Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.     

重症肌无力小鼠模型的建立

目的建立重症肌无力小鼠模型。方法电鳗乙酰胆碱受体(TnAChR)和完全弗氏佐剂(CFA)混合物免疫C57BL/6J小鼠,经二次加强免疫后,检测肌力、腓肠肌肌电图、膈肌终板电镜、血清抗体水平等指标。结果与对照小鼠相比,发病小鼠表现出肌力减弱的症状,肌电图显示小鼠腓肠肌复合动作电位振幅幅度显著下降,电镜证实神经肌接头处突触后膜变平、皱褶减少、空泡样变性,发病小鼠血清抗体水平明显升高。结论成功建立了重症肌无力小鼠模型,有助于探讨其发病机理及探索治疗自身免疫病新的途径和方法。     

甲基强的松龙联合中剂量环磷酰胺治疗重症肌无力危象的临床效果

目的:探究甲基强的松龙联合中剂量环磷酰胺(CTX)治疗重症肌无力危象(MGC)的临床效果及安全性。方法:选取2012年1月~2015年6月在我院神经内科就诊的MGC患者90例,随机分为对照组(45例)和观察组(45例),对照组患者接受甲基强的松龙治疗,观察组采用甲基强的松龙联合中剂量CTX治疗。比较两组患者的临床疗效及不良反应的发生情况。结果:观察组患者呼吸困难消失时间、吞咽功能恢复时间和肢体无力症状改善时间均明显短于对照组(P0.05);观察组用药7 d内治疗有效率86.7%明显高于对照组(P0.05),肺部感染发生率20%,较对照组显著降低,差异均具有统计学意义(P0.05)。结论:甲基强的松龙联合中剂量CTX可有效提高MGC的治疗效果,促进患者早日康复,且肺部感染的发生率降低。     

利用黑斑双鳍电鳐构建实验性自身免疫性重症肌无力小鼠模型

目的建立符合国际化的临床前实验标准的实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis, EAMG)动物模型。方法参照文献报道的方法并改进后,从电鳐电器官提取乙酰胆碱受体(acetylcholine receptor,AchR)蛋白纯品,并采用SDS凝胶电泳蛋白定性鉴定及BCA法蛋白定量;用纯化的蛋白主动免疫C57BL/6小鼠,共免疫3次(分别于第1天、第30天、第60天),进行EAMG小鼠临床评分、体重、血清AchR抗体含量、新斯的明试验、肌电图等综合评价。结果 EAMG模型组与佐剂组比较,自第三周开始发病,平均临床评分显著上升(P<0.01);发病小鼠体重显著减轻(P<0.01);新斯的明试验阳性;血清AchR抗体含量明显增加(P<0.01);肌电图重复电刺激实验阳性。结论从黑斑双鳍电鳐的电器官提取、纯化AchR蛋白成功诱导C57BL/6 EAMG小鼠模型,为进一步研究重症肌无力创造了良好条件。     

激素联合丙种球蛋白治疗小儿重症肌无力的疗效及对患儿免疫球蛋白和补体的影响

目的：探讨激素联合丙种球蛋白治疗小儿重症肌无力的临床疗效及对患儿免疫球蛋白和补体的影响。方法：回顾性分析在我院治疗的70例重症肌无力患儿的临床资料,采用随机序号的方式将其分为观察组和对照组各35例,观察组给予甲泼尼龙联合丙种球蛋白,对照组仅给予甲泼尼龙,观察两组的临床疗效及免疫球蛋白和补体变化情况。结果：观察组总有效率为94．3％明显优于对照组74．3％,两组比较有统计学意义（P〈0．05）;观察组症状明显缓解时间（6．55±1．35）d以及总住院天数（17．15±3．65）d较对照组明显缩短,两组比较差异均有统计学意义（P〈0．05）。结论：采用激素联合丙种球蛋白治疗小儿重症肌无力,可以明显改善患者肌无力症状,获得较为满意的临床疗效,值得进一步推广使用。     

参苓白术辅助治疗对老年溃疡性结肠炎伴脓血便患者临床疗效及免疫功能的影响

目的:探讨参苓白术辅助治疗对老年溃疡性结肠炎伴脓血便患者临床疗效及免疫功能的影响。方法:选取于我院进行治疗的老年溃疡性结肠炎伴脓血便患者50例,随机分为实验组与对照组,每组25例。对照组患者给予柳氮磺吡啶结肠溶胶囊进行治疗,实验组患者在对照组的基础上联合使用参苓白术丸进行治疗,两组患者均治疗3个月,比较两组患者治疗前后血清白介素-2(IL-2)、γ-干扰素(IFN-γ)及白介素-10(IL-10)水平,并评价两组患者的临床疗效。结果:与治疗前相比,两组患者治疗后的血清IL-2、IFN-γ水平均显著降低,IL-10明显升高;与对照组相比,实验组患者血清IL-2、IFN-γ水平较低,IL-10水平更高(P0.05);且与对照组相比,实验组患者的临床总有效率较高(P0.05)。结论:参苓白术散能有效改善老年溃疡性结肠炎伴脓血便患者的免疫功能并提高其临床疗效。     

全反式维甲酸对乙酰胆碱受体特异性淋巴细胞的免疫调控作用

目的:观察全反式维甲酸(ATRA)对乙酰胆碱受体(AChR)特异性淋巴细胞的体外调控作用,探讨其治疗重症肌无力(MG)的可能机制。方法:建立完全弗氏佐剂(CFA)对照组及实验性自身免疫性重症肌无力(EAMG)组大鼠,并获取淋巴结单个细胞悬液,以ACh R97-116多肽片段以及不同浓度的ATRA体外培养72 h,采用流式细胞仪法、CCK-8法、ELISA法分别检测活细胞比例、细胞凋亡和周期的改变以及Th亚群的格局和B细胞抗体分泌能力的变化。结果:ATRA显著降低活细胞比例(P0.001);不同浓度的ATRA均促进了特异性细胞群的凋亡(P0.001),且呈剂量依赖性,而ATRA未改变AChR特异性淋巴细胞的生长周期;ATRA处理后,CFA和EAMG组的淋巴细胞增殖均受到明显抑制,且ATRA对ACh R特异性的淋巴细胞的抑制明显(EAMG组,P0.01)于CFA组(P0.05);ATRA干预后,ACh R特异性CD4+T淋巴细胞的比例下降(P0.01),且ATRA促进了Th2、Treg细胞亚群百分比(P_(IL-4)0.001,P_(Foxp3)0.001),而抑制了促炎性的Th17、Th1细胞亚群百分比(P_(IL-17)0.05,P_(IFN-γ)0.001);ATRA能够降低ACh R特异性B细胞的抗体分泌能力(P0.01)。结论:ATRA不仅能抑制ACh R特异性T细胞功能,同时也能抑制ACh R特异性B细胞功能,其在MG的临床治疗中可能起治疗作用。     

AMP-Deaminase from Thymus of Patients with Myasthenia Gravis

Myasthenia gravis (MG) is characterized clinically by skeletal muscle fatigue following the excessive exercise. Interestingly most of MG patients manifest parallely also some abnormalities of the thymus.AMP-deaminase (AMPD) from human thymus was not a subject of studies up to now. In this paper, mRNA expression and some physico-chemical and immunological properties of AMPD purified from the thymus of MG patients were described. Experiments performed identified the liver isozyme (AMPD2) as the main isoform of AMPD expressed in this organ. The activity of AMPD found in this organ was higher than in other human non-(skeletal) muscle tissues indicating on role the enzyme may play in supplying of guanylates required for the intensive multiplication of thymocytes.     

肝豆状核变性类似杂合丢失现象的报告及可能机制探讨

对39个家系45个病人及60例正常人的ATP7B基因的几个外显子采用8～10%的非变性丙稀酰胺胶进行SSCP分析, 并对异常者测序（放射自显影）, 发现一个家系的8号外显子上同时存在两个突变(C2250G和G2273T),患者属纯合子,其父为杂合子,母亲和妹妹为正常, 类似“杂合丢失现象”。提示在除了肿瘤之外的体细胞遗传病中,二次突变理论也可能是突变发生的机制之一。 Abstract：　 Screen for mutation in many exons with 45 Wilson disease patients in 39 Chinese families by SSCP and nucleotide sequence analysis by autoradiograph. There are two mutations in exon 8 of a patients family: C2250G and G2273T. Found in these two mutation points, the patients father is a heterozygote, patients mother and sister are normal sequences, and patient is a homozygous. It just like a loss of heterogyzosity in this family with Wilson disease. The patient and her parent sibship were confirmed by taternity test with microsatellite vWF SE33 AR and D9S112. The result suggested that Loss of heterozygosity (LOH) is probable mutate mechanism of hereditary disease besides tumor and cancer.     

Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.