Salivary Biomarkers for Graft versus Host Disease

描述(由申请人提供)：这项工作的目标是开发唾液生物标记物用于慢性移植物抗宿主病(CGVHD)。这将使用基于iTRAQ标记物的定量质谱仪(MS)方法来检测接受造血细胞移植(HCT)并随后发展为cGVHD的患者的静息全唾液样本中cGVHD相关蛋白的表达变化。唾液之所以被提出用于这项研究，不仅仅是因为它很容易获得，而且主要是因为唾液腺是粘膜免疫系统中淋巴细胞归巢的目标，我们假设GVHD在粘膜免疫系统中被激活(Murai等人，NAT免疫4：154-160,2003)。因此，唾液蛋白质组成的变化应该发生在疾病的早期，并且应该与疾病的强度相关。GVHD的病因预测唾液炎性细胞因子和相关细胞信号蛋白含量增加。我们的研究将包括发现和确认/确认阶段。在发现阶段，基于iTRAQ的定量MALDI TOF/TOF质谱学技术将用于从患有或不患有cGVHD的[HCT接受者]的静息全唾液样本中识别cGVHD的潜在候选细胞因子/趋化因子和其他蛋白质生物标记物。]在验证阶段，将使用[ELISA]检测显示cGVHD相关表达差异最大的8个候选生物标记蛋白，以确认混合样本中的MS结果，并建立单个患者样本中表达差异的大小和流行率。个体唾液样本中蛋白质表达的变化将被用来构建一个由5种蛋白质组成的生物标志物小组，该小组将被评估预测cGVHD的能力。MS分析将由我们的合作者，UW Shaw神经病理学教授张静博士指导，他开发了MS技术来识别中枢神经系统疾病的脑脊液生物标记物。我们建议应用他的技术来寻找cGVHD的唾液生物标记物。我们已经有了初步的结果，表明对照的整个唾液可以通过建议的MS程序进行分析。拟议的最先进的MS蛋白质组学方法应该产生特定的GVHD生物标志物，使GVHD能够更及时地检测到，从而更早地进行治疗，从而降低cGVHD口腔和其他靶组织的疾病严重程度。[由于许多疾病生物标记物来自潜在的病理生理机制，对这里发现的生物标记物阵列的功能分析应该有助于深入了解cGVHD的发病机制。]移植物抗宿主病(GVHD)是一种常见的、严重的、潜在威胁生命的造血细胞移植并发症，用于治疗血液病和其他疾病。移植物抗宿主病可作为急性或慢性疾病发生，目前很难预测谁会发展成广泛性疾病。这种疾病的病因学建议唾液腺应该早期参与GVHD，唾液蛋白的变化可以及早发现和治疗，这可以降低疾病的严重性和死亡率，[以及对疾病病因的洞察]。这项提议的目标就是开发这样一种唾液检测方法。

DESCRIPTION (provided by applicant): The goal of this work is to develop salivary biomarkers for chronic Graft Versus Host Disease (cGVHD). This will be done using an iTRAQ marker-based, quantitative mass spectrometry (MS) approach to detect cGVHD-associated protein expression changes in resting whole saliva samples from patients who received hematopoietic cell transplants (HCT) and subsequently developed cGVHD. Saliva is proposed for this study not simply because it is convenient to obtain, but primarily because salivary glands are a lymphocyte-homing target in the mucosal immune system, where we postulate GVHD is activated (Murai et al., Nat Immunol 4:154-160, 2003). Hence, salivary protein composition changes should occur early in the disease and should correlate with disease intensity. The etiology of GVHD predicts increases in salivary inflammatory cytokine and related cell signaling protein contents. Our study will consist of discovery and confirmation/validation phases. In the discovery phase, an iTRAQ-based quantitative MALDI TOF/TOF mass spectrometry technique will be used to identify potential candidate cytokine/chemokine and other protein biomarkers for cGVHD in pooled samples of resting whole saliva from [HCT recipients with or without cGVHD.] In the validation phase, the eight candidate biomarker proteins showing the greatest cGVHD-associated expression differences will be measured using [ELISA] assays to confirm the MS results in the pooled samples, and to establish expression difference magnitudes and prevalence in the individual patient samples. The protein expression changes in the individual saliva samples will be used to construct a biomarker panel of 5 proteins that will be evaluated for an ability to predict cGVHD. The MS analyses will be directed by our collaborator, Dr. Jing Zhang, Shaw Professor of Neuropathology at the UW, who has developed MS techniques for identifying CSF biomarkers for central nervous system diseases. We propose to apply his techniques to find salivary biomarkers for cGVHD. We have preliminary results showing control whole saliva can be analyzed by the proposed MS procedures. The proposed state-of-art MS proteomics approach should yield specific GVHD biomarkers that would allow more timely detection of GVHD, leading to earlier treatment, and consequently less disease severity in the oral and other target tissues of cGVHD. [Since many disease biomarkers arise from underlying pathophysiologic mechanisms, function-analysis of the array of biomarkers found here should give insight into the pathogenesis of cGVHD.] Graft Versus Host Disease (GVHD) is a frequent, serious, potentially life-threatening complication of hematopoietic cell transplants used to treat hematologic disorders as well as other diseases. GVHD can occur as acute or chronic disease, and it is currently difficult to predict who will develop extensive disease. The proposed etiology of the disease suggests salivary glands should have an early involvement in GVHD, and salivary protein changes could allow early detection and treatment, which could decrease the seriousness and mortality of the disease, [as well as give insight into disease etiology]. The goal of this proposal is to develop such a salivary assay.