血清抗幽门螺旋杆菌抗体和抗AQP4 抗体在中枢神经系统脱髓鞘病中的相关性研究

目的：探讨血清抗幽门螺旋杆菌抗体（HP-IgG）和抗AQP4抗体在多发性硬化（MS）、视神经脊髓炎（NMO）中的相关性。方法：对33例MS患者、7例NMO患者和35例健康体检者采用间接酶联免疫吸附法（ELISA法）检测血清中抗HP的IgG抗体,采用细胞间接免疫荧光法（CBA）检测血清标本抗AQP4抗体;分析MS、NMO患者中HP-IgG及抗AQP4抗体的阳性率,并对比抗AQP4抗体阳性与抗AQP4抗体阴性患者间HP-IgG阳性率的差别。结果：MS组、NMO组和正常对照组血清中抗血清HP-IgG抗体阳性率分别为69.70%、85.71%、42.86%,差别有统计学意义（P＜0.05）,其中MS组、NMO组与正常对照组血清中抗HP-IgG抗体阳性率差别均有统计学意义（P＜0.05）;但MS组与NMO组血清中抗HP-IgG抗体阳性率差别无统计学意义（P＞0.05）。MS组、NMO组和正常对照组血清中抗AQP4抗体阳性率分别为4.2%、85.71%、0%,差别有统计学意义（P＜0.05）。MS组和NMO患者组中抗AQP4抗体阳性患者与抗AQP4抗体阴性患者抗HP-IgG抗体阳性率分别为72.73%、79.31%,差别无统计学意义（P＞0.05）。结论：HP感染是引起MS及NMO疾病发生的危险因素,而与MS及NMO患者是否含有抗AQP4抗体无关。     

血清水通道蛋白4抗体测定对视神经脊髓炎的诊断及预后判断价值

 目的:探讨血清水通道蛋白4（AQP4）抗体检测对视神经脊髓炎（NMO）诊断及预后判断的临床价值。 方法:  收集2010年1月至2013年12月在温州医科大学附属第一医院神经内科住院或门诊就诊的48例NMO、33例脊髓炎（TM）［包括26例长节段脊髓炎(LETM)和7例复发性脊髓炎(rLETM)］、30例视神经炎（ON） ［包括25例双眼视神经炎（BON）和5例复发性视神经炎（RION）］、52例多发性硬化（MS）及16例其它神经系统疾病（OND）的患者血清及相关临床资料。采用细胞免疫荧光法检测血清AQP4抗体。分析各组患者血清AQP4抗体的阳性率及抗体滴度,比较AQP4抗体阳性NMO患者与AQP4抗体阴性患者的临床特征。通过绘制受试者工作特征曲线（ROC曲线）,评价AQP4对NMO诊断的敏感性和特异性。应用Kaplan-Meier曲线分析血清AQP4抗体阳性患者进展为NMO的累计概率。 结果: NMO组AQP4抗体阳性率最高（87.50％）,其后依次为rLETM组（85.71%）、RION组（80.00%）、LETM组（30.70%）、 BON组（8.00%）和MS组（3.85%）, OND组抗体检测为阴性。NMO组中血清AQP4抗体阳性患者在性别构成比、确诊为NMO时间及伴有严重神经炎的比例等方面,与抗体阴性患者相比差异有统计学意义（P<0.05）。ROC曲线提示血清AQP4抗体对诊断NMO的敏感性为87.5%,特异性为85.4%。Kaplan-Meier曲线提示AQP4抗体阳性患者进展为NMO的累积概率明显高于抗体阴性的患者（P<0.05）。 结论: 血清AQP4抗体检测对诊断NMO具有较高的敏感性及特异性,且有助于NMO预后和转归的判断。     

模拟抗原ELISA检测视神经脊髓炎患者血清水通道蛋白-4抗体

目的 初步探讨以人工合成多肽模拟抗原酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测视神经脊髓炎(neuromyelitis optica,NMO)患者血清水通道蛋白-4(aquaporin-4,AQP4)抗体.方法根据AQP4蛋白的分子构成和三级结构,应用生物信息学和结构生物学的方法设计3段AQP4蛋白膜外段多肽片段,分别为AQP456-69、AQP4135-155、AQP4209-230;以其为抗原ELISA检测9例NMO及神经科其他疾病7例患者血清,并与免疫荧光法检测结果进行对比分析.结果 以AQP4135-155、AQP4209-230为抗原ELISA检测结果显示,9例NMO患者中,7例经免疫荧光法证实AQP4抗体阳性患者吸光度A值平均值比阴性对照患者显著提高(P＜0.05);当血清稀释4倍和8倍时具有较高的A值,与对照组相比差异具有统计学意义(P＜0.05).结论 AQP4蛋白2段膜外段多肽,即AQP4135-155和AQP4209-230可能是NMO患者血清中AQP4抗体所针对的主要抗原表位或抗原表位的主要部分,初步表明模拟抗原ELISA检测NMO患者血清AQP4抗体的可行性.     

利妥昔单抗治疗视神经脊髓炎谱系疾病的疗效分析

目的：观察利妥昔单抗（RTX）治疗视神经脊髓炎谱系疾病（NMOSD）的疗效。方法：回顾性观察2008年1月至2018年11月中国人民解放军总医院129例NMOSD患者的治疗，其中55例血清水通道蛋白-4抗体（AQP4-Ab）阳性，74例AQP4-Ab阴性患者。通过90个月的长期随访，评估利妥昔单抗（RTX）、硫唑嘌呤（AZA）及吗替麦考酚酯（MMF）的治疗效果。AQP4-Ab阳性患者中，14例接受低剂量利妥昔单抗RTX治疗，13例接受AZA治疗，8例接受MMF治疗。结果：接受免疫治疗的AQP4-Ab阳性患者中，治疗后的年复发率（ARR）明显低于治疗前的ARR(RTX治疗组ARR从1.00降至0.70,AZA治疗组从0.80降至0.30,MMF治疗组从0.85降至0.35)。同时在AQP4-Ab阴性患者中，治疗后的ARR也低于治疗前（RTX治疗组从0.30降至0.20,AZA治疗组从0.90降至0.50,MMF治疗组从0.90降至0.40）。免疫治疗后的残疾进展（EDSS）有所下降，AQP4-Ab阳性患者的EDSS评分下降（RTX治疗组从4.00分降至2.75分，AZA组从3.50分降至...     

CD1d~(hi)CD~(5+)CD19~+调节性B细胞在视神经脊髓炎患者外周血中的数量及意义

目的检测视神经脊髓炎(NMO)患者外周血中CD1dhiCD5+CD19+调节性B细胞的比例,探讨CD1dhiCD5+CD19+调节性B细胞能否作为NMO和多发性硬化(MS)鉴别诊断的生物标志物。方法采用流式细胞术检测44例NMO患者,38例MS患者和30例健康正常人外周血中CD1dhiCD5+CD19+调节性B细胞的比例。采用间接免疫荧光法检测NMO患者水通道蛋白4抗体(AQP4-Ab)的表达情况。结果 NMO患者外周血中CD1dhiCD5+CD19+调节性B细胞占CD19+B细胞及淋巴细胞的比例较MS患者和健康正常人明显减低,而MS患者与健康正常人比较,差异无统计学意义。AQP4-Ab阳性NMO患者较AQP4-Ab阴性NMO患者CD1dhiCD5+CD19+调节性B细胞占CD19+B细胞及淋巴细胞的比例明显减低。结论外周血中CD1dhiCD5+CD19+调节性B细胞数量是NMO和MS鉴别诊断的一种生物标志物。     

稳定表达水通道蛋白-4 HEK293细胞株的建立及应用

目的:建立稳定表达水通道蛋白-4(AQP4)的HEK293细胞株,并以其检测12例视神经脊髓炎患者血清中是否存在AQP4抗体。方法:从颞叶癫痫患者脑组织提取总RNA,RT-PCR扩增AQP4的cDNA,构建AQP4-绿色荧光蛋白(GFP)融合表达重组体pEGFP-N1-AQP4,转染HEK293细胞,抗生素筛选联合流式细胞仪分选筛选稳定表达细胞株,RT-PCR和间接免疫荧光法鉴定AQP4的表达,激光共聚焦显微镜观察AQP4在细胞中的定位,并以已建立的细胞为底物间接免疫荧光法检测12例视神经脊髓炎患者血清中是否存在AQP4抗体。结果:经PCR、双酶切、测序鉴定证实成功获取AQP4的cDNA,AQP4-GFP融合表达重组质粒pEGFP-N1-AQP4构建成功,抗生素筛选联合流式细胞仪分选筛选稳定表达细胞株,稳定表达率达90%以上,RT-PCR和间接免疫荧光法鉴定存在AQP4的表达,激光共聚焦显微镜进一步确认AQP4-GFP融合蛋白主要表达于细胞膜上,11例典型视神经脊髓炎患者血清中检出AQP4抗体,对视神经脊髓炎诊断敏感性为91.7%,特异性为94.7%。结论:成功建立稳定表达AQP4的HEK293细胞株,以其作为检测底物检出视神经脊髓炎患者血清中存在AQP4自身抗体。     

视神经脊髓炎患者NMOIgG抗体阳性与阴性患者临床症状对比及预后分析

目的:对NMOIgG抗体表达阳性的视神经脊髓炎患者与其表达阴性患者的不同临床症状与其治疗后预后的情况进行分析与探究。方法:收集在我院接受治疗的100例视神经脊髓炎患者,采集患者的血清学标本,并且对其血清标本进行NMOIgG抗体检测,根据检测的结果将其分为抗体阳性组与抗体阴性组,每组患者50例,对比分析两组患者的临床症状以及治疗的效果与预后的情况。结果:抗体阳性组患者的病程(3.8±0.8)年、脑脊液克隆带数(37.1±6.9)个以及脊髓病灶节段数(67.2±10.8)个与抗体阴性组患者的病程(1.9±0.5)年、脑脊液克隆带数(28.3±5.7)个以及脊髓病灶节段数(34.6±9.5)个相比明显较高,结果有统计学意义(P0.05)。两组患者经治疗后,病程、脑脊液克隆带数以及脊髓病灶节段数等临床症状中,抗体阳性组患者的EDSS评分与抗体阴性组患者的EDSS评分相比明显较高,结果有统计学意义(P0.05)。结论:NMOIgG抗体在视神经脊髓炎患者体内表达阳性时,其病程、脑脊液克隆数等临床症状相对较为显著,且其治疗的预后效果较差,可以用于临床上对该疾病病情变化的判断与研究等。     

视神经脊髓炎特异性自身抗体NMO-IgG的诊断价值研究

目的:初步探讨视神经脊髓炎特异性自身抗体NMO-IgG的诊断价值。方法:入选患者共120例,视神经脊髓炎患者(Neuromyelitis optica,NMO)45例,包括高危NMO(High-risk NMO,HR-NMO)患者9例,多发性硬化(Multiple sclerosis,MS)患者65例,其他神经系统疾病患者10例,采用细胞间接免疫荧光法检测所有患者血清NMO-IgG水平,比较NMO与MS患者临床特点及NMO-IgG水平,初步评估HR-NMO的预后。结果:血清NMO-IgG诊断NMO的灵敏度达67%,特异性达92%;NMO组与MS组患者男女比例、发生严重视神经炎、横贯性脊髓炎、颅脑MRI正常表现、脊髓病灶超过3个椎体节段、合并其他免疫性疾病及血清NMO-IgG水平均有统计学差异(P0.05)。HR-NMO组中血清NMO-IgG阳性的4例中有3例在一年后的随访中有横贯性脊髓炎的再次发作。结论:血清NMO-IgG的检测对视神经脊髓炎的诊断、鉴别诊断及预后评估有一定的价值。     

视神经脊髓炎谱系疾病患者神经性瘙痒的临床特征及其意义

目的 探讨视神经脊髓炎谱系疾病(NMOSD)患者神经性瘙痒(NP)的临床特征及意义。方法 回顾性分析2016年7月-2019年3月蚌埠医学院第一附属医院35例NMOSD患者的临床资料,其中男9例、女26例,年龄13~85岁。依据是否合并NP,分为单纯NMOSD组(25例)和合并NP组(10例),比较两组患者的临床特征,包括水通道蛋白4抗体(AQP4-Ab)阳性、性别、发病年龄、受累脊髓节段、脊髓病变分布及扩展残疾状态量表(EDSS)评分等;分析NP的临床特点,NP发作与病程、病变部位的关系,NP的治疗效果。结果 35例NMOSD患者中10例(28.6%)有NP表现,男4例、女6例。合并NP组患者AQP4-Ab阳性(10/10)较单纯NMOSD组(60%,15/25)高,差异有统计学意义(P=0.033);两组患者性别分布、发病年龄、受累脊髓节段及EDSS评分等差异均无统计学意义(P值均＞0.05)。10例NP患者均为10 d内先后出现NP及其他脊髓炎或脑干症状,其中2例NP为NMOSD首次发病的首发症状,3例为复发的首发症状。NP常发生于颈枕部、上肢、肩胛部、面部及其他部位,呈中重度;9例(9/10)NP的发生部位经MRI证实与受累的脊髓节段有明确的关系。结论 NP可能是NMOSD患者被忽视的一种较为常见的首发症状之一,可能与AQP4-Ab阳性有关,对脊髓的病变定位有一定的提示意义,也可能提示NMOSD急性脊髓炎的发作,了解其临床特征具有一定的诊断、定位诊断及指导治疗等临床价值。     

流式微球阵列法检测肺结核患者血清部分细胞因子的临床意义

目的:探讨流式微球阵列法(CBA)检测不同类型肺结核患者血清中细胞因子IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ水平的临床意义。方法:采用BD CBA Flex Set检测试剂盒和流式微球阵列法(CBA)检测84例肺结核患者(46例活动性肺结核患者及38例非活动性肺结核患者)和30例正常人的6种细胞因子(IL-2、IL-4、IL-6、IL-10、TNF-α、IFN-γ)的水平。结果:肺结核患者血清IL-2、IL-6、IL-10和IFN-γ的水平显著高于正常对照组(P<0.01或P<0.05),且活动性结核组高于非活动性结核组(P<0.01或P<0.05)。血清IL-4与TNF-α水平肺结核患者与正常对照组无显著性差异。结论:CBA法能快速、灵敏、多参数批量同步定量检测血清中6种细胞因子;这些细胞因子在肺结核的免疫发病中具有重要的意义。     

Usefulness of serum S100B as a marker for the acute phase of aquaporin-4 autoimmune syndrome

The aquaporin-4 (AQP4) water channel antibody is used in the diagnosis of neuromyelitis optica (NMO) due to its high sensitivity and high specificity. However, some patients are reported to have neither optic neuritis nor myelitis despite being positive for the AQP4-autoantibody (AQP4-Ab). Therefore, recent reports suggest that such patients should be diagnosed as having 'AQP4-autoimmune syndrome'. In this study, we quantified the levels of glial fibrillar acidic protein (GFAP) and S100B by enzyme-linked immunosorbent assay (ELISA) in CSF and serum samples simultaneously obtained in the acute phase of ten AQP4-autoantibody (AQP4Ab)-positive and seven AQP4Ab-negative patients. Serum levels of S100B were significantly higher in the acute phase of the AQP4Ab-positive patients (2.92±1.22pg/ml) than in the AQP4Ab-negative patients (0.559±0.180pg/ml, p=0.0250), while serum levels of GFAP were not different between the two groups (AQP4Ab-positive vs. AQP4Ab-negative: 0.120±0.113ng/ml vs. 0.00609±0.00609ng/ml, p=0.193). Furthermore, the CSF and serum levels of S100B had a significant positive correlation in AQP4Ab-positive patients (n=10, r=0.673, p=0.0390). Our results raise the possibility that serum levels of S100B, but not GFAP, examined in the acute phase of the disease might be a useful biomarker for the relapse of AQP4 autoimmune syndrome.     

Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes

Background

In 70-80% of cases, neuromyelitis optica (NMO) is associated with highly specific serum auto-antibodies to aquaporin-4 (termed AQP4-Ab or NMO-IgG). Recent evidence strongly suggests that AQP4-Ab are directly involved in the immunopathogenesis of NMO.

Objective

To assess the frequency, syndrome specificity, diagnostic relevance, and origin of cerebrospinal fluid (CSF) AQP4-Ab in patients with NMO spectrum disorders (NMOSD).

Methods

87 CSF samples from 37 patients with NMOSD and 42 controls with other neurological diseases were tested for AQP4-Ab in a cell based assay using recombinant human AQP4. Twenty-three paired CSF and serum samples from AQP4-Ab seropositive NMOSD patients were further analysed for intrathecal IgG synthesis to AQP4.

Results

AQP4-Ab were detectable in 68% of CSF samples from AQP4-Ab seropositive patients with NMOSD, but in none of the CSF samples from AQP4-Ab seronegative patients with NMOSD and in none of the control samples. Acute disease relapse within 30 days prior to lumbar puncture, AQP4-Ab serum titres >1:250, and blood-CSF barrier dysfunction, but not treatment status, predicted CSF AQP4-Ab positivity. A positive AQP4-specific antibody index was present in 1/23 samples analysed.

Conclusions

AQP4-Ab are detectable in the CSF of most patients with NMOSD, mainly during relapse, and are highly specific for this condition. In the cohort analysed in this study, testing for CSF AQP4-Ab did not improve the sensitivity and specificity of the current diagnostic criteria for NMO. The substantial lack of intrathecal AQP4-Ab synthesis in patients with NMOSD may reflect the unique localisation of the target antigen at the blood brain barrier, and is important for our understanding of the immunopathogenesis of the disease.     

Strategy for anti-aquaporin-4 auto-antibody identification and quantification using a new cell-based assay

NMO-IgG is a specific biomarker of neuromyelitis optica (NMO) that targets the aquaporin-4 (AQP4) water channel protein. The current gold standard for NMO-IgG identification is indirect immunofluorescence (IIF). Our aim in this study was to develop a new quantitative cell-based assay (CBA) and to propose a rational strategy for anti-AQP4 Ab identification and quantification. We observed an excellent correlation between the CBA and IIF for NMO-IgG/anti-AQP4 detection. The CBA appeared more sensitive than IIF but on the other hand, IIF allows the simultaneous detection of various auto-Abs, underlining the complementarity between both methods. In conclusion, we propose to use IIF for the screening of patients at diagnosis in order to identify auto-Abs targeting the central nervous system. A highly sensitive, AQP4 specific and quantitative assay such as our CBA could be used thereafter to specifically identify the target of the Ab and to monitor its serum concentration under treatment.     

Aquaporin‐4 Antibodies (NMO‐IgG) as a Serological Marker of Neuromyelitis Optica: A Critical Review of the Literature

Antibodies to aquaporin‐4 (called NMO‐IgG or AQP4‐Ab) constitute a sensitive and highly specific serum marker of neuromyelitis optica (NMO) that can facilitate the differential diagnosis of NMO and classic multiple sclerosis. NMO‐IgG/AQP4‐Ab seropositive status has also important prognostic and therapeutic implications in patients with isolated longitudinally extensive myelitis (LETM) or optic neuritis (ON). In this article, we comprehensively review and critically appraise the existing literature on NMO‐IgG/AQP4‐Ab testing. All available immunoassays—including tissue‐based (IHC), cell‐based (ICC, FACS) and protein‐based (RIPA, FIPA, ELISA, Western blotting) assays—and their differential advantages and disadvantages are discussed. Estimates for sensitivity, specificity, and positive and negative likelihood ratios are calculated for all published studies and accuracies of the various immunoassay techniques compared. Subgroup analyses are provided for NMO, LETM and ON, for relapsing vs. monophasic disease, and for various control groups (eg, MS vs. other controls). Numerous aspects of NMO‐IgG/AQP4‐Ab testing relevant for clinicians (eg, impact of antibody titers and longitudinal testing, indications for repeat testing, relevance of CSF testing and subclass analysis, NMO‐IgG/AQP4‐Ab in patients with rheumatic diseases) as well as technical aspects (eg, AQP4‐M1 vs. AQP4‐M23‐based assays, intact AQP4 vs. peptide substrates, effect of storage conditions and freeze/thaw cycles) and pitfalls are discussed. Finally, recommendations for the clinical application of NMO‐IgG/AQP4‐Ab serology are given.     

NMO-IgG: a specific biomarker for neuromyelitis optica

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease that principally targets the optic nerves and spinal cord and often leads to severe disability and occasionally life threatening respiratory failure. Although its clinical manifestations overlap with those of multiple sclerosis (MS), in established cases these two conditions can be distinguished on the basis of clinical, radiological, and routine spinal fluid studies. The diagnosis in early cases or limited forms of NMO is difficult. We recently discovered a unique IgG autoantibody (NMO-IgG) that is highly specific to patients with NMO and thus a valuable diagnostic aid. Its antigen, aquaporin-4 (AQP4), is the central nervous system's predominant water channel protein. This antibody has not yet been proven to be pathogenic, but several facts suggest that it might be, including the similarity of the immunohistochemical pattern of NMO-(AQP4) IgG binding to mouse CNS tissues to the pattern of immune complex deposition in autopsied patients' spinal cord tissue. The spectrum of diseases identified by NMO-IgG is broader than has previously been recognized clinically and includes incomplete forms of NMO, such as recurrent transverse myelitis without optic neuritis and recurrent optic neuritis without myelitis.     

Persistence of Inoue-Melnick virus and antibody in cerebrospinal fluid.

Serial cerebrospinal fluid samples were obtained from 14 multiple sclerosis patients over a period of several months and were tested under code for Inoue-Melnick virus. In five of the positive patients, virus was present in 28 of 32 specimens collected over a period of 2 to 5 months. Four patients, from whom a total of 34 specimens were taken, yielded only a single isolate each. Five patients contributed 28 specimens, all of which were negative. In six patients (three virus positive and three virus negative), neutralizing antibody was detected in serum and in cerebrospinal fluid.     

Aquaporin-4 autoantibodies in neuromyelitis optica spectrum disorders: comparison between tissue-based and cell-based indirect immunofluorescence assays

Background  

Neuromyelitis optica spectrum disorders (NMOSD) are severe central nervous system inflammatory demyelinating disorders (CNS IDD) characterized by monophasic or relapsing, longitudinally extensive transverse myelitis (LETM) and/or optic neuritis (ON). A significant proportion of NMOSD patients are seropositive for aquaporin-4 (AQP4) autoantibodies. We compared the AQP4 autoantibody detection rates of tissue-based indirect immunofluorescence assay (IIFA) and cell-based IIFA.