■高达46%的假定的自身免疫性边缘叶脑炎患者对所有目前已知的中枢神经系统(CNS)抗原是血清阴性的。我们开发了一种基于细胞的测定法(CBA),以使用源自人诱导的多能干细胞(hiPSCs)的神经元和星形胶质细胞筛选血清和脑脊液(CSF)中的新型神经抗体。
■将人iPSC衍生的星形胶质细胞或神经元与来自99名患者的血清/CSF一起孵育[42名患有炎性神经疾病(IND)和57名患有非IND(NIND)]。IND组包括11名先前已建立神经抗体的患者,6例血清阴性视神经脊髓炎谱系障碍(NMOSD),12例疑似自身免疫性脑炎/副肿瘤综合征(AIE/PNS),和13与其他IND(OIND)。使用荧光标记的抗体检测IgG与固定的CNS细胞的结合,并通过自动荧光测量进行分析。通过流式细胞术进一步分析IgG神经元/星形胶质细胞反应性。外周血单核细胞(PBMC)用作与CNS无关的对照靶细胞。使用稳健回归和异常值去除测试将反应性曲线定义为阳性,在每个单独的读出后具有10%的错误发现率。
■使用我们的CBA,我们在19/99例受试者中检测到识别hiPSC来源神经细胞的抗体.9例患者中特异性结合星形胶质细胞的抗体,八例中的神经元,在两种情况下,两种细胞类型,通过显微镜单细胞分析证实。强调了我们全面的96孔CBA测定的重要性,神经特异性抗体结合在IND(42个中的15个)中的频率高于NIND患者(57个中的4个)(Fisher精确检验,p=0.0005)。具有细胞内反应性抗体的四个AQP4NMO中的两个和七个确定的AIE/PNS中的四个[1GFAP星形细胞病,2胡+,1Ri+AIE/PNS)],如诊断实验室所确定,对我们的CBA也很积极。最有趣的是,我们在六个血清阴性NMOSD中的两个中显示了抗体反应性,12个可能的AIE/PNS中有6个,13个OIND之一。使用hiPSC衍生的CNS细胞或PBMC检测的抗体结合的13例患者与0例患者的流式细胞术,分别,建立检测到的抗体对神经组织的特异性。
■我们独特的基于hiPSC的CBA允许在疑似免疫介导的神经综合征患者中测试新型神经元/星形胶质细胞反应性抗体,在既定的常规实验室中进行阴性测试,为建立这种复杂疾病的诊断开辟了新的视角。
UNASSIGNED: Up to 46% of patients with presumed autoimmune limbic encephalitis are seronegative for all currently known central nervous system (CNS) antigens. We developed a cell-based assay (CBA) to screen for novel neural antibodies in serum and cerebrospinal fluid (CSF) using neurons and astrocytes derived from human-induced pluripotent stem cells (hiPSCs).
UNASSIGNED: Human iPSC-derived astrocytes or neurons were incubated with serum/CSF from 99 patients [42 with inflammatory neurological diseases (IND) and 57 with non-IND (NIND)]. The IND group included 11 patients with previously established neural antibodies, six with seronegative neuromyelitis optica spectrum disorder (NMOSD), 12 with suspected autoimmune encephalitis/paraneoplastic syndrome (AIE/PNS), and 13 with other IND (OIND). IgG binding to fixed CNS cells was detected using fluorescently-labeled antibodies and analyzed through automated fluorescence measures. IgG neuronal/astrocyte reactivity was further analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were used as CNS-irrelevant control target cells. Reactivity profile was defined as positive using a Robust regression and Outlier removal test with a false discovery rate at 10% following each individual readout.
UNASSIGNED: Using our CBA, we detected antibodies recognizing hiPSC-derived neural cells in 19/99 subjects. Antibodies bound specifically to astrocytes in nine cases, to neurons in eight cases, and to both cell types in two cases, as confirmed by microscopy single-cell analyses. Highlighting the significance of our comprehensive 96-well CBA assay, neural-specific antibody binding was more frequent in IND (15 of 42) than in NIND patients (4 of 57) (Fisher\'s exact test, p = 0.0005). Two of four AQP4+ NMO and four of seven definite AIE/PNS with intracellular-reactive antibodies [1 GFAP astrocytopathy, 2 Hu+, 1 Ri+ AIE/PNS)], as identified in diagnostic laboratories, were also positive with our CBA. Most interestingly, we showed antibody-reactivity in two of six seronegative NMOSD, six of 12 probable AIE/PNS, and one of 13 OIND. Flow cytometry using hiPSC-derived CNS cells or PBMC-detected antibody binding in 13 versus zero patients, respectively, establishing the specificity of the detected antibodies for neural tissue.
UNASSIGNED: Our unique hiPSC-based CBA allows for the testing of novel neuron-/astrocyte-reactive antibodies in patients with suspected immune-mediated neurological syndromes, and negative testing in established routine laboratories, opening new perspectives in establishing a diagnosis of such complex diseases.