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慢性移植物抗宿主病（cGVHD）的唾液生物标志物。这将使用基于iTRAQ标记的定量质谱（MS）方法来检测接受造血细胞移植（HCT）并随后发展为cGVHD的患者静息全唾液样本中cGVHD相关蛋白的表达变化。在本研究中提出唾液并不仅仅是因为唾液腺方便获取，主要是因为唾液腺是粘膜免疫系统中的淋巴细胞归巢靶点，我们假设GVHD是在这里被激活的（Murai等人，Nat Immunol 4:154-160, 2003）。因此，唾液蛋白组成的改变应在疾病早期发生，并应与疾病强度相关。GVHD的病因预测了唾液炎症细胞因子和相关细胞信号蛋白含量的增加。我们的研究将包括发现和确认/验证阶段。在发现阶段，基于itraq的定量MALDI TOF/TOF质谱技术将用于鉴定cGVHD的潜在候选细胞因子/趋化因子和其他蛋白质生物标志物，这些细胞因子/趋化因子和其他蛋白质生物标志物来自患有或不患有cGVHD的HCT受体静息全唾液。在验证阶段，将使用[ELISA]检测8种候选生物标志物蛋白，以确认合并样本中的质谱结果，并确定个体患者样本中的表达差异幅度和患病率。单个唾液样本中的蛋白质表达变化将用于构建5种蛋白质的生物标志物面板，以评估预测cGVHD的能力。质谱分析将由我们的合作者张静博士指导，张静博士是华盛顿大学神经病理学肖教授，他开发了用于识别中枢神经系统疾病脑脊液生物标志物的质谱技术。我们建议应用他的技术寻找cGVHD的唾液生物标志物。我们有初步的结果表明，控制整个唾液可以分析所提出的质谱程序。提出的最先进的MS蛋白质组学方法应该产生特异性的GVHD生物标志物，这将允许更及时地检测GVHD，从而导致早期治疗，从而降低GVHD在口腔和其他靶组织中的疾病严重程度。由于许多疾病的生物标志物产生于潜在的病理生理机制，因此对这里发现的一系列生物标志物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）是用于治疗血液病和其他疾病的造血细胞移植中常见的、严重的、可能危及生命的并发症。GVHD可作为急性或慢性疾病发生，目前很难预测谁将发展为广泛的疾病。该疾病的病原学建议唾液腺应该在GVHD的早期参与，唾液蛋白的变化可以允许早期发现和治疗，这可以降低疾病的严重性和死亡率，[以及了解疾病的病因]。本提案的目标是发展这样一种唾液测定。

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.