Effects of antibodies to Torpedo acetylcholine receptor on the acetylcholine receptor--ionic channel complex of Torpedo electroplax and rabbit intercostal muscle.

An electrophysiological, biochemical, and ultrastructural study of the acetylcholine (ACh) receptor-ionic channel complex was undertaken in electric organ membranes from the electric ray, Torpedo ocellata, and intercostal muscle of the rabbit. Rabbits immunized with receptor purified from Torpedo ocellata were paralyzed as is characteristic of experimental autoimmune myasthenia gravis. Incubation of Torpedo membranes with antisera from six paralyzed rabbits, but not control rabbits, resulted in inhibition of [³H]ACh and [¹²⁵I]α-BGT binding by 19 to 95% and 43 to 86%, respectively, and did not significantly affect the binding of [³H]H₁₂-HTX. Anti-ACh-receptor antibodies bound to Torpedo microsacs were identified in electron micrographs by specific binding of ferritin-labeled goat anti-rabbit antibodies. Treatment of Torpedo microsacs with ACh-receptor antisera from rabbits inhibited their carbamylcholine-induced ²²Na efflux, which would suggest direct effect of the antibodies on Torpedo ACh-receptor function. However, addition of the same anti-Torpedo-ACh-receptor antisera for 240 min to inter-costal muscles of rabbits produced only a small reduction in the amplitude of the response to microiontophoretically applied ACh, miniature end-plate potentials, and end-plate potentials. It is suggested that the muscle weakness observed in rabbits immunized with Torpedo ACh-receptor protein may be due to time-requiring action on receptors such as their accelerated degradation, and the amount of antimuscle receptor in rabbit antisera against Torpedo receptor may be too small.     

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.     

Effects of cerebellar nuclei stimulation on the electrical activity of the neurons in the centrum medianum nucleus in cats

The responses evoked in the neurons of the centrum medianum nucleus of the thalamus by stimulation of the deep cerebellar nuclei were studied. A marked predominance of activation effects during single shocks and lowfrequency stimulation was revealed. This was most striking with stimulation of the dentate nucleus. Parallel with increases in stimulation repetition rates, there was a noticeable rise both in unit responsiveness and in depressive responses. In general the percentage of unidirectional responses (either activation or depression) was considerably greater than that of responses which changed in direction with increases in stimulus rate. A predominance of tonic post-stimulation effects was also revealed. This was more marked after dentate or fastigial stimulation. In some centrum medianum neurons single shocks to the cerebellar nuclei evoked phasic responses. One type of these responses had a constant latency of 2- to 12-ms duration. A second type showed somewhat longer, although inconstant, latencies (≤20 ms). Single shocks to the dentate nucleus produced two temporal classes of latency (2 to 6 and 13 to 17 ms). In another type of centrum medianum neuron single shocks to the cerebellar nuclei, especially dentate and interpose, elicited depression of spontaneous firing for periods of 50 to 100 ms. A clear synchronization of spontaneous neuronal activity was evoked by low-frequency stimulation as well. Nearly 50% of the responsive neurons could be driven by excitation of the cerebellar nuclei. Convergent effects occurred more often with higher frequencies than with single-shock stimulation.     

Myasthenogenicity of a human acetylcholine receptor alpha-subunit peptide: morphology and immunology.

Each of 10 rats inoculated with a synthetic peptide comprising residues 125-147 (without a disulfide bond) of human acetylcholine receptor (AChR) alpha-subunit (H alpha) had deposits of IgG and C3 (immune complexes) and showed morphological changes in the fine structure at the motor end-plates 5 weeks after a single immunization. Antibody to the H alpha peptides was elevated 1 week after immunization, but, antibody levels to solubilized human or rat AChR were very low in 8 of the 10 rats. These results suggest that the immune response to peptide H alpha is the myasthenogenic site, which induces morphological change at the end-plates.     

Characterization of acetylcholine receptor antibodies in a patient with primary biliary cirrhosis

Antibodies against the acetylcholine receptor were found in a patient with primary biliary cirrhosis. The patient had no clinical or electrophysiological evidence of disturbed neuromuscular function. The antibodies were of both IgG and IgM isotype. Following passive transfer, these antibodies showed the same capacity to bind in vivo to mouse muscle receptors as immunoglobulins from patients with myasthenia gravis. The affinity of the antibodies was high and comparable to that found in myasthenia gravis patients.     

重症肌无力被动转移动物模型的研究

目的 :探讨血清阳性 (SPMG)和阴性重症肌无力 (SNMG)被动转移动物模型 (P EAMG)的异同。方法 :用ELISA法将重症肌无力 (MG)患者分为SNMG和SPMG两组 ,然后分别用两组患者血清制作P EAMG ,观察两组小鼠的临床表现、电生理及神经肌接头(NMJ)的改变。结果 :SPMG和SNMG组小鼠均表现出明显的肌无力症状 ,低频重复电刺激出现明显衰减反应 ,但SNMG组小鼠肌无力症状较SPMG组明显为轻 ,SPMG和SNMG组小鼠NMJ处棕黄色沉积物明显减少、变细短。结论 :SNMG和SPMG均是自身抗体介导的自身免疫性疾病 ,但两者不完全相同     

设计耐受原并用鼻黏膜耐受治疗EAMG的方法学研究

目的　设计特异性耐受原 ,通过其鼻黏膜耐受治疗对实验性自身免疫性重症肌无力 (EAMG)发病过程的影响 ,以探讨该疗法在模型应用中的可行性。方法　用 Lewis大鼠建立 EAMG模型 ,选取双类似物作为耐受原在致敏同时给予鼻黏膜耐受治疗 ,动态观察大鼠体重及临床评分改变。结果　虽然治疗组大鼠发病率不降低 ,但是在 EAMG急性期和慢性期 ,其临床症状均较对照组减轻 (P <0 .0 5 )。结论　用双类似物鼻黏膜耐受可以达到治疗 EAMG的目的 ,其结果为抗原特异性治疗重症肌无力 (MG)和其他自身免疫性疾病 (AID)提供了依据。     

Myasthenia gravis animal model: quantitative analysis of the efficiency of pre- and postjunctional regions of intercostal muscles in rabbits immunized with Torpedo acetylcholine receptor.

Rabbits were immunized with acetylcholine (ACh) receptor from Torpedo ocellata electric organ, and when paralyzed and moribund, an extensive study was undertaken of neuromuscular transmission in the intercostal muscles of these experimental autoimmune myasthenia gravis (EAMG) rabbits. The resting membrane potential was not significantly changed. The end-plate potentials were large enough to trigger an action potential in only 40% of the surface fiber of EAMG rabbits and miniature end-plate potentials (MEPPs) were greatly reduced in magnitude. Muscle fibers in each EAMG rabbit differed in the levels of inhibition of MEPPs and junctional ACh sensitivity. Extrajunctional ACh sensitivity did not significantly differ between control and EAMG rabbits, and inhibition of acetylcholinesterase with diisopropylfluorophosphate increased the MEPP amplitude in each end plate of EAMG rabbits, but not to the value of the control. Average junctional ACh sensitivity was 3094 ± 701 mV/nC in control rabbits and 898 ± 551 mV/nC in EAMG rabbits, which suggested that there were reduced numbers of functional ACh-receptor-ionic channel complexes in the postsynaptic membrane. In end-plates where no MEPPs could be recorded, junctional ACh sensitivity averaged 28% of control, being 13.5% of control in half the fibers and 43% of control in the other half. Because this last ACh sensitivity was as high as that of other fibers where MEPPs could be recorded, yet none were observed, we suggest that in these few fibers there was an additional defect that was other than postsynaptic, more likely due to detachment of entire end-plates from muscle fibers.     

放射免疫法定量检测乙酰胆碱受体抗体与重症肌无力患者临床特征的相关性

目的探讨乙酰胆碱受体抗体(AChR-Ab)与重症肌无力(MG)临床特征的相关性。方法采用放射免疫法检测115例MG患者及92例对照组(非MG神经系统疾病患者42例,健康体检者50名)血清AChRAb浓度,应用临床绝对评分记录MG患者病情严重程度。分析各组血清AChR-Ab浓度的差异,以及AChR-Ab浓度与MG患者临床特征的相关性。采用ROC工作特征曲线探讨AChR-Ab诊断MG的敏感度和特异度。结果MG患者血清AChR-Ab浓度中位数(四分位数间距,下同)为3.45(39.38)nmol/L,较非MG神经系统疾病患者[0(0)nmol/L]和健康体检者[0(0)nmol/L]增高(P0.01)。全身型MG(GMG)患者AChR-Ab浓度[25.45(46.14)nmol/L]较眼肌型MG(OMG)患者[0.58(3.56)nmol/L]增高(P0.01)。用ROC曲线法分析显示,以血清AChR-Ab浓度≥0.50nmol/L作为诊断MG界值时灵敏度为72.17%,特异度为100%,曲线下面积(AUC)=0.895(95%CI:0.849~0.941)。AChR-Ab浓度与发病年龄、病程及改良Osserman分型呈正相关(r=0.220,P0.05;r=0.184,P0.05;r=0.382,P0.01),但相关性较弱(均r0.5),与临床绝对记分无相关性(r=0.147,P0.05)。结论用放射免疫法检测血清AChR-Ab浓度诊断MG的灵敏度和特异度均高,有助于减少MG的漏诊率及误诊率,值得临床推广。     

Correlating extent of neuromuscular instability with acetylcholine receptor antibodies

In a retrospective study of 86 patients with myasthenia gravis (MG), we correlated the acetylcholine receptor (AChR) antibody titers with single‐fiber EMG studies to explore whether a relationship exists between these parameters. We found that the AChR antibody titers correlated significantly with the mean of the mean consecutive difference of orbicularis oculi (OO, P < 0.0001) and extensor digitorum communis (EDC, P < 0.0001). The correlation was found to be stronger in OO. The antibody titers also correlated with the percentage of potential pairs with increased jitter in both muscles and, again, the correlation was more significant in OO (P < 0.0001) than in EDC (P = 0.001). We speculate that this relationship is stronger in OO than in the limb muscles, because the architectural and immunological differences in the motor unit render OO more vulnerable and sensitive to disturbances in neuromuscular transmission. Muscle Nerve, 2009     

抗乙酰胆碱受体单克隆抗体A7诱导实验性自身免疫性重症肌无力作用

目的探讨抗乙酰胆碱受体单克隆抗体A7(抗AChRmAbA7)对实验性自身免疫性重症肌无力(EAMG)的作用。方法经Lewis大鼠腹腔注射5mL20倍浓缩的含抗AChRmAbA7培养上清液,建立EAMG被动转输模型。对照组经腹腔注射同体积的PBS。每8h监测体重和临床症状评分。48h后处死实验动物。采用放射免疫法检测AChR,并计算出AChR损失率。结果抗AChRmAbA7诱导的EAMG模型AChR损失率高达243%~452%,而且AChR损失率与临床症状评分相关(r=074,P<001)。抗AChRmAbA7攻击位点在终板的AChR上。结论抗AChRmAbA7可用于诱导EAMG模型及有关研究。     

与霍乱毒素B亚单位结合的重组人AChR片断经鼻腔治疗实验性自身免疫性重症肌无力

目的 探讨与霍乱毒素B亚单位结合的重组人AChRα亚单位片断(CTB-Hα1-205)治疗实验性自身免疫性重症肌无力(EAMG)的有效性和基本作用机制.方法 将用AChR诱导的Lewis EAMG鼠随机分为3组:CTB-Hα1-205治疗组、Hα1-205组和CTB-HGG对照组,在诱导EAMG后第14天分别按上述3组从鼻腔滴入CTB-Hα1-205、Hα1-205和CTB-HGG.对各组进行临床分数评估、测定鼠血清中抗AChR特异抗体和淋巴细胞增殖反应.结果 CTB-Hα1-205组和Hα1-205组平均临床分数均较对照组有明显减少,差异有统计学意义(均为P＜0.01),同时CTB-Hα1-205组较Hα1-205组也有明显减少(P＜0.05);与对照组比较,CTB-Hα1-205组和Ha1-205组血清中鼠抗AChR特异抗体IgG、IgG2a、IgG2b和IgG2c的产生均明显被抑制(P＜0.01),CTB-Hα1-205治疗组的IgG、IgG2b和IgG2c较Hα1-205组低,且差异有统计学意义(P＜0.05);另一方面,CTB-Hα1-205组和Hα1-205组的IgG1较对照组却有明显升高(P＜0.05),同时,CTB-Hα1-205组和Hα1-205组对AChR特异性抗原的淋巴细胞增殖反应也被明显抑制(P＜0.05).结论 CTB-Hα1-205比Hα1-205能更加有效治疗EAMG,其作用机制是抑制了自身抗体的产生、IgG亚型的转换和特异的淋巴细胞增殖反应.     

预防性双类似物鼻粘膜耐受对EAMG的影响

目的选择双类似物(Lys262-Ala207)通过不同时间点对实验性自身免疫性重症肌无力(EAMG)模型进行鼻粘膜耐受预防性给药,观察其临床及免疫指标变化,并评价疗效,探讨预防性鼻粘膜耐受在EAMG中的预防作用机制。方法应用乙酰胆碱受体(AChR)加CFA致敏Lewis大鼠建立EAMG模型,并在致敏前10 d(预防耐受A组)及致敏当日(预防耐受B组)给予耐受肽Lvs262-Ala207及相应对照组CA、CB采用相同剂量对照肽MBP-p83-99鼻腔给药。检测给药后A、B组及相应对照组大鼠的体重、临床评分、肌电图、肌肉中AChR含量丢失变化及致敏第42 d血清抗AChR抗体IgG含量。结果急性期和慢性期A、B组体重明显超过相应对照组,临床症状明显轻于相应对照组,慢性期A组体重明显超过B组、病情明显轻于B组;A、B组低频重复电刺激出现衰减反应D5阳性率低于相应对照组;A、B组肌肉AChR含量丢失分别明显低于相应对照组,而A组低于B组;慢性期42 d A、B组IgG含量明显低于相应对照组,同时A组明显低于B组。结论本实验表明,预防耐受的疗效与自身免疫启动时间有关,启动前优于启动时耐受;双类似物鼻粘膜耐受预防不仅可有效地抑制临床症状,且可特异性减低致病性循环抗体含量和减少神经肌接头AChR含量丢失,为采用双类似物鼻粘膜耐受防治人类重症肌无力(MG)提供了依据。     

Effect of myasthenic IgG on degradation of junctional acetylcholine receptor

We investigated the effect of the lgG from patients with myasthenia gravis (MG) on the degradation of normal rat junctional acetylcholine receptor (AChR) labeled with ¹²⁵l-α-bungarotoxin (BuTx) and calculated the degradation rate (DR). The DR for the lgG from these patients was significantly higher than that from healthy volunteers and patients with other autoimmune diseases. For MG, DR was significantly correlated with the severity of the disease but not with anti-AChR antibody titer. DR was accelerated by lgG from patients with generalized MG whose antibody titers were in the normal range and by lgG from patients with ocular MG. These results indicate that measurement of the DR of junctional AChR in normal rats is more closely correlated with the severity of the disease than is measurement of anti-AChR antibody and that the former is a sensitive and confirmatory method for evaluating MG. © 1993 John Wiley & Sons, Inc.     

Myasthenia gravis and acetylcholine receptor antibodies: A clinico immunological correlative study on South Indian patients

Acquired myasthenia Gravis (MG), a disorder of impaired neuromuscular transmission is recognized as an autoimmune disorder, with a majority of the patients having antibodies against acetylcholine receptor (AChR antibodies) in the serum.

Aim and Objectives:

To correlate the AChR antibody status with the clinical characteristics of patients with myasthenia gravis.

Study Design:

Retrospective and prospective study.

Materials and Methods:

This study was carried out in patients with definite MG, attending the Neurology services of the National Institute of Mental Health and Neurosciences, Bangalore, India, during the period 1999-2003. The AChR antibody status was determined using the direct and indirect enzyme linked immunosorbent assay (ELISA) technique.

Results:

There were 165 patients in this study (M : F :: 1.5 : 1). The overall seropositivity rate was 59.4%. Seropositive patients had higher age of onset and presentation, and more frequent occurrence of crises, both at presentation and at any time during the course. Other parameters, viz. gender of the patient, Osserman staging, thymic enlargement on CT and remission during follow-up did not differ between the two groups.

Conclusion:

This communication reports the result of AChR antibody assay in a large cohort of patients, using a simple diagnostic tool, namely direct and indirect ELISA technique. In addition, the characteristics of a large cohort of patients with seronegative myasthenia gravis are described.     

Induction of the morphologic changes of both acute and chronic experimental myasthenia by monoclonal antibody directed against acetylcholine receptor

Summary To investigate pathogenic mechanisms in experimental autoimmune myasthenia gravis (EAMG) and myasthenia gravis (MG), we studied the acute and chronic effects in rats of injection of rat monoclonal antibodies (MCABs) directed against the acetylcholine receptor (AChR). Animals were severely weak 12 h after a single injection, at which time macrophages were found invading endplate regions of muscle and cholinesterase-stained regions were separted from the underlying muscle fibers. Ultrastructural studies showed findings identical to the acute phase of EAMG: degenerating postsynaptic membranes and invasion and phagocytosis of endplate regions by macrophages. Animals receiving sublethal doses of MCAB recovered clinically by 4–5 days after injection. Recovery was accompanied by a progressive decrease in the number of macrophages associated with endplates and reapposition to the myofibers of the cholinesterasestained regions. Animals injected once, or repeatedly over several months, remained clinically and electromyographically normal after recovery from the initial episode of weakness, but their endplate ultrastructure was highly simplified with blunted or absent synaptic folds and shallow or absent secondary synaptic clefts. These studies demonstrate that anti-AChR MCABs can induce the changes of both acute and chronic EAMG. There is good correlation between the inflammatory changes and the acute clinical disease but poor correlation between morphological and clinical parameters in the chronic syndrome. The latter observation suggests that severe ultrastructural changes, similar to those seen in chronic EAMG and MG, cannot account, at least in rats, for the clinical and electrophysiologic abnormalities of MG.Supported in part by grants from the Muscular Dystrophy Association and the National Institutes of Health (NS15462, A19268)     

Further electrophysiological studies of neuromuscular transmission in an animal model of myasthenia gravis

The development of experimental autoimmune myasthenia gravis (EAMG) in rats produces a significant reduction in the amplitude of spontaneously occurring miniature end-plate potentials (MEPPs) and impulse-evoked end-plate potentials (EPPs). This junctional abnormality, however, does not impair the ability of the affected fibers to produce propagated muscle action potentials of normal amplitude. Quantal content analysis indicates that in Mg²⁺-blocked EAMG muscle the mean number of acetylcholine (ACh) quanta released per nerve impulse closely approximates the corresponding normal value. At EAMG end-plates, the depolarization produced by either saturating or nonsaturating doses of carbamylcholine was significantly less than that seen at normal end-plates, suggesting a reduced acetylcholine receptor (AChR) content. When the depolarizing response to 250 μm carbamylcholine was examined at various EAMG end-plates with different sizes of MEPPs, there was a direct correlation between the carbamylcholine-induced depolarization and the MEPP amplitude; this correlation, however, was much less pronouced in normal end-plates, which supports the concept that the MEPP size in the receptor-immunized muscle reflects the content of the functional postsynaptic AChR. In control solution, the indirect twitch tension produced in EAMG muscle was normal. The twitch tension in EAMG muscle however, was almost completely abolished by a dose of d-tubocurarine that reduced the tension in normal muscle by only 50%. When 4-aminopyridine, a drug known to increase the quantum content of EPPs, was applied to curarized normal or EAMG muscles, normal muscle contraction was restored and the decremental response to repetitive nerve stimulation was abolished. We conclude that the major defect of neuromuscular transmission in EAMG results from postsynaptic abnormalities at the end-plates, presumably secondary to reduction of the number of functional AChRs. Chronic EAMG in rats is a reasonable model of human myasthenia gravis in which a similar defect of neuromuscular transmission is present.     

A comparison of nerve transection and chronic application of β-bungarotoxin on acetylcholine receptor distribution and other nerve-muscle properties

β-Bungarotoxin (β-BuTX), a snake venom neurotoxin which acts presynaptically to inhibit acetylcholine (ACh) release at the neuromuscular junction, was applied to the rat phrenic nerve-diaphragm muscle preparation to determine its effectiveness to mimic denervation. The distribution of junctional and extrajunctional ACh receptors on the muscle were assayed biochemically by [¹²⁵I]α-bungarotoxin ([¹²⁵I]α-BuTX) binding and electrophysiologically by iontophoretic application of ACh. Spontaneous transmitter release and muscle membrane potential were measured under conditions of denervation, β-BuTX treatment, and bee venom phospholipase A₂ exposure. Within 7 days after treatment with a single dose (5μg/kg) of enzymatically active β-BuTX, extrajunctional [¹²⁵I]α-BuTX binding increased fivefold, and there was a decrease in miniature end-plate potential (MEPP) frequency and in resting membrane potential (RMP) to values less than those of control muscles but greater than those of denervated muscles. The same dose of enzymatically inactive β-BuTX or snake venom phospholipase A₂ was without effect, but a fivefold greater dose of enzymatically inactive β-BuTX resulted in changes in extrajunctional binding and RMP similar to those of muscles exposed to the enzymatically active toxin. However, unlike muscles treated with active toxin, those treated with inactive toxin had MEPP frequencies similar to control muscles and exhibited contraction elicited by phrenic nerve stimulation. These results taken together indicate that the nerve exerts a trophic influence on muscle independent of ACh and muscle activity.