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     

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.     

Anti-alkaline phosphatase antibody positive myasthenia gravis

Anti-alkaline phosphatase antibody (AP Ab) was specific in 9% of 249 anti-acetylcholine receptor (AChR) Ab-positive myasthenia gravis (MG) (SPMG) patients but not in patients with AChR Ab-negative MG (SNMG), other neurological and immunological diseases, or healthy volunteers. No cross-reactivity and no significant titer correlation were found between AP Ab and AChR Ab. We confirmed immunologically by radioimmunoassay and western blot analysis the presence of antibodies directed against AP. AP Ab-positive SPMG patients were characterized clinically as having female predominance and a more severe form of generalized MG than AP Ab-negative SPMG patients, and about half required artificial ventilation at maximum severity. AP Ab's pathogenic role in MG is yet unclarified, but our findings show AP to be a novel antigen among the various autoantigens present in MG patients and in whom AP Ab may modify clinical symptoms.     

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.     

Myasthenia gravis with anti-acetylcholine receptor antibody or anti-muscle specific kinase antibody

Patients with myasthenia gravis (MG) are divided into 3 groups: anti-acetylcholine receptor antibody-positive MG (AChR MG), anti-muscle specific kinase antibody-positive MG (MuSK MG), and AChR-and MuSK-negative MG (double seronegative MG; double SNMG). A recent study on the detection of low-affinity antibodies binding to AChR showed the presence of AChR antibodies in about 70% of double SNMG patients. There is accumulating evidence that double SNMG patients are similar to AChR-MG patients with respect to clinical features and thymic pathology. Since most MG patients are thought to belong to the AChR-MG or MuSK-MG group of patients, this article reviews the pathophysiology, clinical features, and treatments in these 2 groups of MG patients. The pathophysiology of AChR-MG is closely related to the abnormal thymic pathology, such as thymic hyperplasia or presence of thymoma, and thy(mo)mectomy is recommended in patients with generalized AChR-MG. On the contrary, little thymic abnormality in patients with MuSK-MG discourages thymectomy; however, MuSK-MG patients do respond to thymectomy and therefore studies to define the indications of thymectomy in MuSK-MG patients are required. The responses to cholinesterase inhibitors are poor in patients with MuSK-MG, and these patients tend to show hyperactivity to the Tensilon test, such as fasciculation of facial muscles and stuffy sensation in the throat. The adverse responses to a small dose of intravenous edrophonium chloride injection may support the clinical diagnosis of MuSK-MG. Further, only small doses of acetylcholinesterase inhibitors are administered to patients with MuSK-MG in order to avoid cholinergic hyperactivity. In general, both types of MG patients respond well to treatments with immunosuppressants, including steroids, but some patients with MuSK-MG show persistent bulbar symptoms.     

Analysis of immunoglobulin secretion by lymph organs with myasthenia gravis

OBJECTIVES: To investigate in vitro anti-acetylcholine receptor (AChR) antibody secretion by lymph organs with myasthenia gravis (MG) with special attention to clinical status and stages. MATERIALS AND METHODS: Twenty MG patients before therapy were included in this study. Four patients were negative for serum anti-AChR antibodies (AChRAb). All patients received extended thymectomy. Thymic cells, bone marrow (BM) cells and peripheral blood mononuclear cells (PBMC) were cultured for 1 week. AChRAb secretion and IgG secretion in the culture medium were determined by immunoprecipitation assays and ELISA respectively. RESULTS: PBMC secreted AChRAb and IgG most efficiently, followed by BM cells, and then thymic cells. Even in patients whose diseases were of short duration (less than 2 months), the tendency was not changed. AChRAb secretion by BM cells and PBMC significantly correlated with serum AChRAb levels. Thymectomy did not change AChRAb or IgG secretion by PBMC within 3 months. AChRAb secretion by PBMC paralleled the clinical status. Some seronegative patients turned positive by culturing PBMC. CONCLUSION: PBMC is the most efficient site at producing AChRAb, even in patients of short duration (equal to, or less than, 2 months). Monitoring AChRAb secretion by PBMC is useful to estimate the autoimmune activity of MG.     

重症肌无力患者酪氨酸激酶抗体的检测

目的探讨酪氨酸激酶抗体（MuSKAb）在血清阴性重症肌无力（SNMG）发病中的作用。方法采用放射免疫法检测198例重症肌无力（MG）患者血清中抗乙酰胆碱受体抗体（AChRAb）水平，筛选出SNMG血清样本再行MuSKAb水平检测。结果MG患者血清AChRAb浓度明显高于对照组（P〈0．05），其阳性率为81．3％，SNMG患者血清MuSKAb均为阴性。结论MuSKAb可能在中国SNMG患者中的检出率较低。     

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.     

Fewer thymic changes in MuSK antibody-positive than in MuSK antibody-negative MG

In generalized myasthenia gravis (MG) patients without detectable acetylcholine receptor (AChR) antibodies (SNMG), the thymus is often reported as "normally involuted." We analyzed thymic compartments in 67 patients with generalized MG, with AChR antibodies (AChR+, n = 23), with muscle-specific kinase (MuSK) antibodies (MuSK+, n = 14) or with neither (MuSK-, n = 30), and in 11 non-MG controls. Four of 14 MuSK+ thymi had rare small germinal centers, but overall they were not different from age-matched controls. However, approximately 75% MuSK- samples showed lymph node-type infiltrates similar to those in AChR+ patients, but with fewer germinal centers. These variations may explain some apparent differences in responses to thymectomy in SNMG.     

AChR phosphorylation and indirect inhibition of AChR function in seronegative MG

BACKGROUND: Approximately 10% to 20% of patients with autoimmune MG do not have antibodies to the acetylcholine receptor (AChR), so-called seronegative MG (SNMG). IgG antibodies from up to 70% of SNMG patients bind to the muscle-specific receptor tyrosine kinase, MuSK. The plasmas and non-IgG fractions from SNMG patients (and some with AChR antibodies) also contain a factor, perhaps an IgM antibody, that inhibits AChR function, but it is not clear how this factor acts and whether it is related to the MuSK IgG antibodies. METHODS: The authors studied 12 unselected SNMG plasmas and their non-IgG fractions; seven were positive for MuSK IgG antibodies. Ion flux assays, electrophysiology, phosphorylation, and kinase assays were used to look at mechanisms of action. RESULTS: Eight of the 12 plasmas and their non-IgG fractions inhibited AChR function, but the inhibitory activity was transient and did not correlate with the presence of MuSK IgG antibodies. Two of three plasmas added outside of a cell-attached patch pipette inhibited AChR function within the patch, and these two plasmas also increased AChR phosphorylation. CONCLUSIONS: The authors propose that a plasma factor(s) in SNMG patients, distinct from MuSK IgG antibodies, binds to a muscle membrane receptor and activates a second messenger pathway leading to AChR phosphorylation and reduced AChR function. Identifying the target for this factor should lead to improved diagnosis of MG in MuSK antibody-negative patients and may provide new insights into the function of the neuromuscular junction and pathophysiological mechanisms in MG.     

Clinical, electrophysiological and immunological remissions after thymectomy in myasthenia gravis.

OBJECTIVES: Approximately 50% of patients treated with thymectomy have a chance for symptom-free life. However, immunological and neurophysiological abnormalities may be detected in patients with clinical remission. Although improvement usually parallels decrease in acetylcholine receptor antibody (AChRAb) levels and jitter values, there is a question what factors influence immunological and electrophysiological remission in a population of myasthenia gravis (MG) patients. METHODS: We analyzed retrospectively clinical data of 32 MG patients operated for generalized MG, followed-up at our department for 17.2 (4-31) years. They were in clinical remission for 12.8 (2-25) years. All of them had single fiber electromyograhy (SFEMG) of extensor digitorum communis muscle (EDC) muscle and estimation of AChRAb level at the end of follow-up. Their age at onset of MG was 17 years (6-48) and at thymectomy 19 (6.4-58) years. Tensilon test was positive in 30, repetitive nerve stimulation in 29 cases. RESULTS: Clinical remission was reached on average 4.2 years after thymectomy. SFEMG jitter value normalized in 60% of cases. AChRAb were negative only in 34% of patients. Jitter values correlated with AChRAb levels (P=0.006, r=0.5) but were not related to clinical factors. Only time to thymectomy correlated with time from thymectomy to clinical remission (P=0.001, r=0.5). CONCLUSIONS: Clinical remission is not always accompanied by normalization of SFEMG and AChRAb. Although normalization of neuromuscular transmission in patients with remission of MG is individual, short duration of MG before thymectomy increases the chance of early remission.     

Pseudo-myasthenia gravis and thymic hyperplasia in Graves' disease.

BACKGROUND: Diagnostic confusion between thyroid disease and myasthenia gravis (MG) can arise because the two may have similar clinical features, and also because of the more frequent coexistence of these autoimmune disorders in the same individual. In MG, autoantibodies directed against the acetylcholine receptor result in muscle weakness. Thymic pathology is well recognized in MG, with thymic hyperplasia frequent in early onset MG and thymoma more common in later onset MG. In Graves' disease, autoantibodies against thyroid antigens result in hyperthyroidism. A seldom-recognized feature of Grave's disease is the occurrence of an enlarged thymus (thymic hyperplasia) on chest CT, or of thymic lymphoid hyperplasia pathologically. CASE STUDY: This report describes a case in which the discovery of a mediastinal mass during imaging of the thyroid, and the presence of myasthenic-like symptoms, in a patient with Graves' disease prompted investigations into whether the patient also had MG. RESULTS: Despite symptoms which strongly suggested MG, subsequent investigations did not confirm the diagnosis, and treatment of Grave's lead to a resolution of the symptoms and regression of the thymic enlargement seen on CT. CONCLUSIONS: The case study highlighted clinical similarities between Grave's disease and myasthenia gravis which might cause diagnostic confusion, and also the investigations which are useful in order to differentiate the two diseases. In addition to common clinical features, the autoimmune diseases Grave's disease and myasthenia gravis may both produce radiological thymic enlargement.     

Detection and characterization of MuSK antibodies in seronegative myasthenia gravis

Antibodies to rat muscle specific kinase, MuSK, have recently been identified in some generalized "seronegative" myasthenia gravis (SNMG) patients, who are often females with marked bulbar symptoms. Using immunoprecipitation of (125)I-labelled-human MuSK, 27 of 66 (41%) seronegative patients were positive, but 18 ocular SNMG patients, 105 AChR antibody positive MG patients, and 108 controls were negative. The antibodies are of high affinity (Kds around 100 pM) with titers between 1 and 200 nM. They bind to the extracellular Ig-like domains of soluble or native MuSK. Surprisingly they are predominantly in the IgG4 subclass. MuSK-antibody associated MG may be different in etiological and pathological mechanisms.     

Investigation for RAPSN and DOK-7 mutations in a cohort of seronegative myasthenia gravis patients

Introduction: Myasthenia gravis (MG) is an autoimmune disease. Patients without detectable antibodies against the nicotinic acetylcholine receptor or the muscle‐specific tyrosine kinase are referred to as seronegative MG (SNMG). Because late‐onset congenital myasthenic syndromes (CMSs) due to RAPSN or DOK7 mutations may be mistaken for SNMG, we investigated their frequency in a nationwide SNMG cohort. Methods: We performed sequencing of RAPSN and DOK7 in all Norwegian SNMG patients (n = 74) and 37 healthy controls, examining for the N88K and c.1124_1127dupTGCC mutations, respectively. Results: We found 1 patient homozygous for N88K and 2 carriers of the N88K mutation. Sequencing of DOK7 revealed no mutations. Conclusions: This study confirms that rapsn CMS can be mistaken for SNMG. In addition, the frequency of rapsn CMS in our nationwide SNMG cohort was found to be low. SNMG patients with an atypical clinical presentation and pediatric cases should be tested for the N88K mutation before initiation of immunosuppressive drug treatment or thymectomy. Muscle Nerve, 2011     

Increased binding of anti-acetylcholine receptor antibodies to thymic antigen in patients with myasthenia gravis and other autoimmune diseases, as compared to those with myasthenia gravis alone

We compared the binding activity against acetylcholine receptors solubilized from human muscle (AChRM) and human thymus (AChRT), of sera from patients with myasthenia gravis (MG) alone, to those of sera from patients with myasthenia gravis and associated autoimmune diseases (MG AD). The sera of the MG AD group bound relatively better to thymic antigen (86% vs. 62%). This group was found to contain a higher proportion of women over 40 years of age (more than 50% of the group). The expression of a particular AChR antigen in normal human thymus may be one of the factors involved in the pathogenesis of MG, especially when this disease is associated with other autoimmune disorders.     

MG患者AChRAb对培养大鼠神经元胞浆钙的影响

目的　本研究从离体细胞水平探讨重症肌无力 (MG)患者 CNS受损的可能机制。方法　从 MG患者和正常人血中提取 Ig G。体外培养新生 SD大鼠皮层神经元 ,用免疫组化法观察 ACh RAb与神经元 n ACh R免疫结合反应 ,并行神经元胞浆钙测定。结果　 MG患者 Ig G可与离体培养的皮层神经元免疫结合并引起异常胞浆钙变化 ,而正常人血中提取的 Ig G则否。结论　 MG患者外周 Ig G(ACh RAb)可与离体培养的神经元 n ACh R结合并引起神经元生理活性变化 ,是 MG患者 CNS受损的可能机制。     

重症肌无力患者血清补体和乙酰胆碱受体抗体的浓度变化及其相关性

目的探讨重症肌无力(MG)患者血清补体和乙酰胆碱受体抗体(AChRAb)的浓度变化及其相关性。方法对22例MG患者进行糖皮质激素规范化治疗,在治疗前、后检测其血清补体、AChRAb的浓度,并与14例非MG对照组(NC组)作对照,分析两组血清补体和AChRAb变化之间的关系。结果MG组在糖皮质激素治疗前补体C3浓度明显低于NC组(P<0.01),治疗后MG组补体C3明显增高(P<0.05),但仍低于NC组;AChRAb浓度在治疗前MG组明显高于NC组(P<0.01),治疗后MG组AChRAb浓度明显降低(P<0.05),但仍高于NC组。C5浓度MG组和NC组差异无显著性(P>0.05)。但补体(C3、C5)浓度和AChRAb浓度在治疗前(r=0.326、r=0.102,均P>0.05)和治疗后(r=0.114、r=-0.0146,均P>0.05),以及二者治疗前后变化值之间(r=-0.346、r=0.058,均P>0.05)相关性不显著。结论糖皮质激素治疗后MG患者C3浓度增加、AChRAb浓度下降,二者之间无明显相关性,补体和AChRAb在MG发病过程中可能起协同作用。     

枸橼酸钠使ELISA的检测值升高

目的血浆交换（ＰＰ）前血乙酰胆碱受体抗体（ＡＣｈＲＡｂ）滴度高,重症肌无力（ＭＧ）病情重;交换后病情减轻,但血中此抗体反而更高。抗体测定中,交换前用的是血清,交换后用血浆。本研究旨在探讨抗凝剂枸橼酸钠对用ＥＬＩＳＡ法测定血中ＡＣｈＲＡｂ滴度是否有干扰作用。方法对４５例未经ＰＰ的ＭＧ患者血清、血浆和加枸橼酸钠血清中的ＡＣｈＲＡｂ滴度用ＥＬＩＳＡ进行检测、比较,并对３例ＰＰＭＧ患者在ＰＰ前后及不同时间采血进行检测并比较。结果血浆及加枸橼酸钠血清中ＡＣｈＲＡｂ滴度均显著高于血清中者（Ｐ＜０．０１）,且ＰＰ前后及不同采血时间血清中的ＡＣｈＲＡｂ滴度有所改变。结论枸橼酸钠可使ＥＬＩＳＡ的背景着色加深,有正向干扰作用。建议凡ＥＬＩＳＡ检测均应尽量用血清而非血浆。