Table1_Identification of novel autoantigens as potential biomarkers in juvenile idiopathic arthritis associated uveitis.xlsx

BackgroundMany children with juvenile idiopathic arthritis (JIA) have autoantibodies, targeting nuclear components (anti-nuclear antibodies, ANA). ANA in JIA is associated with uveitis, an eye inflammation which may cause permanent vision impairment if not detected and treated. However, ANA-testing is neither specific nor sensitive enough to be a clinically reliable predictor of uveitis risk, and the precise autoantigens targeted by ANA in JIA are largely unknown. If identified, specific autoantibodies highly associated with uveitis could be used as biomarkers to facilitate identification of JIA patients at risk.
MethodsAntibodies from six ANA-positive, oligoarticular JIA patients, with and without uveitis, were explored by two large-scale methods: (1) screening against 42,100 peptides on an autoimmunity profiling planar array, and (2) immunoprecipitations from cell lysates with antigen identification by mass spectrometry. Three hundred thirty-five peptide antigens, selected from proteins identified in the large-scale methods and the scientific literature were investigated using a bead-based array in a cohort of 56 patients with oligoarticular- or RF-negative polyarticular JIA, eight of which were having current or previous uveitis.
ResultsIn the planar array, reactivity was detected against 332 peptide antigens. The immunoprecipitations identified reactivity towards 131 proteins. Only two proteins were identified by both methods. In the bead-based array of selected peptide antigens, patients with uveitis had a generally higher autoreactivity, seen as higher median fluorescence intensity (MFI) across all antigens, compared to patients without uveitis. Reactivity towards 17 specific antigens was significantly higher in patients with uveitis compared to patients without uveitis. Hierarchical clustering revealed that patients with uveitis clustered together.
ConclusionThis study investigated autoantigens in JIA and uveitis, by combining two exploratory methods and confirmation in a targeted array. JIA patients with current or a history of uveitis had significantly higher reactivity towards 17 autoantigens and a generally higher autoreactivity compared to JIA patients without uveitis. Hierarchical clustering suggests that a combination of certain autoantibodies, rather than reactivity towards one specific antigen, is associated with uveitis. Our analysis of autoantibodies associated with uveitis in JIA could be a starting point for identification of prognostic biomarkers useful in JIA clinical care.

背景
许多幼年特发性关节炎（juvenile idiopathic arthritis, JIA）患儿体内存在靶向核组分的自身抗体，即抗核抗体（anti-nuclear antibodies, ANA）。JIA患者体内的抗核抗体与葡萄膜炎（uveitis）相关，该眼内炎症若未及时检出并治疗，可导致永久性视力损害。然而，抗核抗体检测既缺乏足够的特异性，也无足够的敏感性，无法作为临床可靠的葡萄膜炎风险预测指标，且JIA患者体内抗核抗体所靶向的精确自身抗原尚未明确。若能鉴定出与葡萄膜炎高度相关的特异性自身抗体，即可将其作为生物标志物，助力识别存在患病风险的JIA患者。

方法
本研究针对6名抗核抗体阳性的寡关节型幼年特发性关节炎（oligoarticular JIA）患者（其中部分患者合并葡萄膜炎，部分无葡萄膜炎）的抗体展开探索，采用两种大规模实验方法：（1）在自身免疫profiling平面芯片（autoimmunity profiling planar array）上针对42100条肽段进行筛选；（2）从细胞裂解液中进行免疫沉淀，通过质谱（mass spectrometry）鉴定抗原。研究人员从大规模实验方法及科学文献中筛选得到335个肽段抗原，随后采用基于磁珠的芯片对56名寡关节型或类风湿因子（rheumatoid factor, RF）阴性多关节型JIA患者进行检测，其中8名患者当前存在或既往有葡萄膜炎病史。

结果
在平面芯片检测中，研究人员检测到针对332个肽段抗原的抗体反应；免疫沉淀实验则鉴定出针对131种蛋白质的反应性，且两种方法仅共同鉴定出2种蛋白质。在针对筛选肽段抗原的磁珠芯片检测中，与无葡萄膜炎的患者相比，葡萄膜炎患者的整体自身反应性更高，表现为所有抗原的中位荧光强度（median fluorescence intensity, MFI）更高。相较于无葡萄膜炎的患者，葡萄膜炎患者针对17种特定抗原的反应性显著升高。层次聚类（hierarchical clustering）分析显示，葡萄膜炎患者可聚为一类。

结论
本研究结合两种探索性方法，并通过靶向芯片验证，对JIA及葡萄膜炎相关自身抗原进行了研究。与无葡萄膜炎的JIA患者相比，当前存在或既往有葡萄膜炎病史的患者针对17种自身抗原的反应性显著更高，且整体自身反应性更强。层次聚类分析提示，葡萄膜炎的发生与特定自身抗体的组合相关，而非仅针对某一种抗原的反应性。本研究针对JIA合并葡萄膜炎相关自身抗体的分析，可为鉴定可用于JIA临床诊疗的预后生物标志物提供起点。