Proteomics Core Facility, University of Gothenburg, Gothenburg, Sweden.
J Pathol. 2013 Apr;229(5):719-28. doi: 10.1002/path.4166.
Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5-D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross-reactivity of clone 5-D8/1 with normal islet proteins. Western blotting, two-dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5-D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B-type and ATP synthase beta subunit. Immunohistochemistry using the clone 5-D8/1 revealed a granular cytoplasmic staining pattern in mitochondria-rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent-onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5-D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5-D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent-onset type 1 diabetes patients could reflect cross-reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes.
当前或近期的肠病毒感染与 1 型糖尿病有关。然而，这些证据主要是使用一种特定的鼠单克隆抗体（克隆 5-D8/1）产生的，该抗体结合病毒衣壳蛋白 VP1。使用其他独立方法难以证实这些发现，这导致人们担心这可能是人为的。为了解决这个问题，我们研究了克隆 5-D8/1 与正常胰岛蛋白的潜在交叉反应性。使用 Western blot、二维凝胶电泳和质谱法鉴定了克隆 5-D8/1 结合的人类胰岛蛋白。我们发现该克隆与两种线粒体蛋白，肌酸激酶 B 型和 ATP 合酶β亚基有明显的反应性。使用克隆 5-D8/1 的免疫组织化学显示，在富含线粒体的细胞中，即肝细胞、胆管上皮细胞、血管内皮细胞、骨骼肌细胞和肿瘤唾液腺嗜酸性细胞瘤细胞中，存在颗粒状细胞质染色模式，而结缔组织和浸润性免疫细胞呈阴性。在近期发生 1 型糖尿病患者的胰岛中发现了克隆 5-D8/1 的染色，但在体外感染肠病毒的分离胰岛中没有发现，在将克隆 5-D8/1 与线粒体蛋白混合后，可以阻断这种染色。总的来说，我们的数据表明，克隆 5-D8/1 除了肠病毒 VP1 外，还检测到两种人类线粒体酶。以前报道的在近期发生 1 型糖尿病患者胰岛中 VP1 阳性的观点，可能反映了对天然胰岛蛋白的交叉反应性，而不是 EV 的存在，这一观点得到了独立技术如 PCR 或原位杂交等难以证明 EV 感染的支持。这些发现呼吁重新审视 1 型糖尿病患者胰岛中肠病毒的存在。
