Zanone M M, Petersen J S, Vergani D, Peakman M
Immunology Department, King's College School of Medicine and Dentistry, London, UK.
J Neuroimmunol. 1997 Sep;78(1-2):1-7. doi: 10.1016/s0165-5728(97)00071-4.
We have previously identified an association between symptomatic diabetic autonomic neuropathy (DAN) and autoantibodies to sympathetic and parasympathetic nervous structures. The antigens identified by these autoantibodies are not known, but glutamic acid decarboxylase (GAD) has been suggested as a candidate target, since anti-GAD autoantibodies are present in patients with long-term diabetes and GAD is expressed in a variety of cell types and structures in the nervous system. The aim of this study was to examine GAD expression in sympathetic ganglia and vagus nerve and to compare the distribution of GAD within these tissues with that of anti-sympathetic ganglia and anti-vagus nerve autoantibodies from patients with DAN, using single and double indirect immunofluorescence on tissue sections. The monoclonal antibody GAD-6, specific for GAD65, gave a granular, peripheral, cytoplasmic staining pattern in sympathetic ganglion cells. Dual immunofluorescence demonstrated that serum from a patient with anti-sympathetic ganglion autoantibodies stained the same cells, but homogeneously throughout the cytoplasm. In the vagus nerve, patient's serum stained the fibres only; GAD-6 stained the cytoplasm of parasympathetic ganglion cells but only occasional fibres. In addition, GAD enzymatic activity was detectable in both sympathetic ganglia and vagus nerve. Incubation of sera or GAD-6 overnight with a crude homogenate of human brain as an antigen source abolished staining of the nervous tissues by GAD-6, but not by patients' sera. The different localisation of GAD and the autoantigens targeted by patients' sera indicates that GAD is not the target of the autoantibodies characteristic of DAN. Moreover, absorption studies using human brain homogenate suggest that the targets of anti-sympathetic ganglion and anti-vagus nerve autoantibodies are absent or represented only at low levels in the central nervous system and may be confined to the periphery.
我们之前已确定有症状的糖尿病自主神经病变（DAN）与针对交感和副交感神经结构的自身抗体之间存在关联。这些自身抗体所识别的抗原尚不清楚，但谷氨酸脱羧酶（GAD）被认为是一个候选靶点，因为抗GAD自身抗体存在于长期糖尿病患者中，且GAD在神经系统的多种细胞类型和结构中均有表达。本研究的目的是检测交感神经节和迷走神经中GAD的表达，并使用组织切片上的单重和双重间接免疫荧光法，比较这些组织中GAD的分布与DAN患者抗交感神经节和抗迷走神经自身抗体的分布。针对GAD65的单克隆抗体GAD - 6在交感神经节细胞中呈现颗粒状、周边性、细胞质染色模式。双重免疫荧光显示，患有抗交感神经节自身抗体患者的血清对相同细胞进行染色，但在整个细胞质中呈均匀染色。在迷走神经中，患者血清仅对纤维进行染色；GAD - 6对副交感神经节细胞的细胞质进行染色，但仅偶尔对纤维进行染色。此外，在交感神经节和迷走神经中均可检测到GAD酶活性。将血清或GAD - 6与人脑粗匀浆作为抗原源一起孵育过夜，消除了GAD - 6对神经组织的染色，但患者血清的染色不受影响。GAD与患者血清所靶向的自身抗原的不同定位表明，GAD不是DAN特征性自身抗体的靶点。此外，用人脑匀浆进行的吸收研究表明，抗交感神经节和抗迷走神经自身抗体的靶点在中枢神经系统中不存在或仅以低水平存在，可能局限于外周。
