChRs in brain.     

Block of recombinant nicotinic receptor channels by IgG from myasthenic patients

In myasthenia gravis (MG), specific antibodies directed against nicotinic acetylcholine receptor (nAChR) channels are produced. Recently, a distinct blockade of native embryonic-type nAChR channels by purified IgG from MG patients has been demonstrated. In the present study, we investigated the interaction of myasthenic IgG with recombinant embryonic- or adult-type nAChR channels expressed on human embryonic kidney (HEK) 293 cells. For the experiments, the patch clamp technique was applied in combination with an ultra-fast application system for agonists. Repetitive 20-ms pulses of 1 mM acetylcholine (ACh) were applied with or without purified IgG added to the perfusion system. The purified IgG from MG patients blocked currents through both, embryonic- and adult-type nAChR channels to a similar extent. A block by myasthenic IgG of adult-type nAChR channels, which are present exclusively at the postsynaptic membrane of the neuromuscular synapse, was shown to occur. The reversible pharmacological blockade of adult-type nAChR channels by myasthenic IgG may be one factor responsible for rapid fluctuations of weakness or rapid recovery of muscle strength after plasmapheresis in MG.     

Enzyme-linked immunosorbent assay for antibody against the nicotinic acetylcholine receptor in human myasthenia gravis

Antibody against acetylcholine receptor (AChR) of human skeletal muscle was measured using enzyme-linked immunosorbent assay and found in 23 (74%) of 31 Japanese patients with generalized myasthenia gravis. In 15 patients with generalized myasthenia gravis who had not undergone thymectomy and who were not receiving adrenocorticosteroids, the antibody was found in 13 (87%). Antibody was also found in 13 (54%) of 24 patients with myasthenia gravis against AChR fractions obtained from fetal calf thymus. Based on the subunit structures of the AChR protein, the double precipitation assay using iodine 125-alpha-bungarotoxin is also capable of detecting antibody against the toxin binding site, by cross reactivity. This is among the first reports of experiments in which enzyme-linked immunosorbent assay was used to measure the antibodies in human myasthenia gravis and provides evidence of anti-AChR antibody against antigens from fetal calf thymus.     

Penicillamine-induced myasthenia gravis associated with antibodies to acetylcholine receptor

A woman with rheumatoid arthritis was treated with penicillamine and developed myasthenia gravis. This drug-induced disease was associated with characteristic autoantibodies to acetycholine receptor. After discontinuing the drug, her symptoms improved and the antibody titers fell. Penicillamine is now being used much more frequently in the treatment of rheumatoid arthritis and it is likely that this complication will become more prevalent.     

Effect of sera from seronegative myasthenia gravis patients on neuromuscular junctions

About 85 % of patients with generalized myasthenia gravis (MG) have anti-nicotinic acetylcholine receptor (nAChR) antibodies in their sera (seropositive MG; SPMG). The other 15 % (seronegative MG; SNMG) are also considered to have antibody-mediated disease, but the nature of the antibodies in SNMG is not fully understood. We investigated the effect of sera from patients with MG on spontaneous muscle action potentials and acetylcholine (ACh)-induced potentials, and we examined the localization of epitopes recognized by SPMG sera or SNMG sera. SPMG sera and SNMG sera inhibited spontaneous muscle action potentials and ACh-induced potentials in the spinal-muscle co-culture system. However, spontaneous muscle action potentials and ACh-induced potentials in the neuromuscular junctions were strongly blocked by SPMG serum, whereas they were weakly blocked by SNMG serum. Both types of sera reacted strongly with the neuromuscular junctions in normal rat muscles, as shown by double immunostaining with serum and α-bungarotoxin. The SPMG epitope remained in the neuromuscular junctions, whereas that of SNMG disappeared after denervation of the sciatic nerve. Therefore, we suggest that the skeletal muscle weakness in SNMG may be due to an interaction with presynaptic neuromuscular transmission and nAChR.     

重症肌无力病人乙酰胆碱受体抗体的测定及临床意义

用ＥＬＩＳＡ（固相酶联免疫吸附）法测定１７２例ＭＧ病人血清乙酰胆碱受体抗体（ＡｃｈＲａｂ），结果显著高于健康献血员组和非ＭＧ病人组。不同性别、病程及临床类型与ＡｃｈＲａｂ无相关性，但４１～５０岁组的显著高于其他年龄组。６７例类固醇激素治疗组、２２例大剂量两种球蛋白治疗组、１２例胸腺切除术组及３例ＭＧ危象病人２４次血浆交换疗法（ＰＥ）组，治疗后伴随肌无力症状的好转，ＡｃｈＲａｂ均显著低于治疗前。结果表明：ＡｃｈＲａｂ测定为ＭＧ诊断提供了可靠的实验依据，为类固醇激素、大剂量丙种球蛋白、胸腺切除术和ＰＥ等治疗ＭＧ提供理论依据和疗效评定的实验指标，进一步证实了ＭＧ免疫学发病机理。     

Antibodies to synthetic peptide (125-148) of the alpha-subunit of human nicotinic acetylcholine receptor in sera from patients with myasthenia gravis

We measured the amount of antibodies to a synthetic peptide that corresponds to the alpha-subunit residues Lys125-Thr148 of human acetylcholine receptor (AChR) in myasthenic sera. We detected anti-peptide antibodies in 52% (89/171) of the patients with myasthenia gravis (MG), but none in any of the healthy controls. Anti-peptide antibodies should provide a valuable immunologic parameter for the clinical evaluation of MG, but no apparent correlation was observed between the titers of anti-peptide and anti-AChR antibodies.